Biology of moderately halophilic aerobic bacteria

MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, 1092-2172/98/$04.0010June 1998, p. 504–544 Vol. 62, No. 2 Copyright © 1998, American Society for Microbiology Biology of Moderately Halophilic Aerobic Bacteria ANTONIO VENTOSA, 1 * JOAQUI ´N J. NIETO, 1 AND AHARON OREN 2 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain, 1 and Division of Microbial and Molecular Ecology, Institute of Life Sciences, and the Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel 2 INTRODUCTION.......................................................................................................................................................505 TAXONOMY AND PHYLOGENY ...........................................................................................................................506 ECOLOGY...................................................................................................................................................................509 Numerical Taxonomy Approaches to the Analysis of Natural Communities of Moderately Halophilic Bacteria.........................................................................................................................................509 (i) Salt lakes and brines....................................................................................................................................509 (ii) Saline soils....................................................................................................................................................511 (iii) Cold saline habitats....................................................................................................................................512 (iv) Alkaline saline habitats..............................................................................................................................513 (v) Salted fish, meat, and other foods .............................................................................................................513 (vi) Unusual habitats .........................................................................................................................................513 Morphological and Physiological Diversity.........................................................................................................513 Measurement of In Situ Activities........................................................................................................................514 Molecular Approaches to the Elucidation of the Community Structure of Halophilic Bacteria in Hypersaline Environments............................................................................................................................514 Competition between Halophilic Bacteria and Archaea....................................................................................514 Biogeochemical Importance...................................................................................................................................515 Bacteriophages ........................................................................................................................................................515 PHYSIOLOGY ............................................................................................................................................................516 Requirement for Salt..............................................................................................................................................516 Minimal and Defined Media.................................................................................................................................516 Tolerance to Heavy Metal Ions.............................................................................................................................517 Internal Ion Concentrations..................................................................................................................................517 (i) Sodium............................................................................................................................................................519 (ii) Potassium......................................................................................................................................................519 (iii) Magnesium...................................................................................................................................................519 (iv) Calcium.........................................................................................................................................................519 (v) Manganese.....................................................................................................................................................519 (vi) Chloride ........................................................................................................................................................519 Ion Pumps in the Cell Membranes......................................................................................................................519 Transport of Organic Compounds .......................................................................................................................521 Organic Osmotic Solutes.......................................................................................................................................521 (i) Amino acids....................................................................................................................................................522 (ii) Glycine betaine.............................................................................................................................................522 (iii) Ectoine and hydroxyectoine.......................................................................................................................523 (iv) Other organic osmotic solutes...................................................................................................................523 (v) Distribution of organic osmotic solutes within moderately halophilic bacteria..................................523 (vi) Uptake and action of exogeneously supplied glycine betaine................................................................523 (vii) Accumulation of other osmotic solutes by transport from the medium.............................................524 ENZYMES ...................................................................................................................................................................524 Cytoplasmic Enzymes.............................................................................................................................................525 Stress Proteins ........................................................................................................................................................526 Membrane-Bound Enzymes...................................................................................................................................526 Extracellular Enzymes............................................................................................................................................526 Amino Acid Composition of Proteins from Moderately Halophilic Bacteria.................................................527 CELL ENVELOPES...................................................................................................................................................527 Polar Lipids.............................................................................................................................................................527 (i) Salinivibrio costicola.......................................................................................................................................528 (ii) Halomonas elongata.......................................................................................................................................528 (iii) Halomonas israelensis ..................................................................................................................................528 * Corresponding author. Mailing address: Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain. Phone: 34-5-455 6765. Fax: 34-5-462 8162. E- mail: [email protected]. 504 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from (iv) “Pseudomonas halosaccharolytica”..............................................................................................................528 (v) Other gram-negative halophilic bacteria...................................................................................................529 (vi) Gram-positive moderate halophiles..........................................................................................................529 (vii) Adaptation of the polar lipid composition to changing salinities.......................................................529 Effect of Salt Concentration on Fatty Acid Composition..................................................................................529 (i) Gram-negative halophilic bacteria..............................................................................................................529 (ii) Gram-positive halophilic bacteria .............................................................................................................530 GENETICS ..................................................................................................................................................................530 Genome Analysis and Physical Maps..................................................................................................................531 Native Plasmids and Derived Cloning Vectors...................................................................................................531 Genetic Transfer .....................................................................................................................................................532 Reporter Genes........................................................................................................................................................533 Isolation of Mutants...............................................................................................................................................534 Genetic Basis of Osmoregulation .........................................................................................................................534 BIOTECHNOLOGICAL APPLICATIONS .............................................................................................................535 Fermented Foods.....................................................................................................................................................535 Enzymes....................................................................................................................................................................535 Compatible Solutes.................................................................................................................................................535 Polymers...................................................................................................................................................................536 Degradation of Toxic Compounds........................................................................................................................536 Other Potential Applications.................................................................................................................................536 CONCLUSION AND FUTURE DIRECTIONS......................................................................................................537 ACKNOWLEDGMENTS ...........................................................................................................................................537 REFERENCES ............................................................................................................................................................537 INTRODUCTION Compared to the extensive literature on the physiology, biochemistry, and ecology of the aerobic red halophilic archaea (family Halobacteriaceae), the aerobic halophilic bacteria have been relatively little studied. Research on the halophilic and halotolerant bacteria often seems to be less glamorous than the study of the archaea, with their unique adaptations, including a highly saline cytoplasm, specialized salt-requiring proteins, and the unique light-driven proton and chloride pumps bacteriorhodopsin and halorhodopsin (171). During early research on the microbiology of hypersaline environments, the halophilic bacteria were often neglected, even though they inhabit a wide range of habitats such as saline lakes, saltern ponds, desert and hypersaline soils, and salted foods, a range much less restricted than the habitats in which the halophilic archaea thrive (276, 284, 285). However, Kushner (168) clearly states: “Though they are less exciting at first glance than the extreme halophiles the moderately halophilic bacteria, and solute-tolerant microorganisms in general, pose quite sufficiently interesting questions, especially those implied by their ability to grow over wide ranges of solute concentrations. Further work on these relatively little-studied microorganisms may be expected to bring dividends in the form of insight on the relation of internal and external solute concentrations, and on the state of cell-associ- ated ions within the cytoplasm. If the last decade has been that of the extreme halophiles, we can hope that the next one will see their more modest, moderate cousins (in the spiritual sense only) take their proper place in the scientific canon.” The moderately halophilic bacteria pose specific questions to the scientist, many of them related to their adaptability to a wide range of salinities. Thus, species such as Salinivibrio costicola and Halomonas halodenitrificans are able to grow over a range of water activities between 0.98 (close to freshwater) to 0.86 (close to saturated NaCl) (168). This in itself is a feat that may be much more difficult to achieve than the rigid, salt-requiring metabolism of the halophilic archaea, which lyse the moment the salt concentration in their environment drops below 10 to 15%. The occurrence of nonpigmented halotolerant bacteria was probably first mentioned in 1919 by LeFevre and Round in their study of the microbiology of cucumber fermentation brines. One of the bacterial groups isolated grew in 0 to 15% NaCl, whereas other bacteria studied exhibited growth over the range of 5 to 25% (178). An early classic study of halophilic bacteria is the work of Hof (127), who inoculated salt mud from a solar salt facility on Java onto a variety of media of different salinities. In addition to red archaeal types, different types of white colonies were isolated, including endosporecontaining Bacillus species able to grow at 24% NaCl. Using media containing between 12 and 18% salt, she isolated a Pseudomonas-type bacterium from salted beans preserved in brines varying in salt concentration from 6 to 29%. This organism, designated Pseudomonas beijerinckii, grew from 3 to 18% salt but not at 0.5%, showing its obligate halophilic character. To quote from Hof’s paper: “it may be concluded that most of the important groups of bacteria are able to live in concentrations up to about 15% salt and that many groups are physiologically active even at much higher salt concentrations.” To describe microorganisms according to their behavior toward salt, different classification schemes have been devised. Although several classifications or categories have been proposed (274, 329, 351), the most widely used is that of Kushner, who defined moderate halophiles as organisms growing optimally between 0.5 and 2.5 M salt (168). Bacteria able to grow in the absence of salt as well as in the presence of relatively high salt concentrations (e.g., 8% in the case of Staphylococcus aureus) are designated halotolerant (or extremely halotolerant if growth extends above 2.5 M). A rare case of a bacterium that requires 2 M salt at least (optimal growth at 3.4 M), such as is exemplified by the actinomycete Actinopolyspora halophila (95), is considered a borderline extreme halophile (137, 168). It should be pointed out that the salt requirement and tolerance of many species vary according to growth conditions such as temperature and medium composition. The growth temperature should be specified, especially for the definition of the lower salt range enabling growth. Thus, Marinococcus halophilus grows at NaCl concentrations as low as 0.01 M at 20°C but at least 0.5 M is required at 25°C (228). Similarly, S. cos- VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 505 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from ticola can grow between 0.5 and 4 M NaCl at 30°C but can grow down to 0.2 M at 20°C (168). TAXONOMY AND PHYLOGENY Moderately halophilic bacteria constitute a heterogeneous physiological group of microorganisms which belong to different genera. Our current knowledge of the taxonomic status of these bacteria contrasts with early studies, since in 1980 only six moderately halophilic species were included in the Approved Lists of Bacterial Names (312) and most strains used in physiological and biochemical studies were isolated from salted cured foods or unrefined salt or were even laboratory culturecontaminants. Thesemoderatehalophiles wereVibrio (Salinivibrio)costicola (314), Micrococcus (Nesterenkonia)halobius (237), Paracoccus (Halomonas)halodenitrificans (159), Flavobacterium (Halomonas)halmephilum (60), Planococcus (Marinococcus)halophilus (228), and Spirochaeta halophila (103). During the last decade, the extensive studies on hypersaline environments that have been carried out in many geographical areas have permitted the isolation and taxonomic characterization of a large number of moderately halophilic species. Thus, moderate halophiles are represented by several methanogenic archaea as well as strictly anaerobic bacteria that have been reviewed recently (182, 232) and are not included in this article. However, most species are gram-negative or gram-posi- tive aerobic or facultatively anaerobic moderately halophilic bacteria (340, 341). Although some gram-negative species were considered members of different genera (Halomonas,Deleya, Volcaniella,Flavobacterium,Paracoccus,Pseudomonas,Halovibrio,orChromobacterium), phenotypic and phylogenetic data support their close relationship, and they are currently included in the family Halomonadaceae as members of two genera: Halomonas and Chromohalobacter (53, 76). Table 1 shows the featuresthatdifferentiatethevalidlypublishedmoderatelyhalophilic species included in these two genera. Besides members of the family Halomonadaceae, several other gram-negative strictly aerobic or facultatively anaerobic species have been described as moderate halophiles belonging to genera that include nonhalophilic species as well, such as Pseudomonas,Flavobacterium,orSpirochaeta, while others are placed in genera represented, at least until now, exclusively by halophilic species: Salinivibrio,Arhodomonas,orDichotomicrobium. The differential features of these species are shown in Table 2. Three organisms have been extensively used in physiological and biochemical studies dealing with the mechanisms of haloadaptation and adaptability: Salinivibrio costicola,Halomonas elongata, and Halomonas israelensis. S. costicola was originally isolated from rib bones in a sample of Australian bacon (91, 314). Similar strains have been isolated from saltern ponds near Alicante (96) and other locations in Spain and the Canary Islands (91). H. elongata was isolated from a solar salt facility on Bonaire, Netherlands Antilles. It is an extremely versatile organism, able to grow at a very wide range of salt concentrations. It can also grow anaerobically with nitrate as the electron acceptor, TABLE 1. Main characteristics of moderately halophilic gram-negative members of the Halomonadaceae a Feature H. elongata (357) H. subglaciescola (74) H. halodurans (115) H. halmophila (54) H. eurihalina (200, 266) H. halophila (269) Morphology Rods Rods Rods Rods Short rods Rods Size (mm) ND 0.5–1.1 35–10 0.4–0.6 31.5–2.0 0.3–0.6 30.9–1.3 0.8–1.0 32.0–2.5 0.5–0.7 31.5–2.0 Pigmentation None Cream None Cream Cream Cream Motility 11 112 1 Facultative anaerobe 12 222 2 Oxidase 11 112 1 NaCl range (%) 3.5–20 0.5–20 3.5–20 0.5–20 5–20 2–30 NaCl optimum (%) 3.5–8 ND 8 ND 7.5 7.5 Temp range (°C) 15–45 0–25 4–37 ND 15–45 15–45 pH range 5–9 5–9 5.5–8.5 ND 5–10 5–10 Acid production from: Arabinose 22 1 ND 22 Glucose 11 112 1 Lactose 21 222 2 Trehalose ND ND ND ND 2ND Mannitol 22 212 2 Hydrolysis of: Gelatin d 2 221 2 Casein 2dND22 2 Starch 22 222 2 Esculin 22 121 1 Tween 80 22 ND 21 2 DNA 22 ND 11 2 H 2 S production 22 2 ND 11 Nitrate reduction 11 221 1 Nitrite reduction 12 222 2 Phosphatase ND 2 221 1 Type strain ATCC 33173 UQM 2927 ATCC 29686 ATCC 19717 ATCC 49339 CCM 3662 Habitat Salterns Antarctic saline lakes Estuarine water Dead Sea Saline soils, salterns Saline soils G1C content (mol%) 60.5 60.9–62.9 63.2 63 59.1–65.7 66.7 Phylogenetic branch Gamma Proteobacteria Gamma Proteobacteria Gamma Proteobacteria Gamma Proteobacteria Gamma Proteobacteria Gamma Proteobacteria a 1, positive; 2, negative; d, differs among strains; ND, not determined. 506 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from forming nitrite, and it has also been reported to grow fermentatively on glucose (357). However, glucose fermentation was not confirmed in later studies (83). H. israelensis (previously designated strain Ba 1 ) was originally obtained from unrefined solar salt obtained from the Dead Sea (272, 273) and was only recently assigned a species name (128). The gram-positive moderately halophilic aerobic bacteria, with the exception of two Bacillus species, belong to genera that include only species with halophilic requirements: the genera Halobacillus,Marinococcus,Salinicoccus,Nesterenkonia, and Tetragenococcus. The validly described species currently accepted as moderate halophiles and their characteristics are shown in Table 3. Finally, there are some moderately halophilic actinomycetes that have been recently isolated from different saline soil samples and have been characterized as species of the genera Actinopolyspora or Nocardiopsis. Table 4 shows the differential characteristics of these four moderately halophilic species. Besides these moderately halophilic species that have been characterized taxonomically and, according to the rules of the code of nomenclature of bacteria, are considered validly described species, there are several other moderately halophilic aerobic bacteria that have been used for other purposes, including physiological, biochemical, and biotechnological studies, but have not been studied taxonomically in detail. Typical examples are “Pseudomonas halosaccharolytica” (120, 121) and “Micrococcus varians subsp. halophilus” (241). Recent studies based on 16S rRNA sequence analysis have permitted a determination of the phylogenetic position of most moderately halophilic bacteria. During last decade, the old technique of comparison of 16S rRNA oligonucleotide catalogs showed that Spirochaeta halophila belongs to the spirochete phylum and that some Halomonas and Deleya species are members of the gamma subclass of the Proteobacteria (341). They were placed in a new family, Halomonadaceae (76). More recently, complete 16S rRNA sequence analysis confirmed that Spirochaeta halophila is within the Spirochaeta cluster of the spirochete phylum, related to Spirochaeta isovalerica,S. litoralis,S. bajacaliforniensis, and S. aurantia (average similarity, 87.4%) (253). In addition, several studies have identified the phylogenetic position of most gram-negative moderately halophilic aerobic species currently described. Members of the genera Halomonas,Deleya,Halovibrio, and Volcaniella, as well as Paracoccus halodenitrificans, form a monophyletic group within the gamma subclass of the Proteobacteria (53, 56, 200, 204, 290). The levels of 16S rRNA sequence similarity among these species ranged from 91.5 to 100%; although several subgroups, which might represent separate genera, were resolved, they could not be differentiated on the basis of phenotypic or chemotaxonomicfeatures.Forthesereasons,DobsonandFranzmann (53) proposed placing all members of the above four genera and P. halodenitrificans in a single genus, the genus Halomonas, and emended the description of the family Halomonadaceae. This family now comprises the species of Halomonas and Zymobacter and the moderate halophile, originally isolated from the Dead Sea, Chromohalobacter marismortui (200). All have 15 signature characteristics in their 16S rRNA TABLE 1—Continued H. salina (53, 336) H. halodenitrificans (53, 159) H. variablis (53, 65) H. canadensis (128) H. israelensis (128) H. pantelleriense (290) Chromohalobacter marismortui (344) Short rods Short rods Curved rods Rods Rods Rods Rods 0.7–0.8 32.0–2.5 0.5–0.9 30.9–1.2 0.5–0.8 31.0–3.0 ND ND 0.4–0.7 31.4–2.6 0.6–1.0 31.5–4.0 Yellowish or cream Cream Cream White Cream Cream-pink Brown-yellow 2221ND 11 2222222 1112222 2.5–20 3–20 7–28 3–25 3.5–20 1.2–15 1–30 5 5–9 10 8 8 10 10 15–40 5–32 15–37 15–30 15–45 10–44 5–45 6–10 ND 6.5–8.4 5–9 5–9 7.5–11 5–10 2ND ND ND ND ND 1 22ND ND ND ND 1 2ND ND 22ND 1 2ND ND ND ND ND 1 2ND ND ND ND ND ND 2222ND 22 2ND ND 2ND 22 2222ND 22 221ND ND ND 2 22ND ND ND ND 2 2ND ND ND ND ND 2 12222ND 2 1121111 21222ND 2 22ND ND ND ND 2 ATCC 49509 ATCC 13511 DSM 3051 ATCC 43984 ATCC 43985 DSM 9661 ATCC 17056 Saline soils, salterns Meat-curing brines Great Salt Lake Unknown (contaminant) Dead Sea Alkaline saline lake Dead Sea, salterns 60.7–64.2 64–66 61 57 64 65 62.1–64.9 Gamma Proteobacteria Gamma Proteobacteria Gamma Proteobacteria ND ND Gamma Proteobacteria Gamma Proteobacteria VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 507 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from TABLE 2. Main characteristics of other aerobic or facultatively anaerobic gram-negative moderately halophilic bacteria a Feature Salinivibrio costicola (91, 201) Pseudomonas halophila (65) Flavobacterium gondwanense (52) Flavobacterium salegens (52) Arhodomonas aquaeolei (4) Spirochaeta halophila (103) Dichotomicrobium thermohalophilum (124) Morphology Curved rods Rods Rods Rods Short rods Helicoidal Prosthecate Size (mm) 0.5 31.5–3.2 0.8–1.0 31.5–5.0 0.4–0.8 31.7–11.7 0.5–0.8 31.2–11.5 0.8–1.0 32.0–2.5 0.4 315–30 0.8–1.8 30.8–2.0 Pigmentation Cream Reddish-brown Orange Yellow None Red Reddish-brown Motility 1 1221 12 Facultative anaerobe 1 2222 12 Oxidase 1 1111 ND 1 NaCl range (%) 0.5–12 0.1–20 0–5 0–20 6–20 0.5–8 0.8–22 NaCl optimum (%) 10 5 5 5 15 5 8–14 Temp range (°C) 5–45 4–37 0–30 ND 20–45 25–40 20–60 pH range 5–10 4.5–9.6 5–9 5–9 6–8 5.8–9.5 Acid production from: Arabinose 2ND 212 ND ND Glucose 1ND 212 12 Lactose 2ND 212 ND ND Trehalose 1ND ND ND 2ND ND Mannitol 1ND ND 12 ND ND Hydrolysis of: Gelatin 11d12 ND 2 Casein 1 1222 ND 2 Starch 2 2112 ND 2 Esculin 1 1112 ND ND Tween 80 d ND 111 ND ND DNA d ND 112 ND 2 H 2 S production d 2222 ND 2 Nitrate reduction 2 2211 12 Nitrite reduction 2 2222 ND 2 Phosphatase 2ND 112 ND ND Type strain NCIMB 701 DSM 3050 DSM 5423 DSM 5424 ATCC 49307 ATCC 29478 DSM 5002 Habitat Salterns Great Salt Lake Hypersaline Antarctic lake Hypersaline Antarctic lake Petroleum reservoir producing fluid Solar Lake Solar Lake G1C content (mol%) 49.4–50.5 57.0 35–39 39–41 67 62 62–64 Phylogenetic branch Gamma Proteobacteria ND Flavobacterium- Bacteroides Flavobacterium- Bacteroides Gamma Proteobacteria Spirochetes ND a 1, positive; 2, negative; d, differs among strains; ND, not determined. 508 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from sequences, including a distinctive cytosine residue at position 486 (53). Arhodomonas aquaeolei represents a deeply branching lineage in the gamma subclass of the Proteobacteria, most closely related to purple sulfur bacteria (particularly species of the genera Ectothiorhodospira and Chromatium). The 16S rRNA sequence analysis supports the placement of this single species in a separate genus (4). Very recently, the phylogenetic position of six (Salini)vibrio costicola strains revealed that this moderate halophile constitutes a monophyletic branch that is distinct from other Vibrio species and from other species belonging to the gamma subclass of the Proteobacteria (202). Since other phenotypic and genotypic data supported these differences, placement of this species in a separate genus, Salinivibrio, has been proposed (201). The 16S rRNA sequences of Flavobacterium gondwanense and F. salegens, two moderate halophiles isolated from a hypersaline Antarctic lake, contain the definitive flavobacterial signatures that unequivocally place them in the Flavobacterium- Bacteroides phylum (52). These species cluster with a group of organisms that contains the type species of the genus Flavobacterium,F. aquatile (with 89 and 90% sequence similarity between them and this species) (52). Recent studies have determined the phylogenetic relationships of moderate halophiles within the gram-positive branch. Farrow et al. (64) showed that Marinococcus halophilus (formerly Planococcus halophilus) forms a distinct line of descent and is only distantly related to the genera Planococcus,Sporosarcina, and Bacillus. The 16S rRNA sequence data confirm the placement of M. halophilus in a separate genus. In addition, they indicated that Sporosarcina halophila, an endospore-form- ing motile gram-positive moderate halophile, was not closely related to Sporosarcina ureae, the type species of this genus. Later studies permitted the placement of S. halophila in a new genus, Halobacillus,asH. halophilus, closely related to other moderately halophilic species isolated from the Great Salt Lake, H. litoralis and H. trueperi (315). This study also confirmed the placement of Salinicoccus roseus in a separate genus, since it constitutes a deep branch not closely related to other gram-positive bacteria. Bacillus salexigens is closely related to Bacillus pantothenticus, a species that belongs to phylogenetic group I of the genus Bacillus, as well as to Halobacillus halophilus,H. litoralis, and the halotolerant species Bacillus dipsosauri (89). Tetragenococcus muriaticus, a recently described species isolated from a traditionally fermented Japanese fish sauce, is closely related to the halotolerant species Tetragenococcus halophilus (formerly Pediococcus halophilus), which showed a closer phylogenetic relationship to other lactic acid bacteria of the enterococci and lactobacilli than to pediococci (304). While all these moderately halophilic species belong to the low-G1C group of the gram-positive phylum, only Nesterenkonia halobia (formerly Micrococcus halobius) is within the high-G1C group (218, 316), constituting a cluster that is clearly separate from other species of the genera Micrococcus, Arthrobacter, and Kocuria. The recent phylogenetic studies of moderate halophiles have been based on the comparison of 16S rRNA sequence data. These studies have been very helpful for improving the classification of moderate halophiles according to a natural (phylogenetic) approach. In particular, results show that they are represented in many of the major bacterial phyla: spirochetes, Proteobacteria,Flavobacterium-Bacteroides, and low-G1C and high-G1C gram-positive organisms. A recent study of African soda lakes showed a wide phylogenetic diversity within the alkaliphilic (and in some cases halophilic) isolates (58). As with the moderate halophiles, these authors concluded that the alkaliphile phenotype is also polyphyletic and might have evolved many times (58). ECOLOGY Numerical Taxonomy Approaches to the Analysis of Natural Communities of Moderately Halophilic Bacteria (i) Salt lakes and brines. The communities of moderately halophilic bacteria in thalassohaline (seawater-derived) hypersaline environments, such as saltern ponds for concentrating seawater, may to a large extent resemble the communities present in seawater. This is not too surprising, since many marine bacteria have a broad salt tolerance. It was even reported that the majority of 30 isolates of marine aerobic heterotrophic bacteria tested could grow at up to 20% NaCl and that some could even multiply in media containing 30% NaCl (72). Moderately halophilic bacteria could also be enriched from seawater by gradual salinity increases: when seawater was periodically amended with salt and nutrients, moderately halophilic bacteria outcompeted the slightly halophilic marine bacteria, which completely disappeared above 15% salt (347). A variety of halophilic bacteria were also isolated from sea sands and seaweeds (235). Thus, the sea contains many moderately halophilic or at least extremely halotolerant bacteria. In a study of Spanish saltern ponds of intermediate salinity (between 15 and 30% sea salts) (Alicante on the Mediterranean coast, Huelva on the Atlantic coast), the dominant types of colonies developing on agar plates were assigned by numerical taxonomy to the genera Salinivibrio, the Pseudomonas-Alteromonas- Alcaligenes group, Acinetobacter, and Flavobacterium (284– 286). Most of these isolates should probably be reclassified in the family Halomonadaceae. Most isolates grew optimally at about 10% salts (the concentration that also yielded the largest number of CFU on agar plates) and could be found at salt concentrations up to about 25%. (Salinivibrio species dominated below 15% salt, while bacteria assigned to the Pseudomonas-Alteromonas-Alcaligenes group were especially abundant above 15%. Flavobacterium and Acinetobacter were found in smaller numbers and were evenly distributed up to 30%, while gram-positive cocci were found mostly above 25% salt (286). Most of the 140 isolates from the Huelva salterns clustered in eight phenons, two of which were identified as Halomonas (Deleya) (53), four resembled S. costicola, and the others were tentatively assigned to the genera Flavobacterium and Acinetobacter (187). Similar Acinetobacter-like bacteria (probably to be assigned to the Halomonadaceae) have been found in other hypersaline habitats (265). Numerical taxonomic studies have been performed in inland, athalassohaline salterns near Granada, Spain (39, 41, 274), and Chile (262). The La Mala´ saltern near Granada is fed by brines from a subterranean well which is lower in Cl 2 and higher in Mg 21 ,Ca 21 , and K 1 than seawater. Of the 174 strains isolated, 74 were assigned to the genus (Salini)vibrio,22 were assigned to Alteromonas, 43 were assigned to Halomonas (Deleya) (53), 7 were assigned to Acinetobacter, 13 were assigned to Pseudomonas, and 9 were assigned to Flavobacterium (37). An even more unusual inland saltern is the Salar de Atacama, Chile, located in the Atacama desert at 2,700 m above sea level. The most abundant isolates obtained from this environment were (Salini)vibrio strains, followed by Acinetobacter,Marinomonas, and Alteromonas (262). Lake Assal in Djibouti (French Somaliland) contains 27.7% salts in its surface layers, increasing to 39.8% at a depth of 20 m. Of the 164 isolates obtained, 11 were moderate halo- VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 509 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from philes requiring 3 to 15% salt, 7 were extremely halophilic archaea, 2 did not tolerate salt, and the great majority were slightly halophilic, requiring 1 to 5% NaCl (16). The authors concluded that “it is clear that the bacteria were, on the whole, common bacteria belonging to the same species which are currently isolated from large and small rivers and from seawater.” This result is unexpected in view of the high salt concentrations prevailing in Lake Assal and the high temperature of the water (33 to 34°C), which seem more suitable to the development of extremely halophilic archaea than of marine-type slightly or moderately halophilic bacteria. Regretably, additional studies of this interesting but poorly accessible environment have not been reported. A sadly neglected hunting ground for interesting new types of moderately halophilic bacteria is the Great Salt Lake, Utah, which has seen drastic changes in its salinity during the last decades. Most studies of its microbiology have centered around the red halophilic archaea and the Dunaliella communities (244, 258, 261). Attempts to enumerate viable bacteria in complex medium with 5, 13, and 20% NaCl yielded 3.6 310 5 , 9.3 3 10 5 , and 2.4 310 6 CFU/ml, respectively, with surface water from the north arm of the lake (22% salt), and 7.5 310 5 , 1.1 3 10 5 , and 1.5 310 3 cells/ml, respectively, when surface water from the south arm (8.5% salt) served as the inoculum (65). The data presented do not allow a differentiation between colorless bacteria and red archaea, but it was stated that moderately halophilic bacteria make up the predominant population in the south arm whereas extremely halophilic archaea predominate in the north arm. The Great Salt Lake yielded a number of new species: Pseudomonas halophila,Halomonas variabilis (53, 65), Halobacillus litoralis, and Halobacillus trueperi (315). Although halophilic bacteria can easily be isolated from the Dead Sea by means of enrichment cultures (242, 349), no systematic studies have been performed on their abundance in this extremely hypersaline (presently around 34%), athalassohaline (around 1.8 M Mg 21 ,1.7MNa 1 ,0.4MCa 21 , and 0.14 MK 1 ) chloride lake. The first prokaryotes isolated from the TABLE 3. Main characteristics of aerobic gram-positive moderately halophilic bacteria a Feature Halobacillus Bacillus H. halophilus (33, 315) H. litoralis (315) H. trueperi (315) B. halophilus (342) B. salexigens (89) Morphology Coccoid Rods Rods Rods Rods Size (mm) 1.0–2.0 32.0–3.0 0.7–1.1 32.0–4.5 0.7–1.4 32.0–4.5 0.5–1.0 32.5–9.0 0.3–0.6 31.5–3.5 Pigmentation Orange Orange Orange None None Spore shape S E/S E/S E E Spore position C/ST C/ST C/ST C ST/C Sporangium swollen 2ND ND 21 Motility 1 111 Facultative anaerobe 2 222 2 Oxidase 1 111 1 NaCl range (%) 2–20 0.5–25 0.5–30 3–30 7–20 NaCl optimum (%) 10 10 10 15 10 Temp range (°C) 15–40 10–43 10–44 15–50 15–45 pH range 7–9 6–9.5 6–9.5 6–8 6–11 Acid production from: Arabinose 2ND ND 2ND Glucose 2 111 1 Lactose ND ND ND 22 Trehalose 2 111 2 Mannitol 2 122 1 Hydrolysis of: Gelatin 1 112 1 Casein 1 222 1 Starch 1 222 2 Esculin 2 221 1 Tween 80 222ND 2 DNA 1 111 1 H 2 S production ND ND ND 21 Nitrate reduction 2 222 2 Nitrite reduction 2 222 2 Phosphatase d 222 d Voges-Proskauer 2 222 2 Menaquinone system MK-7 ND ND MK-7 ND Cell wall type Orn–D-Asp Orn–D-Asp Orn–D-Asp m-Dpm m-Dpm Type strain DSM 2266 DSM 10405 DSM 10404 ATCC 49085 ATCC 700290 Habitat Saline soils, salterns Great Salt Lake Great Salt Lake Unknown (rotting wood from seawater) Salterns, saline soils G1C content (mol%) 40.1–40.9 42 43 51.5 36.3–39.5 Phylogenetic branch Low-G1C grampositive Low-G1C grampositive ND ND Low-G1C grampositive a S, spherical; E, ellipsoidal; C, central; ST, subterminal. 1, positive; 2, negative; d, differs among strains; ND, not determined. MK-9, MK-8, MK-7, MK-6, menaquinone with nine, eight, seven, and six isoprene units, respectively. m-Dpm, meso-diaminopimelic acid. 510 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from lake (which at the time contained 28 to 29% salt) were probably colorless bacteria (362). Some of the early isolates were characterized further and described as Chromohalobacter (originally Chromobacterium)marismortui (343), “Pseudomonas halestorgus” (subsequently lost), and Halomonas (originally Flavobacterium)halmophila (54, 60, 350). Another colorless isolate from the Dead Sea is Halomonas israelensis (also known as strain Ba 1 ) (128, 170). Subterranean brines have also been subject to microbiological studies. Several euryhaline microorganisms belonging to the Halomonadaceae, possibly including some novel species, were isolated from Permian underground salt formations in the United States which were penetrated by meteoric waters which slowly solubilize the salt (356). A subterranean brine associated with an oil field in Oklahoma yielded Arhodomonas aquaeolei, an aerobic, organic acid-metabolizing, gram-nega- tive, motile rod, growing in the range of 6 to 20% NaCl with an optimum of 15% (4). (ii) Saline soils. The soil habitat is inherently inhomogeneous, and it can be expected that a wide range of salinities might be present in any one saline soil (101). Saline soils appear to yield mostly halotolerant rather than halophilic microorganisms, presumably reflecting adaptation to periodic episodes of relatively high dilution (267, 268). One early study stated that microorganisms may be unable to multiply in saline soil and that the microbiota of saline soil habitats are passive inhabitants brought by the wind. In this study, soils near the Red Sea, with a salt content varying from 25 to 30% on the surface to 1.5 to 2% at a depth of 50 cm, were examined. The highest bacterial counts were obtained in surface soil, using media without NaCl. The numbers obtained were much smaller than those commonly found in nonsaline soils. The authors stated that “the accepted assumption about the widespread distribution of salt-resistant and halophilic microorganisms in saline soils requires reconsideration” (116). Later studies have unequivocally confirmed the abundance of halophilic bacteria in saline soils. The species composition in soils differs greatly from that of the aquatic environments discussed above: while Salinivibrio abounds in salt lakes and salterns, the dominant types encountered in saline soils belong to genera such as Bacillus,Pseudomonas,Alcaligenes (last two probably to be reclassified as members of the Halomonada- TABLE 3—Continued Marinococcus Salinicoccus Nesterenkonia halobia (218, 316) Tetragenococcus muriaticus (304) M. halophilus (111, 189) M. albus (111) S. roseus (344) S. hispanicus (188, 345) Cocci Cocci Cocci Cocci Cocci Cocci 1.0–1.2 1.0–1.2 1.0–2.5 1.0–2.0 ND 0.5–0.8 Yellow-orange None Pink red Reddish-orange None or yellow White 222222 222222 222222 112222 222221 211112 2–25 2–25 0.9–25 0.5–25 3–25 1–25 10 10 10 10 10 7–10 15–37 15–37 15–40 15–37 20–40 15–40 ND ND 6–9 5–9 5–10 5–9.6 222222 122111 222222 122221 122121 12111ND 12121ND 22121ND 12211ND 22122ND 21111ND 22222ND 212122 222122 221d2ND 22222ND MK-7 MK-7 MK-6 MK-6 MK-9, MK-8, MK-7 ND m-Dpm m-Dpm L-Lys–Gly 5 L-Lys–Gly 5 L-Lys–Gly–L-Glu Lys–D-Asp ATCC 27964 CCM 3517 ATCC 49258 ATCC 49259 ATCC 21727 JCM 10006 Salterns, saline soils Salterns Salterns Salterns, saline soils Salterns Fermented squid liver sauce 46.4 44.9 51.2 45.6–49.3 ND 36.5 Low-G1C grampositive ND Low-G1C grampositive ND High-G1C grampositive ND VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 511 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from ceae), and Micrococcus (possibly Nesterenkonia) (268, 285). A saline soil near Alicante, Spain, with a Cl 2 content between 2.4 and 12.7% yielded a high proportion of nonmotile rods on media containing 10 or 20% salt; this type of bacterium is only rarely isolated from aquatic hypersaline environments (265). The most abundant types were assigned to the genera Alcaligenes,Acinetobacter,Flavobacterium, and Pseudomonas. Many halotolerant gram-positive bacteria were also found. Facultative anaerobes were rare. Most isolates required moderately high salt (5 to 10%) for optimal growth but were also able to grow at low salinities (0.5 to 2%) (267). This probably reflects the ecological difference between water and soil environments, with water being relatively homogeneous and constant and soil being heterogeneous and affected by factors such as rainfall (267, 268). A study was made of the rhizosphere soil near xerophytic plants growing in hypersaline soils (5 to 10.7% NaCl) near Alicante, showing that the range of salt concentrations allowing growth of the organisms isolated did not correlate with the salinity of the soil from which they were isolated. Most isolates showed salt optima between 5 and 15% NaCl, but about half of the strains also grew at 0.9% NaCl. Plating on agar media containing 10% salt yielded mostly gram-positive rods, whereas gram-negative rods dominated between 10 and 20% salt and Gram-positive cocci developed above 20% salt. The gram-positive bacteria thus isolated were assigned to the genera (in order of abundance) Bacillus,Micrococcus,Arthrobacter,Staphylococcus,Planococcus,Corynebacterium,Nocardia, and Actinomyces (268). Saline soils have been somewhat neglected compared to hypersaline aquatic environments. The recent isolation of novel halophilic Actinopolyspora and Nocardiopsis species from salty soils in Death Valley (Calif.), Alicante, and Iraq (6, 291, 370, 373) suggests that a wealth of interesting unknown halophilic microorganisms may be present in these soils. (iii) Cold saline habitats. Extensive microbiological studies in the Antarctic, especially the cold saline lakes in the Vestfold Hills region and the saline soils of the Dry Valleys, have contributed some interesting insights into the extreme conditions under which halophilic bacteria may occur and thrive. The Vestfold Hills region is a coastal, ice-free area in east Antarctica, which contains in excess of 300 lakes and ponds. These are relics of seawater catchments isolated some 6,000 years ago by uplift and trapped in valleys and depressions. Some of these lakes are hypersaline; the most saline lakes have a total salt concentration of up to 28%. Salinity may show seasonal variations due to meltstream influx and ice cover melt in the Austral summer. The best studied is Organic Lake, a meromictic lake with a maximum depth of 7.5 m. The lake is stratified, with salt concentrations increasing from 0.8 to 21%, and is anoxic below a depth of 4 to 5 m. The ice cover excludes wind-induced turbulence throughout winter. Thermal profile and the increasing salinity with depth prevent turnover in the ice-free summer period. Temperatures range from 214 to 115°C (73, 195, 196). Many strains of moderate halophiles, belonging to genera including Halomonas,Flavobacterium, and Cytophaga, were isolated from the lake. Hexadecane- and phenanthrene-degrading bacteria were also found (196). Most isolates were able to grow from 0.5 to 20% NaCl and at temperatures as low as 0 to 5°C (55, 74). The species isolated include Halomonas subglaciescola, growing at salt concentrations between 0.5 and 20%, with a predicted minimum temperature of 23.3 to 29.2°C, an op- TABLE 4. Main characteristics of moderately halophilic actinomycete species a Feature Actinopolyspora Nocardiopsis A. mortivallis (374) A. iraquiensis (292) N. lucentensis (371) N. halophila (6) Spore chain morphology Short (no more than 10) Short (no more than 15) Short Very long Pigmentation Yellowish white Yellow-brownish Yellowish brown Yellow-coral red Spore Oval to cylindrical Spheroidal Cylindrical Cylindrical NaCl range (%) 5–30 5–20 ND 5–20 NaCl optium (%) 10–15 10–15 5–10 15 Temp range (°C) 10–50 16–40 ND 16–40 Acid production from: Glucose 1ND 12 Mannitol 2111 Xylose 1221 Nitrate reduction 2ND 1ND Hydrolysis of: Gelatin 1ND 1ND Casein 1ND 1ND Esculin ND 212 Starch ND 112 Tyrosine ND 112 Tween 80 11ND 1 Urea 2221 Xanthine 1111 Menaquinone system MK-9 (H 4 ), MK-10 (H 4 ) MK-9 (H 4 ) MK-10 (H 8 ) MK-10 (H 6 ,H 8 ) Cell wall type IV IV III III Phospholipid type PIII PIII PIII PIII Mycolic acids 2222 Type strain JCM 7550 A.S.4.1193 DSM 44048 A.S.4.1195 Habitat Saline soil Saline soil Saline soil Saline soil a 1, positive; 2, negative; ND, not determined. MK-10 (H 8 ,H 6 , and H 4 ), octa-, hexa-, and tetrahydrogenated menaquinones with 10 isoprene units; MK-9 (H 4 ), tetrahydrogenated menaquinone with 9 isoprene units. 512 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from charolytica,” the apparent internal salt concentration was relatively independent of the salt concentration in which the cells were grown, but in this organism the measured sum of the intracellular concentrations was quite high (1.4 to 1.9 M Na 1 1K 1 in cells grown between 1 and 3 M NaCl) (192). In S. costicola, the intracellular Na 1 1K 1 concentration was significantly lower than outside only at the highest medium salinities. However, upon treatment with cetyltrimethylammonium bromide, the cellular Na 1 and K 1 concentrations did not equilibrate with the external medium, possibly indicating that the ions may partially occur in a bound state (308). Below we summarize a few data on the intracellular abundance of individual ions. (i) Sodium. The apparent intracellular Na 1 concentrations are often far too high to enable the generally salt-sensitive cytoplasmic enzymes to be active (see below). However, the assessment of the true intracellular Na 1 concentration is problematic, as discussed above. In addition, Na 1 and other ions may be bound to the outer cell layers, in amounts increasing with external salinity (133). It was thus suggested that much of the cell-associated Na 1 in “P. halosaccharolytica” is not cytoplasmic (192). (ii) Potassium. In most halophilic bacteria, K 1 is accumulated to a few tenths of 1 M (133). H. elongata seems to be an exception, with K 1 concentrations as low as 20 mM (359). In any case, in contrast to some archaeal halophiles, cytoplasmic potassium contributes relatively little to the achievement of an osmotic balance. (iii) Magnesium. Intracellular Mg 21 concentrations have been determined only seldom in halophilic bacteria. In H. elongata grown in medium containing 24 mM Mg 21 , the intracellular Mg 21 concentrations varied from 9 to 23 mM depending on the growth conditions (359), which is not particularly high compared to 30 mM in Escherichia coli and 102 mM in Halobacterium salinarum (cutirubrum) (45, 46). (iv) Calcium. H. elongata, growing in media with 0.7 mM Ca 21 and in the presence of 0.05 to 3.4 M NaCl, was reported to contain between 3.1 and 12 mM Ca 21 intracellularly, most of it probably bound (353). (v) Manganese. A single measurement of intracellular Mn 21 concentrations in S. costicola (by using 54 Mn added to the medium as label) gave a value of 0.6 mM Mn 21 /kg of cell water (46). (vi) Chloride. Estimations of intracellular chloride concentrations within cells of moderately halophilic bacteria are greatly variable, from relatively low values (55 and 139 mM in H. halodenitrificans and S. costicola, respectively, grown in 1 M NaCl) (31) to values as high as 0.7 to1Min“P. halosaccharolytica” grown at NaCl concentrations between 1 and 3 M (192). The measured intracellular Cl 2 concentrations are in most cases much lower than the combined Na 1 and K 1 concentrations (133). Thus, S. costicola grown in 1 M NaCl and 6 mM KCl will have a combined intracellular Na 1 and K 1 concentration of about 0.6 M but only about 0.1 to 0.2 M Cl 2 (171). When the cells are grown at higher salt concentrations, the apparent intracellular Cl 2 concentration may be increased (1.5 M in cells grown in 3 M) (141). The old assumption that Cl 2 is the main counterion for the intracellular cations in moderate halophiles is not necessarily true, and since no other anions have been detected at high concentrations within the cells, it has been speculated that most of the cellular cations may be associated with negative charges present on proteins, cell envelopes, and other macromolecules (170–172). To verify the theory that part of the Na 1 and other cations may be tightly bound to different cellular structures, bulk analysis of ions is insufficient (160). Indications that a significant fraction of the intracellular Na 1 does not occur freely dissolved in the cytoplasm but is rather strongly bound, came from a number of studies in which 23 Na nuclear magnetic resonance spectroscopy (NMR) was used to obtain information on the state of the Na 1 ions. Sodium NMR relaxation times can indicate changes in the intracellular environment in which the Na 1 is embedded. The need for thick cell suspensions, easily leading to the development of anoxic conditions, is a problem inherent in this method (94). By using NMR to probe the ionic environment inside H. israelensis cells, three types of cell-associated Na 1 were detected. One fraction (about 40% of the total) is free, dissolved in the cytosol, and can exchange with the extracellular Na 1 . The other 60% is bound; part of it could exchange with the “free” intracellular Na 1 , while another part could not exchange and was “invisible” to the NMR. Since the shift reagent [dysprosium bis(tripolyphosphate)] used to discriminate between extracellular and cell-associated Na 1 was not effective at the highest NaCl concentrations, the approach could be applied up to about 2 M salt only. In the concentration range tested, the “NMR-visible” intracellular Na 1 concentration never exceeded 15 to 20% of the medium concentration (97, 299). It should be noted that the use of shift reagents may lead to faulty results as they form complexes and therefore disturb the equilibrium across the membrane. The state of intracellular lithium was also investigated by NMR. Li 1 can replace part of the intracellular Na 1 . The interaction of Li 1 ions with the bacteria is different in cells grown in high salt and in low salt: in low salt (0.5 M), almost all (92%) of the Li 1 was “seen” by the NMR, while in 3 M salt-grown cells, only 55% of the Li 1 was detected. Goldberg et al. suggested that three types of Li 1 ions existed: free, weakly bound, and strongly bound (98). NMR methods were also applied to study S. costicola grown at NaCl concentrations between 0.6 and 2 M. Apparent intracellular Na 1 concentrations (excluding strongly bound Na 1 which is not detected by the NMR) were always below 25% of the medium concentration (94). It was calculated that the fraction of the free intracellular Na 1 was about 40% of the total, which yielded an estimate of the total intracellular Na 1 concentrations as being between 0.14 and 0.435 M (94), much lower than those obtained by bulk analyses in cell pellets (308). Additional evidence for a substantial binding of Na 1 ions to cellular structures came from a study in which cell envelopes of “P. halosaccharolytica” were incubated in 2 M NaCl and 5.5 mM KCl. It was found that 149 mg of Na 1 and 14 mg of K 1 were bound per g of protein, about twice as much Na 1 and eight times less K 1 as found per gram of protein in whole cells (192). It has often been speculated whether the exclusion of salt and the maintenance of a steep ionic gradient by the moderately halophilic bacteria is achieved through the constant pumping of ions by active, energy-dependent mechanisms (see below) or by tightening of the permeability barrier of the membrane. This important question does not seem to have been pursued in depth. Continuous-culture experiments with H. elongata strongly indicate that the maintenance energy of these organisms is relatively independent of the salinity of the medium, providing evidence in favor of an ion-tight membrane (82). Ion Pumps in the Cell Membranes A low intracellular ionic environment can be achieved only by energy-dependent mechanisms. This was recognized as early as 1954 by Baxter and Gibbons: “...adaptation at the VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 519 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from cellular level, in a mechanism that is probably energy-depen- dent that maintains the intracellular salt concentration at a level considerably below that of the environment” (9). To achieve low intracellular Na 1 ion concentrations against a constant influx of Na 1 ions leaking inside through a not completely impermeable membrane, in addition to Na 1 entering the cells during cotransport with amino acids and other substrates (see below), mechanisms of Na 1 extrusion have to be present in the cell membrane. Two possible mechanisms have been suggested: activity of Na 1 /H 1 antiport and presence of a primary respiration-driven Na 1 pump. It is generally accepted that Na 1 /H 1 antiport activity is an important mechanism for maintaining low intracellular sodium concentrations. The Na 1 /H 1 antiporter activity of S. costicola has been relatively well studied (105, 106, 332). However, there is still considerably controversy about the occurrence and relative importance of primary Na 1 pumps in different representatives of the very heterogeneous group of moderate halophiles. S. costicola cells maintain a proton motive force (the combined action of the pH gradient over the membrane and the membrane potential) varying from 170 to 100 mV (inside negative) in cells grown at pH values varying from 5.7 to 9.0. Cells maintain their intracellular pH close to 7.5, and thus the reversed pH gradient is compensated at least in part by an increased membrane potential at high medium pH. The primary respiration-driven outward proton pump supplies the driving force for the Na 1 / H 1 antiport activity for Na 1 efflux. Protonophores, such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 3,39,49,4- tetrachlorosalicylanilide (TCS), respiration inhibitors, and monensin, which facilitates Na 1 /H 1 exchange, dissipate the proton motive force, thereby abolishing Na 1 /H 1 antiport activity (105). When S. costicola cells, kept anaerobically at pH 6.5, were given a pulse of oxygen, protons were extruded, both in the presence and in the absence of Na 1 ions. At pH 8.5, an acidic response was observed in absence of Na 1 but alkalinization was noted in its presence, suggesting that an Na 1 /H 1 antiport mechanism is functional at pH 8.5 and that the establishment of an Na 1 gradient participates in pH homeostasis. All these effects are abolished by protonophores. At pH 8.5, growth is prevented by CCCP (at the rather high concentration of 50 mM [see below]) and by TCS (106). During growth on lactate as the energy source, Na 1 translocation in S. costicola was inhibited by CCCP but little affected by valinomycin. Amiloride, an inhibitor of the eukaryotic Na 1 /H 1 antiporter system, inhibited lactate-dependent Na 1 translocation. It was concluded that lactate-dependent H 1 extrusion enables the generation of a Na 1 gradient mediated by the secondary Na 1 /H 1 antiporter (332). All of the evidence reviewed above supports the idea that H 1 extrusion is the primary process, enabling the secondary conversion of the proton motive force into a sodium motive force via the antiporter. However, primary Na 1 pumps have been postulated to be involved in the extrusion of Na 1 from certain moderate halophiles ever since the first respiration-driven Na 1 pumps were identified in Vibrio alginolyticus and the moderate halophile S. costicola (328). The evidence for the existence of such a pump is based in part on the fact that growth of this organism in alkaline media is not inhibited by the protonophore CCCP. Thus, growth was postulated to occur in the absence of proton circulation across the cell membrane. The Na 1 -specific pump, with an optimum in the alkaline pH range, then maintains the intracellular cation environment, in addition to the Na 1 /H 1 antiporter, which is operative at low pH (328). The primary outward Na 1 pump in S. costicola was identified to be the NADH oxidase (NADH:quinone reductase) segment of the respiratory chain. At pH 8.5, the formation of a membrane potential was inhibited by 2-heptyl-4-hydroxyquinoline (NQNO), which inhibits the Na-motive NADH oxidase. When NADH served as electron donor, Na 1 translocation was resistant to CCCP and stimulated by valinomycin (as noted above, when lactate was used as the electron donor, Na 1 translocation was inhibited by CCCP and little affected by valinomycin). Amiloride had little effect on NADH-dependent Na 1 translocation (332). In a comparative study of eight moderate halophiles, all six gram-negative species tested (H. variabilis,H. halophila,H. canadensis,S. costicola,“P. beijerinckii,” and “P. halosaccharolytica”) showed Na 1 -dependent NADH:quinone reductase activity. Buildup of a membrane potential linked to NADH oxidation in inverted membrane vesicles was not completely inhibited by CCCP alone but was abolished by a combination of CCCP and the Na 1 -conducting ionophore monensin. Succinate oxidation and the terminal oxidase step of the respiratory chain did not show any specific requirement for Na 1 ions. No Na 1 -depen- dent NADH oxidase could be demonstrated in the gram-pos- itive Marinococcus halophilus and “Micrococcus varians subsp. halophilus,” since in these organisms the membrane potential generated during NADH oxidation was completely dissipated by CCCP (334). However, Nikolayev and Matveyeva (225) also postulated the presence of a primary Na 1 pump in “Micrococcus varians subsp. halophilus,” based on the low level of inhibition of alanine transport by CCCP at alkaline pH. Extensive studies on ion metabolism in H. israelensis also suggested the importance of a primary Na 1 pump in the regulation of intracellular Na 1 concentrations (152–154, 156). The presence of an uncoupler-stimulated Na 1 pump was postulated to explain the drop in respiration rate of up to 80% observed when the pH was increased from 6.5 to 8.5 in the absence of Na 1 . Catalytic amounts of Na 1 prevented this drop. The rate-limiting step in the electron transport at high pH was located in the NADH-oxidoreductase section of the respiratory chain, and Na 1 was suggested to release the kinetic control (152). In the presence of Na 1 , a pH gradient of reversed polarity was formed, in both the absence and presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydra- zone (FCCP), driven by the Na 1 /H 1 antiport activity. Inverted membrane vesicles responded as expected from the behavior of intact cells. It was concluded that extrusion of Na 1 ions is the primary event and that proton counterflow and Na 1 /anion symport phenomena are secondary events which minimize the buildup of a membrane potential generated by the primary Na 1 pump (156). Respiration initiates efflux of Na 1 from preloaded cells, driven by an Na 1 pump insensitive to uncouplers such as FCCP and the ATPase inhibitor N,N9-dicy- clohexylcarbodiimide (DCCD) (153, 154). Movement of Na 1 is thus directly coupled to electron transport. The uncouplerinsensitive primary Na 1 pump may play an important role in the regulation of intracellular salt concentrations. When K 1 ions were omitted from the medium, a shift in pH from 6.5 to 8.5 caused an increase in respiration rate in H. israelensis, in contrast to the above-described drop in respiration in the absence of Na 1 . When both Na 1 and K 1 were present, the pH did not affect the respiration rate (152). Stimulation of respiration by K 1 was most pronounced at acidic pH and was accompanied by an alkalinization of the cytoplasm. The effect of K 1 was observed only in intact cells. Possibly to compensate for the electrogenic entry of K 1 , more protons are extruded by the primary proton pump, leading to internal alkalinization. The step in the electron flux affected by K 1 has not yet been identified (152). MacLeod (184) suggested an alternative explanation not involving a primary respiration-driven Na 1 pump for at least 520 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from part of the observations presented above. Many of the conclusions relating to the nature of the ion pumps are based on the effect of ionophores such as CCCP on cells or membrane preparations. What is often not taken into account is that CCCP functions poorly at alkaline pH and may be altogether ineffective in complex media containing such ingredients as proteose peptone and tryptone. Cells in complex media may not be inhibited by high CCCP concentrations, while 5 mM CCCP causes complete growth inhibition in defined medium (106). The fact that CCCP causes only a partial collapse of the membrane potential in S. costicola at high pH (328) may thus be due to the ineffectiveness of this protonophore under the conditions used. In S. costicola, CCCP proved effective at higher concentrations and TCS was almost as effective at pH 8.5 as at pH 7 (106). MacLeod therefore suggested that NADH oxidation at pH 8.5 in S. costicola leads to proton extrusion, which drives the formation of a membrane potential through an Na 1 /H 1 antiporter, and he argued that no compelling evidence exists that a protonophore-resistant primary electrogenic Na 1 pump operates in S. costicola at alkaline pH (184). To our knowledge, the above studies have not been pursued recently, and therefore the question of the relative importance of Na 1 /H 1 antiporters and primary respiration-driven Na 1 pumps in the extrusion of sodium and the maintenance of low intracellular Na 1 concentrations in moderately halophilic bacteria remains unanswered. Little information is available on transport systems for other inorganic ions in the moderate halophiles. A K 1 transport system was characterized in a Halomonas strain isolated as a contaminant from a Dunaliella culture, enabling cells to accumulate the necessary K 1 concentration following a hyperosmotic shock. The K s of the transporter was 21.5 mM; thus, its affinity is lower than that of the well-characterized Kdp system of E. coli. However, high-affinity K 1 transport systems are probably not essential for Halomonas and other moderately halophilic bacteria, which generally live in seawater-derived brines, in which K 1 can hardly be considered limiting (36). A potassium extrusion system was described in H. israelensis, probably based on a K 1 /H 1 antiporter. K 1 and Rb 1 ions were found to rapidly penetrate the cell membrane. Under energized conditions, a powerful pump extrudes this K 1 and Rb 1 . At low pH values, the presence of Na 1 is not required for K 1 extrusion. However, millimolar concentrations of Na 1 are needed for effective K 1 and Rb 1 extrusion at high pH values, again possibly pointing to the involvement of primary Na 1 pumps. The K 1 pump was postulated to have a function in the regulation of intracellular salt concentrations and/or internal pH (310). Little is known about the mechanisms of extrusion of anions from the cells of moderately halophilic bacteria. No information was found in the literature beyond the assumption that the membrane potential generated by Na 1 efflux may be the driving force causing efflux of permeant anions (153, 154). Transport of Organic Compounds The most widely studied transport system in moderate halophiles is the transport of a-aminoisobutyrate (AIB), a nonmetabolizable amino acid analog. In S. costicola, AIB transport is competitively inhibited by Gly, Ala, and to a lesser extent Met (107, 108). In H. elongata, competition was observed with Ala, Gly, Ser, D-Ala, L-homoserine, and D-Ser and to a lesser extent with Met, Leu, Phe, and His (190). The transport activity is Na 1 dependent. Sodium at concentrations below 0.2 M increased the apparent affinity of the S. costicola transport system. NaCl concentrations between 0.2 and 1 M increased the V max , while at NaCl concentrations above 1 M, the V max decreased without affecting the K m (107, 108). In cells grown in the presence of 1 or 2 M NaCl, AIB transport was active at higher salinities than in cells grown in 0.5 M NaCl. Low-salt- grown cells adapted to AIB transport at high salinities after 6 h of incubation at high salt in a process independent of protein synthesis (175). A similar phenomenon was reported for H. elongata, which showed optimum AIB uptake at an NaCl concentration of 0.38 M in low-salt-grown cells and 1.37 M in high-salt-grown cells (190, 296). Adaptation of AIB transport in S. costicola to the highest salinities (4 M NaCl) required the presence of nutrients, probably related to the accumulation of compatible solutes, and glycine betaine was especially stimulatory (172, 175). Transport showed an optimum at pH 8.5 to 9 and required the presence of Na 1 and a membrane potential (107, 108, 183). Na 1 was required for AIB transport in all the gram-negative bacteria in which a respiration-driven primary Na 1 pump was demonstrated (H. halophila,H. canadensis, H. variabilis,S. costicola,“P. beijerinckii,” and “P. halosaccharolytica”), while two gram-positive bacteria lacking the respiration-driven Na 1 pump (Micrococcus halophilus and “M. varians subsp. halophilus”) did not require Na 1 for AIB uptake (333). NaCl caused a two- to threefold stimulation of the transport activity of proline in H. israelensis. As in the case of AIB transport in S. costicola and H. elongata, the maximum uptake rate of proline was observed at salt concentrations similar to those at which the cells were grown (257). Transport of different amino acids was compared in a variety of moderate halophiles by Nikolayev et al. (225, 226). In S. costicola,Nesterenkonia halobia, and H. halodenitrificans, all amino acid transport was found to be Na 1 dependent, but in some cases Li 1 ,K 1 ,NH 41 ,orCs 1 could replace Na 1 .In S. costicola transport of Asp in the presence of Li 1 was 10% of that with Na 1 , while Ala transport was strictly Na 1 dependent. For Arg transport, K 1 ,NH 41 , and Cs 1 could replace Na 1 to different degrees. In H. halodenitrificans, Arg transport in the presence of Li 1 ,NH 41 ,orK 1 was only 1.5 to 2 times lower than when Na 1 was present. Asp, Arg, and Ala could be transported by “M. varians subsp. halophilus” at salt concentrations of up to 2 M. The transport of Asp and Arg was inhibited by NaCl, while that of Ala was stimulated (226). Organic Osmotic Solutes Since the intracellular ion concentrations measured in the moderate halophiles are generally insufficient to provide osmotic balance with the external medium, much effort has been dedicated to the search for organic compounds accumulated by the cells. Only relatively recently were the principal organic osmotic solutes of the moderately halophilic bacteria identified. Techniques such as natural-abundance 13 C-NMR and high-pressure liquid chromatography are now routinely used to probe the intracellular environment of the moderate halophiles. These techniques led to the identification of the tetrahydropyrimidines ectoine and hydroxyectoine (Fig. 1), and to the recognition of the role of glycine betaine, which is not synthesized by most representatives of the group but can be readily taken up if available in the outside medium. Before the importance of these compounds was realized, unsuccessful attempts to identify intracellular organic compounds at high concentrations led to the view that these bacteria may be able to manage without the traditional osmotic balance and may maintain a hypoosmotic cytoplasm (351). The discovery that ectoine and hydroxyectoine, two compounds that have long resisted detection, may be present inside the cells in molar VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 521 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from concentrations abolished the need to hypothesize a hypoosmotic intracellular space (363). The observation that cells of S. costicola swelled rapidly upon treatment with penicillin (254) is also incompatible with the concept of a hypoosmotic cytoplasm, since it will not support the turgor pressure necessary for cell growth. The organic intracellular solutes are called compatible solutes, since they provide osmotic balance without interfering with the metabolic functions of the cells (17, 132). We do not intend present here an in-depth discussion of the action of organic solutes, including the question why some are more “compatible” than others in enabling activity of salt-sensitive enzymes at low water activities. For a survey of the theories proposed, we refer the reader to the reviews by Galinski (80– 82). Compatible solutes are polar, highly soluble molecules and uncharged or zwitterionic as physiological pH. They are strong water structure formers and as such are probably excluded from the hydration shell of proteins. This “preferential exclusion” probably explains their function as effective stabilizers of the hydration shell of proteins. Water near the interfaces is structurally different (more dense), allowing the osmotic solutes to migrate toward the less dense water fraction. This phenomenon of nonspecific exclusion is often described in terms of increased surface tension of water, with the presence of solutes affecting the forces of cohesion between water, minimization of entropy, and reinforcement of the hydrophobic effect. Compatible solutes display a general stabilizing effect by preventing the unfolding and denaturation of proteins caused by heating, freezing, and drying (79–82, 85). (i) Amino acids. Most early attempts to detect organic osmotic solutes in moderately halophilic bacteria were aimed at the quantitation of intracellular amino acids. The search for amino acids was to some extent inspired by the finding that in vitro protein synthesis by ribosomes of S. costicola is already inhibited by relatively low chloride concentrations but proceeds uninhibited or even somewhat stimulated in the presence of 0.6 M potassium glutamate (141, 173). Also, a number of tricarboxylic acid cycle enzymes are strongly inhibited by Cl 2 but function well in 1 M sodium or potassium glutamate or higher. With the exception of a few specialized groups of grampositive bacteria that accumulate proline (see below), amino acids do not seem to be of great quantitative importance as osmotic solutes in moderate halophiles. In H. elongata, the total intracellular amino acid pool increased with medium salinity (16, 105, and 358 mM in cells grown in 0.05, 1.37, and 3.4 M NaCl, respectively). In the 358 mM amino acid pool, Glu was found at the highest concentration, followed by Ala and Gln (359). In H. halophila, intracellular Gly and Asp concentrations increased with salinity (44). However, the data presented (in micrograms of amino acid per milligram of protein) do not allow an exact calculation of cytoplasmic concentrations. Quite high glutamate concentrations were detected in S. costicola (428, 1,203 and 389 mM in cells grown in 0.51, 1.7, and 3.4 M NaCl, respectively) (170). Proline is the main organic osmotic solute in certain grampositive species (Salinicoccus roseus and Salinicoccus hispanicus) (87, 306). In other gram-positive halophiles, amino acids do not contribute greatly to the osmotic balance. Thus, Bacillus haloalcaliphilus maintains an intracellular amino acid concentration of about 70 mmol/kg of water (361). The halotolerant Planococcus strain A4a, isolated from the dry valleys of Antarctica, showed an increase in the concentrations of intracellular amino acids with salinity: cells grown in 0 and 1.5 M NaCl contained intracellular Glu, Asp, and Gln concentrations of 102, 6, and 8 mM and 71, 49, and 39 mM, respectively. In 1.5 M salt-grown cells, the total intracellular amino acid concentration was about 340 mM, in addition to 605 to 640 mM K 1 (207). (ii) Glycine betaine. Glycine betaine was recognized in the early 1980s as the most important organic osmotic solute in photosynthetic purple bacteria and halophilic cyanobacteria. Its possible role in the osmotic stabilization of halophilic heterotrophic bacteria had been suggested as early as 1968 (273). When grown in complex growth media, most halophilic bacteria accumulate glycine betaine or its precursor, choline, from FIG. 1. Biosynthetic pathway for ectoine and hydroxyectoine based on enzymological studies. The proposed formation of hydroxyectoine (dotted line) from ectoine is still hypothetical. Modified from reference 85 with permission of the publisher. 522 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from the medium, and under these conditions glycine betaine is often found as the sole or main osmotic solute (131). However, with the possible exception of the actinomycete Actinopolyspora halophila, none of the halophilic or halotolerant heterotrophic bacteria seem to be able to synthesize the compound de novo (80). Certain halophilic bacteria can use glycine betaine not only as an osmotic stabilizer but also as a carbon and energy source. A number of haloalkaliphilic isolates from Mono Lake, Calif., grew on glycine betaine by sequential demethylation via dimethylglycine to sarcosine, which was then excreted into the medium. At high salinities, glycine betaine catabolism was suppressed and the compound was taken up solely to serve as an osmotic solute, thereby allowing extension of the salt range, enabling growth in 2.5 to 3–3.5 M NaCl (51). An unidentified Halomonas sp. grew on glycine betaine as the sole carbon and energy source by oxidation via dimethylglycine and sarcosine to glycine. Growth on glycine betaine was possible only up to 2 M NaCl. For growth at higher salt concentrations, the presence of another carbon and energy source such as glucose was required, and catabolism of glycine betaine was inhibited, so that it served as an osmotic solute only (36). (iii) Ectoine and hydroxyectoine. Ectoine, first detected as an osmotic solute in photosynthetic purple bacteria of the genus Ectothiorhodospira, is derived from N-acetylated diaminobutyrate by intramolecular dehydration to form a pyrimidine derivative. As a result of the delocalization of the pelectrons, the amino functions are not reactive, and therefore the compound has escaped detection for a long time (82). Ectoine and its b-hydroxy derivative are now recognized as the most widespread compatible solutes found in the domain Bacteria (see Table 6). The biosynthesis of ectoine proceeds in three enzymatic steps, starting with aspartate semialdehyde, with diaminobutyrate and N-g-acetyldiaminobutyrate as intermediates (259) (Fig. 1). All three enzymes, diaminobutyrate transaminase, diaminobutyrate acetyltransferase, and N-acetyldiaminobutyrate dehydratase (“ectoine synthetase”), have been identified in H. elongata (81, 259). Hydroxyectoine was hypothesized to be synthesized in a similar pathway, but the intermediates and enzymes involved are unknown (180). Some of the enzymes involved in ectoine synthesis in H. elongata have been isolated and partially characterized. The 2,4-diaminobutyrate transaminase has an apparent molecular mass of 260 kDa and probably consists of six subunits. The presence of K 1 ions (1 to 50 mM) increased the activity threefold and also stabilized the enzyme (323). An alternative biosynthesis pathway for ectoine, starting with glutamate, was suggested to be operative in Brevibacterium linens (15) and in Streptomyces parvulus (134). Ectoine was found in high concentrations in different Halomonas species and is the dominant solute in cells grown in defined medium lacking glycine betaine or its precursor, choline (24, 363). Intracellular ectoine concentrations can be sufficiently high to balance the osmotic pressure of the medium in combination with the intracellular ions and other dissolved organic compounds present. H. elongata grown in 10% NaCl had an estimated ectoine concentration of 2.25 M, which, in addition to about 0.4 M cations and negative counterions, would give about 3.05 M, a value close to osmotic equilibrium (363). The ectoine concentration in H. israelensis is probably much lower: 0.1 to 0.2 M ectoine was measured in cells grown in 1 to 2 M salt (277). Also, S. costicola produces ectoine, but its intracellular concentrations never approach those of the NaCl in the medium. It was estimated that the “true” intracellular environment of H. israelensis and S. costicola may osmotically balance 60 to 80% of the extracellular solute concentration (160). (iv) Other organic osmotic solutes. Amino acids, glycine betaine, and the tetrahydropyrimidines are by no means the only organic compounds found in moderately halophilic bacteria. The following are also found. (i) Nε-acetyllysine and Ndacetylornithine were identified by 13 C-NMR in a number of gram-positive bacteria such as Halobacillus halophilus and Planococcus spp. The second compound is also known as an intermediate in arginine biosynthesis. Intracellular concentrations may be in the molar range (up to 0.5 mmol/mg [dry weight]). In a strain designated M96/12b, proline is the dominant osmotic solute in the exponential growth phase and is replaced by Nd-acetylornithine in stationary-phase cells (364). (ii) Polyamines cannot be expected to play a significant role in osmoregulation, since they were never found in high concentrations. Certain moderately halophilic bacteria may lack polyamines altogether: no polyamines were detected in S. costicola (109, 139). Halomonas and Chromohalobacter contain spermidine (0.3 to 2.1 mmol/g of wet cells, which is in the concentration range generally found in nonhalophilic bacteria) (109). When grown in a complex medium, S. costicola can incorporate polyamines (putrescine, cadaverine, spermidine, and spermine) from the medium. The presence of these polyamines did not increase the osmotic stability of the cells upon salt downshock (139). (v) Distribution of organic osmotic solutes within moderately halophilic bacteria. An overview of the occurrence of organic osmotic solutes in different moderately halophilic bacteria is given in Table 6. A few general principles emerge from this table and from additional studies (80, 82, 87, 306, 330). (i) Cells grown in 10% salts generally have an organic solute content between 0.6 and 1.3 mmol/mg dry weight, equivalent to about 10 to 20% of the cellular dry weight. (ii) Apart from glycine betaine, all N-containing osmotic solutes are derived from the glutamate or the aspartate branch of amino acid biosynthesis. (iii) Uptake of osmotic solutes from the medium is preferred over de novo synthesis. (iv) All bacteria tested take up glycine betaine, but only Actinopolyspora halophila synthesizes the compound de novo. (v) Ectoine and hydroxyectoine are the predominant osmotic solutes in representatives of the gamma subclass of the Proteobacteria and in many gram-posi- tive species. (vi) Charged amino acids such as glutamate never accumulate at concentrations exceeding 0.4 M. (vii) Proline, Nd-acetylornithine, and Nε-acetyllysine are found in low-G1C gram-positive bacteria such as some Bacillus species and the related genera Salinicoccus and Halobacillus. The interchangeability between proline and Nd-acetylornithine probably points to a common biosynthetic pathway. (viii) In most cases, cells do not accumulate a single osmotic solute but maintain intracellular cocktails of different compounds. Glutamate is often found as a minor compound in organisms that contain primarily ectoines (36, 165). The types of osmotic solutes present in the cells may depend greatly on the growth conditions. Thus, H. israelensis contained mainly trehalose when grown below 0.6 M salt, but at higher salt concentrations ectoine became the major solute (277). (vi) Uptake and action of exogeneously supplied glycine betaine. When grown in complex media, all heterotrophic, aerobic, moderately halophilic bacteria tested were found to contain glycine betaine at concentrations increasing from around 0.2 M to 0.65–0.85 M to over1Mincells grown in 3, 10, and 20% salts, respectively (131, 133). Glycine betaine is either taken up from the medium (171) or derived from choline by means of an O 2 -dependent choline oxidase (producing betaine aldehyde), and an NAD-dependent betaine aldehyde dehydro- VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 523 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from genase. H. elongata and S. costicola possess both enzymes, and hybridization experiments with specific gene probes have detected genes similar to the betA and betB genes of E. coli (22). The choline oxidase of S. costicola is membrane bound, and its activity was strongly induced by the presence of choline and inhibited by Cl 2 ions. The betaine aldehyde dehydrogenase is located in the cytoplasm. Its activity was stimulated by 0.5 M NaCl, and the enzyme could function at up to 2 M salt. The intracellular glycine betaine concentration increased with external salt and with the availability of choline (29, 171). A high-affinity transport system for glycine betaine and the enzymes of the choline-betaine pathway were characterized in H. elongata (23). Thin-layer chromatography and 13 C-NMR analysis showed that exogeneous choline is taken up and transformed in H. elongata, demonstrating the existence of the choline-glycine betaine pathway (23). The first indication of the importance of glycine betaine in the physiology of moderately halophilic bacteria came from a study of respiration in H. israelensis. Glycine betaine was found to improve the salt resistance of the respiration system and to accelerate succinate oxidation. Stimulation was more pronounced the higher the NaCl concentration of the medium (273, 309). Labeled glycine betaine was accumulated by the cells. The authors stated that, “Apparently the prerequisite for the betaine effect is that the cells be free of, or contain little of, a genuine factor associated with salt resistance, and betaine acts by replacing it. Therefore the high-salt-grown cells, not influenced by betaine, presumably contained the genuine factor, which they may have acquired either through accumulation from their environment or by a process of synthesis and retention,” and, “Since it was shown that betaine accumulation was also salt-dependent, it seems that salt resistance was directly related to intracellular betaine concentration.” (273). The real nature of the stimulatory effect of glycine betaine is not yet completely clear, and it was suggested to be due neither to stimulation of substrate uptake nor to counteracting of plasmolysis; instead, it seemed to involve an effect on the cytoplasmic membrane, distinct from its function as a compatible solute (296). Ken-Dror et al. (155) suggested that the effect of glycine betaine on respiration may be explained by isoosmotic adaptation due to its accumulation in the cytosol (deplasmolysis) or by facilitation of penetration of Na 1 into plasmolyzed cells. Na 1 was found to be required for the proper functioning of the respiratory chain, and no glycine betaine accumulation was observed in the absence of Na 1 . (vii) Accumulation of other osmotic solutes by transport from the medium. Glycine betaine is not the only compound that can be taken up from the medium and serve as an osmotic solute. Other more unusual compounds can be accumulated for the same purpose. Two haloalkaliphilic isolates from Mono Lake designated strains ML-D and ML-G, produce ectoine in minimal medium. Strain ML-D also synthesizes hydroxyectoine. Under nitrogen limitation, exogenously supplied dimethylsulfoniopropionate (DMSP) (normally produced by marine algae as an osmotic solute) substituted for ectoine in strain ML-G and became the major intracellular solute. Strain ML-D could accumulate arsenobetaine (produced by the brine shrimp in Mono Lake) for the same purpose (32). Not only can DMSP serve as an osmotic solute in heterotrophic bacteria, but also it may be catabolized. Two moderately halophilic alkaliphiles isolated from Mono Lake could grow on DMSP, one strain by cleaving it to DMS and acrylate, the other by demethylation with 3-methiolpropionate as an intermediate, yielding methanethiol. At high salinities, DMSP catabolism was suppressed and the compound was preferentially taken up and used for osmotic purposes (51). ENZYMES When discussing enzymatic activities of moderately halophilic bacteria, and especially their relation to salt, we must discriminate three distinct categories of activities: (i) intracellular enzymes, which are not exposed to the salt concentration of the medium but sense the “true” intracellular environment—in most cases characterized by low ion concentrations and the presence of organic osmotic solutes; (ii) membrane-bound activities, including transport proteins, which sense both the intracellular environment and the outer medium; and (iii) truly extracellular enzymes, exposed to the external hypersaline con- TABLE 6. Organic osmotic solutes within moderately halophilic bacteria a Species b Glycine betaine Ectoine Hydroxyectoine Trehalose Others Micrococcus halobius 11 1 1 Glucose, glutamate “Micrococcus varians subsp. halophilus”11 1 Glucose, glutamate Marinococcus halophilus 11 11 Glucose Marinococcus albus 11 1 Alanine Planococcus citreus Proline, N-acetylornithine, N-acetyllysine Halobacillus halophilus 11 Proline, N-acetyllysine Salinicoccus roseus* 1Proline Salinicoccus hispanicus* 1Proline Halomonas halophila 11 1 Glutamate Halomonas elongata 11 1 Glutamate, glucose Halomonas halmophila 11 1 Glutamate, alanine Halomonas halodenitrificans 11 Glutamate, glucose Halomonas salina 11 1 Glutamate Halomonas variabilis* 11 11 11 1 Halomonas eurihalina 11 1 Glutamate Salinivibrio costicola 11 Glutamate Chromohalobacter marismortui* 111 “Pseudomonas halosaccharolytica” 11 1 Bacillus haloalkaliphilus 111 N-Acetylornithine Bacillus halophilus 11 Proline, N-acetylornithine a Data were derived from references 43, 87, and 306. b 11, present in high concentrations; 1, present in minor amounts; p, yeast extract was present in the medium, enabling the accumulation of glycine betaine. 524 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from ditions. For earlier reviews on the subject, we refer the reader to the papers by Cazzulo (27), Kamekura (138), and Kushner and Kamekura (176). Cytoplasmic Enzymes The pyruvate kinase of S. costicola has a high percentage of the acidic amino acids Asp and Glu and a low proportion of hydrophobic amino acids and is enriched in Gly and Ser, all typical characteristics of halophilic proteins (47, 48). Stability was improved at high salinities. However, while a low concentration (0.25 M) of NaCl or KCl was somewhat stimulatory, virtually no activity was detected above 1 M salt. This would imply that if the intracellular Na 1 and K 1 concentrations are as high as reported (Table 5), the enzyme can be expected to be strongly inhibited under physiological conditions (48), assuming no sparing effect of compatible solutes. The phosphoenolpyruvate carboxykinase of S. costicola did not show a great excess of acidic over basic amino acid residues, and in this respect it resembled the enzyme from animals and yeast. However, an unusually high content of Gly and Ser was found (16.7 and 10.2%), which supposedly maintains the balance between hydrophilic and hydrophobic forces. Oxaloacetate decarboxylation and 14 CO 2 -oxaloacetate exchange were activated by NaCl and KCl at concentrations up to 1 M (with the activities being about 2.5 times as high as in the absence of salt), while the optimum for carboxylation of phosphoenolpyruvate was found at 0.025 to 0.05 M salt. In view of these properties, it was suggested that under physiological conditions the enzyme probably acts in the direction of phosphoenolpyruvate synthesis (300, 302). Other enzymes from Salinivibrio that have been characterized are the NADP-specific malic enzyme, glycerol dehydrogenase, threonine deaminase, and aspartate transcarbamylase. The malic enzyme was found to be unstable in dilute solutions but stable in 1 M NaCl or KCl or in 25% glycerol. Maximum activation occurred by 0.1 M NH 4 Cl or KCl (not by NaCl), and higher concentrations were inhibitory, with about 23% of the optimal activity remaining at 0.85 M NaCl or KCl (301). The glycerol dehydrogenase was most active at 0.25 to 0.5 M salt, and KCl was preferred over NaCl. The enzyme was very salt tolerant, with about 60% of its maximal activity remaining in 3 M KCl. In this respect, it is not greatly different from a similar enzyme from E. coli (9). The threonine deaminase was most active in the absence of salt, with as little as 30% of the optimal activity remaining in 3 M NaCl or KCl. However, salts did stabilize the enzyme. Regulatory properties were not greatly influenced: feedback inhibition by isoleucine was observed both in the absence of salt and in 3 M NaCl or KCl, so this enzyme is expected to function well over a wide range of salinities (168, 169). The aspartate transcarbamylase showed much more pronounced halophilic properties: it proved stable at 1.5 M NaCl, but activity was rapidly lost at lower salt concentrations. Optimal activity was measured at 1.5 M NaCl or 1.0 M KCl, but high activity was still present at 0.15 M NaCl. The enzyme differs from its nonhalophilic (Saccharomyces) and halophilic archaeal (Halobacterium salinarum [formerly H. cutirubrum]) counterparts in the salt response patterns of activity and regulation: ATP and CTP were inhibitory, with the strongest inhibition in the low-salt range. Above 0.5 M salt, hardly any regulatory effect of CTP was observed. This observation is important since enzymes must not only be active but also be subject to allosteric regulation (5). The properties of the alanine dehydrogenase of H. elongata depend greatly on the salt concentration at which the cells were grown. The enzyme isolated from cells grown at 50 mM salt was optimally active at salt concentrations between 0 and 20 mM, while the enzyme isolated from 1.37 M and 3.4 M salt-grown cells had its optimum at 340 and 500 to 600 mM, respectively (19). The estimated intracellular salt concentrations of the cells grown at the respective salinities were 42, 312, and 630 mM; hence, the enzyme seems to be well adapted to the actual conditions inside the cells. No simple explanation was put forward for the phenomenon, but for cells grown at 0.05 and 1.37 M salt, two bands with alanine dehydrogenase activity were detected on native gels, while for cells grown in 3.4 M, only one band was seen (19). Experiments involving dissociation during different enzyme purification methods and sequence analyses would clarify the effect. Low salt concentrations were optimal for the functioning of isocitrate, malate, succinate and a-ketoglutarate dehydrogenases from H. halodenitrificans, while lactate dehydrogenase and cytochrome oxidase (being membrane-bound enzymes) were more active in the presence of salt (10). Also in the alkaliphilic Bacillus haloalkaliphilus, the intracellular malate dehydrogenase and isocitrate dehydrogenase were inhibited by salt, with about 20% of the optimal activity remaining above 1 to 2 M NaCl (361). Organophosphorus acid anhydrase activity was detected in a halophilic isolate (designated JD6.5, probably belonging to the genus Alteromonas) isolated from a warm, hypersaline (24% salt) spring near the Great Salt Lake, Utah (38). No information was given about the salt requirement or the sensitivity of the enzyme. The complex enzymatic machinery required for protein synthesis by the ribosomes presents an excellent object for the study of the influence of salt on intracellular activities, and therefore extensive research efforts have been dedicated to in vitro protein synthesis by S. costicola ribosomes (30, 141, 171, 173, 368). These studies included the measurement of poly(U)- directed phenylalanine incorporation and [ 14 C]valine incorporation, with E. coli phage R 17 RNA as the template, leading to the formation of a protein with the same electrophoretic mobility as the valine-rich R 17 coat protein. Optimum activity was obtained at low salt concentrations (0.1 to 0.3 M, with ammonium glutamate being the most stimulatory). Choride salts proved especially inhibitory (with NH 4 Cl, KCl, and NaCl being increasingly toxic), and hardly any activity was observed in the presence of 0.6 M Cl 2 . Choride was found to prevent the attachment of the 50S ribosomal subunit to the 30S subunitmRNA complex and also displaced already bound ribosomes. The accuracy of the translation process was not affected. However, the inhibitory effect of Cl 2 could be partially reversed by glycine betaine or glutamate. When Cl 2 was replaced by other anions, such as glutamate, sulfate, or acetate, excellent in vitro protein synthesis activity was found at cation concentrations as high as 0.6 M, both in S. costicola and in H. canadensis (30, 141, 171, 173, 368). Not all components of the protein-synthe- sizing machinery are equally chloride sensitive: the phenylalanyl-tRNA synthetase of S. costicola functions well at 0.5 M NaCl. The ribosomal proteins of S. costicola are slightly more acidic than those of E. coli but much less so than those of the halobacteria.TheS.costicolaribosomes showedanunusualsedimentation behavior in sucrose gradients: association of subunits to monosomes took place only at high salt concentrations (367). A study of the binding of dihydrostreptomycin to the 30S ribosomal subunits of S. costicola showed more binding in cells grown at high salt concentrations, suggesting that some ribosomal properties may vary with the salt concentration in which the cells were grown (161). VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 525 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from Stress Proteins Upon sudden exposure to high NaCl concentrations (2 to 2.5 M), low-salt-grown H. halophila cells showed a transient cessation of cell division. During the initial phase of the adaptation to the new conditions, protein synthesis and amino acid uptake were inhibited. During the adaptation period, changes in the patterns of pulse-labelled proteins were observed. Distinct changes were also seen in the protein profiles upon downshock from 2.5 to 1 M salt, but only minor changes were found upon downshock from 1 to 0.5 M (59). High-salt-related proteins of 39, 24, 20, and 15.5 kDa have been identified in H. elongata, and the synthesis of these proteins increased with increasing salinity. A different set of proteins (60, 42, 15, and 6 kDa) was induced under low-salt conditions (209). A study of protein turnover in S. costicola by monitoring the breakdown of pulse-labeled proteins showed that in cells growing exponentially in 1 or 1.5 M NaCl the turnover is about 5% per h and in cells growing in 0.5 M NaCl the turnover is as high as 9% per h, compared with 1 to 2% per h for E. coli. A shift from high-salt to low-salt conditions gave rise to an increase in the turnover rate of pulse-labeled proteins. It was suggested that the low salt concentration alters the “native” protein conformation and increases the susceptibility of proteins to proteolysis. Hipkiss et al. postulated that the increased protein turnover at low salinity may set a limit to growth at such concentrations (119). The appearance of heat shock proteins was examined in Chromobacterium marismortui. When cells were grown in 1 M salt, transfer to 42°C resulted in complete inhibition of the synthesis of the normal proteins and induced the formation of heat shock proteins. In cells grown in 2.5 M salt, a heat shock at 42°C still allowed the synthesis of some of the normal proteins. The higher the salt concentration at which the cells were grown, the higher the upper temperature limit at which heat shock proteins could be synthesized (150). A similar phenomenon was observed in H. halophila (149). Also, the sensitivity of H. halophila to oxidative stress (sensitivity to H 2 O 2 ) depends on the NaCl concentration of the medium. Cells grown in 1 M salt were much more resistant to peroxide than were 2.5 M salt-grown cells. The effect could not be explained by differences in catalase activity. Exposure to 50 mMH 2 O 2 resulted in the induction of several new proteins. The number of these proteins and the kinetics and extent of their induction were influenced by salinity, with the adaptive response to nonlethal concentrations of H 2 O 2 being faster in 1 M than in 2.5 M NaCl (220). A manganese superoxide dismutase of H. israelensis was also characterized. This enzyme did not require salt for activity (373). Membrane-Bound Enzymes Membrane-bound enzymes are in contact with the high salt concentrations in the medium, and therefore they can be expected to be active at high salt concentrations or even to require high salt concentrations for full activity. A membrane-bound 59-nucleotidase of S. costicola was optimally active at 2 M NaCl or KCl or higher. This enzyme splits ATP, ADP, and AMP, which are dephosphorylated to adenosine, which is subsequently transported into the cells. Its activity is distinct from that of the F 0 F 1 ATPase also present in the membrane and can be differentiated from that of the proton channel ATPase by its higher magnesium requirement and its insensitivity to DCCD (11, 12). Also the DCCD-inhibited F 0 F 1 ATPase of S. costicola requires high salt concentrations for both activity and stability, with optimal activity reported at 0.5 M KCl. KCl concentrations of up to 3 M caused relatively little inhibition, but 3 M NaCl produced 75% inhibition (26, 117). The NADH:quinone oxidoreductase of H. israelensis depends on Na 1 for activity. Na 1 ions increased the rate of quinone reduction severalfold, but oxidation of the quinol with oxygen was not affected by Na 1 (153). Succinate respiration in this organism was Na 1 dependent and showed a maximum rate between 0.2 and 0.8 M Na 1 (272). The membrane-bound nitrate reductase and nitrite reductase activities of H. halodenitrificans were separated by treatment with detergent and ammonium sulfate fractionation. The nitrite reductase was identified as a cd-type cytochrome (99, 100). Both a membrane-bound and a cytoplasmic nitrite reductase activity were detected. When phenazine methosulfate and ascorbate served as electron donors, the membrane-bound enzyme produced nitrous oxide and the cytoplasmic enzyme produced nitric oxide. In the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. After solubilization, the membrane-bound enzyme behaved like the cytoplasmic one and produced nitric oxide or ammonia in the presence of the above-mentioned electron donors. All activities are probably due to one cd-type cytochrome enzyme, whose products depend on its location and on the nature of the electron donors (186). Manometric activity measurements in resting-cell suspensions showed nitrite reduction to be optimally active at 0.38 to 0.75 M NaCl, with about 20% of the maximum activity remaining in the presence of 3 M NaCl (283). A membrane-bound assimilatory nitrate reductase was characterized in H. israelensis. This enzyme accepts electrons from the electron transport chain in a pathway that includes a b-type cytochrome and was found to be very similar to dissimilatory membrane-bound nitrate reductases known from other organisms. The assimilatory nitrite reductase was soluble, repressed by ammonium ions, and induced by nitrate, and it used reduced ferredoxin as its preferred electron donor (125). Other membrane-associated enzymes activated by salt are the lactate dehydrogenase of H. halodenitrificans (optimum concentration, 0.8 M NaCl) (10, 283) and the alkaline phosphatase of “P. halosaccharolytica” (370). Alkaline and acid phosphatases were detected in H. elongata, with both enzymes being located on the cell envelope. In low-salt (50 mM)-grown cells, little activity was detected, and at higher salinities (1.4 and 3.4 M), the activities of both enzymes were optimal at the same salt concentration at which the cells were grown. These enzymes thus seem to “adapt” to the NaCl concentration in the medium in a mechanism that was not further elucidated. The effect was not due to the presence of isozymes, since only one band of each enzyme was detected on gels (18). Extracellular Enzymes A considerable amount of effort has been dedicated to the study of extracellular salt-tolerant enzymes of the moderately halophilic bacteria, especially toward the use of such enzymes in biotechnological processes. Halophilic amylases were characterized from a moderately halophilic Acinetobacter (236), N. halobia (240), “M. varians subsp. halophilus” (158), and other Micrococcus isolates (157, 233, 234). A moderately halophilic Acinetobacter strain isolated from sea sand had two amylases. Both were most active at pH 7 and 50 to 55°C in 0.2 to 0.6 M NaCl or KCl. Activity was lost upon dialysis against water but could be stabilized by the addition of 10 mM CaCl 2 . Cells grown in 1 to 2 M NaCl made the largest amount of enzyme, and only a small amount of amylase was produced during growth in low-salt medium or at 4 M NaCl (236). An amylase was purified from N. halobia.Itwas 526 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from optimally active at pH 6 to 7 in 0.25 M NaCl or 0.75 M KCl and at 50 to 55°C. The products of starch degradation were maltose, maltotriose, maltotetraose, and small amounts of glucose (240). “M. varians subsp. halophilus” produced the highest amylase activity when grown in 2 M NaCl. When the organism was grown at lower salt concentrations, the excreted enzyme was inactivated by the low salt concentration but could be reactivated by dialysis against 2.5 M NaCl. The enzyme was stabilized by 10 mM CaCl 2 . The purified amylase has two components with apparent molecular masses of 86 and 60 kDa. Both were optimally active at 0.75 to 1 M NaCl or KCl and pH 6 to 7 at 55°C (in the absence of CaCl 2 ) or 60°C (in the presence of 50 mM CaCl 2 ) (158). The amylases from two other micrococci showed maximum activity in 1 M NaCl at pH 7.5 and 50°C (157) and in 1.4 to 2 M NaCl at pH 6 to 7 and 50°C (233, 234). Little information has been published on extracellular proteases from moderately halophilic bacteria. An unidentified pseudomonad was reported to secrete a protease with a maximum activity at 18% salt (337). “M. varians subsp. halophilus” produces a nuclease (nuclease H) when grown in 1 to 4 M NaCl or KCl. The purified enzyme has both DNase and RNase activities. Enzyme production is maximal in media containing 2.5 to 3.5 M salt. The enzyme was also synthesized in media containing RbCl, CsCl, or Na 2 SO 4 but not in media containing LiCl, NaNO 3 , NaBr, or NaI (147). Activity was optimal when the organism was grown in 2.9 M NaCl or 2.1 M KCl at 40°C. Activity was lost by dialysis against water but was restored upon dialysis against buffer containing 3.4 M NaCl (142, 143). The protein had a large (21 mol%) excess of acidic over basic amino acids (145). Inclusion of high magnesium (40 mM or higher) or phosphate (over 14 mM) concentrations in the medium abolished extracellular nuclease activity and caused flocculation of the cells. Under these conditions, the enzyme was adsorbed to the surface of the flocs (144, 372). The nuclease shows considerable biotechnological potential (see below). Another halophilic nuclease (an exonuclease, releasing 59- mononucleotides from both DNA and RNA) was produced by Bacillus halophilus. The enzyme was optimally active in 1.4 to 3.2 M NaCl or 2.3 to 3.2 M KCl and was stimulated by Mg 21 and Ca 21 ions. Activity was lost by dialysis against water, but 10 mM Ca 21 stabilized the protein and dialysis against 3.5 M NaCl restored most of the activity. The optimum pH was 8.5, and the optimum temperature was 50 or 60°C when DNA and RNA served as the substrate, respectively (239). Amino Acid Composition of Proteins from Moderately Halophilic Bacteria Halophilic archaea of the family Halobacteriaceae contain high intracellular salt concentrations, and their proteins are adapted to the presence of molar concentrations of K 1 ,Na 1 , and Cl 2 . This adaptation is reflected in the bulk amino acid composition of these microorganisms, showing a high content of acidic amino acids (Glu and Asp), and a low content of basic amino acids. A low proportion of hydrophobic amino acids is offset by a high frequency of amino acids such as Gly and Ser (177). The aerobic halophilic bacteria tolerate high salt concentrations, but organic osmotic solutes provide most of the osmotic balance. However, the apparent intracellular salt concentrations can be fairly high (Table 5). Therefore, one should expect that extracellular and membrane-bound proteins of the halophilic bacteria may display halophilic characteristics with amino acid compositions similar to those found in the halophilic archaea, while there is little a priori reason to assume that the cytoplasmic proteins should show such adaptations. However, in view of reports of high apparent intracellular salt concentrations in certain species (Table 5), some degree of halophilic character may be expected in the intracellular enzymes as well. Comparative studies showed that ribosomal proteins from moderate halophiles often had a slightly higher content of acidic amino acids than did the comparable proteins from E. coli and other nonhalophiles. Thus, the N-terminal sequences of the ribosomal A proteins of S. costicola and H. canadensis were slightly more acidic than that of the E. coli protein but much less so than that of the similar protein from H. salinarum (cutirubrum), with the ratios of basic to acidic amino acids being 0.36 in S. costicola and 0.41 in H. canadensis, compared to 0.54 in E. coli and 0.05 in H. salinarum (62, 63). The values for whole ribosomes of H. canadensis were 1.23 and 1.16, respectively, compared to 1.58 in E. coli and 0.69 in H. salinarum (61). The average hydrophobicity of the ribosomal proteins of S. costicola and H. canadensis did not differ greatly from that for E. coli and was much higher than that for H. halobium (194). Determinations of the abundance of different amino acids in the bulk protein of H. elongata showed an excess of acidic amino acids and a low frequency of basic amino acids, with the values being intermediate between those for E. coli and the archaeon Haloferax mediterranei. Convergent evolution of amino acid usage was suggested to have led to this “halophilic” character of the H. elongata proteins (93). However, this convergent evolution did not lead to changes in the frequencies of the hydrophobic amino acids. The ratio of the occurrence of hydrophobic amino acids (Ala, Val, Leu, Ile, Phe, and Met) to that of the borderline hydrophobic amino acids (Ser and Thr) was 3.56 in H. elongata, compared with 3.58 in E. coli, 2.73 in H. mediterranei, and 2.46 in membranes of H. salinarum (248). CELL ENVELOPES The cytoplasmic membrane forms the barrier between the cytoplasm (generally low in salt) and the environment with its high and possibly fluctuating salinity. It can thus be expected that the properties of this membrane are regulated by the outside salt concentration to adjust such functions as ion permeability and the activity of integral membrane proteins (294). The envelope of the moderate halophiles is involved in regulating the cytoplasmic ionic environment by both binding and pumping ions, and haloadaptation is at least in part a response of the cell envelope to osmotic stress (296). Salt-dependent changes in the properties of the cell membrane have been identified on the levels both of the types of phospholipids that dominate in the membrane structure and of the types of fatty acid chains present in the lipids. Polar Lipids Studies of the effect of salinity on polar lipid composition of the membranes have consistently shown that the higher the salinity, the higher the content of negatively charged phospholipids at the expense of neutral phospholipids. With increased salt concentrations, the amount of phosphatidylethanolamine (PE) (uncharged) is decreased and there is a concomitant increase in the amounts of the negatively charged phosphatidylglycerol (PG) and/or diphosphatidylglycerol (cardiolipin, CL) (294, 351, 354). To explain the shift toward a higher negative-charge density on the membrane at increasing salt concentration, it was first postulated that the increase in the amount of anionic lipids VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 527 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from served to allow charge balance at the membrane surface exposed to high Na 1 concentrations (121, 122). However, a simple calculation showed that millimolar concentrations of salt would suffice to provide the negative-charge shielding (296). Moreover, a similar increase in the amount of negatively charged polar lipids was observed when nonionic solutes were added to the medium to increase its osmotic value. Another idea proposed is that the high content of negative phospholipids may contribute to the regulation of the selective permeability of the membrane to cations (231). It is now assumed that the change in polar lipid composition provides a mechanism for preserving the membrane bilayer structure. PE containing unsaturated fatty acids tends to form nonbilayer phases, while PG forms bilayers. Addition of PG to PE in the appropriate amounts could suppress the formation of a hexagonal-II phase and thus counteract the effect of increased salinity. A functional membrane requires a suitable proportion of bilayer-forming and non-bilayer-forming lipids. Modification of the membrane phospholipid ratio is necessary to preserve the integrity of the membrane bilayer in the face of an increased tendency of PE to form nonbilayer phases as a consequence of raised external salinity. The tendency to form nonbilayer phases could activate specific phospholipid-synthe- sizing enzymes located in the membrane, resulting in a rise in the proportion of lipids such as PG that prefer the lamellar phase, to counteract the disruptive forces of the nonbilayerphase-forming lipids (293, 294, 322). (i) Salinivibrio costicola. The major lipids of S. costicola are PG and PE, with lesser amounts of lyso-PE, CL, a glycolipid, and two other phospholipids, tentatively identified as lyso-CL and lyso-PG. When the medium salinity was increased over the range 0.5 to 3 M, the level of PG increased and that of PE decreased. Thus, the PG/PE ratio increased from 0.5:1 to 1:1 when the medium salinity was increased from 1 to 3 M. The growth phase of the culture had little effect on the lipid composition (110, 322). The altered salt dependence of S. costicola at temperatures below the optimum may be due to a modification in the membrane lipid phase behavior and stability brought about by changes in lipid composition (3). The effect of salinity on the phase behavior of total lipid extracts from S. costicola was compared with the phase behavior of binary mixtures of PG and PE. Although many similarities were detected, certain differences were observed, suggesting that the minor lipid components do play a part in determining the phase behavior of the membrane (322). Salt-sensitive mutants of S. costicola have been isolated with a phospholipid composition similar to the wild type when grown in 1 M NaCl but showing only a partial change in phospholipid composition upon growth at elevated salt concentrations. These data provide evidence that a correct phospholipid composition may be needed for haloadaptation (162). High concentrations of the chaotropic anion SCN 2 (1Mas KSCN but not as NaSCN) caused lysis of S. costicola. The protective effect of Na 1 is probably due to specific interactions of Na 1 with components of the cell membrane, thereby rendering their structure resistant to the action of chaotropic anions, which act by breaking hydrophobic bonds (335). (ii) Halomonas elongata. In cells grown at 3.4 M NaCl, the CL content was four times as high as in cells grown at 0.05 M NaCl; the change was mainly at the expense of PE, while the PG content did not change greatly. Thus, the anionic lipids PG and CL contributed 57% of the polar lipids at high salt concentrations and 45% at low salt concentrations (354). The hydrophobic-hydrophilic cell surface character of H. elongata changed with medium salinity, as shown in phase partitioning studies. Mid-exponential-phase cells growing at 3.4 M NaCl were more hydrophilic than cells at 0.05 M NaCl, and high-salt-grown cells were more hydrophilic than low-salt- grown cells in all stages of growth. Surface hydrophobicity tended to increase as the cells approached the stationary phase. The hydrophobicity of the cell was not influenced by trypsin treatment, in contrast to many other microorganisms in which trypsin treatment converts hydrophobic to hydrophilic cells. The increase in the proportion of charged phospholipids at higher NaCl concentrations may in part explain the hydrophilicity. At the high salinity, the hydrophilic cell surface makes the cell more attractive to water molecules in a water-poor environment and the hydrated cell surface may help the cell obtain cytoplasmic water and thereby prevent desiccation (114). Electron microscopic studies showed that an increasing medium salinity had a profound effect on the cell envelope of H. elongata, with the cell wall becoming more compact internally and coherent at higher salinity (354). The cell wall peptidoglycan is unique in that it contains leucine, which contributes to the overall hydrophobicity of the cell. To study the differences in cell wall composition, rabbits were immunized with live H. elongata cells grown at different NaCl concentrations. Antiserum prepared against low-salt-grown cells reacted with cells grown at all salt concentrations, but antiserum against high-salt-grown cells was specific for this type of cells. Two possible explanations for the finding were put forward: either the low-salt-grown cells possessed a unique antigen, or some type of conformational change in the surface antigens caused the rabbits to respond to a determinant that was not exposed in low-salt-grown cells (355). (iii) Halomonas israelensis. The major lipid components of H. israelensis are PG and PE, with a-glucosyl-PG (256) and an acidic glucuronic acid-containing glycolipid (glucuronosyldiglyceride). In stationary-phase cells, the CL level was increased at the expense of PG (317). The biosynthesis of glucosyl-PG was studied in a cell-free system and was found to proceed from UDP-glucose and PG. Mg 21 and Ca 21 were required for activation, and KCl and NaCl were inhibitory, even at the low concentration of 0.1 M (319). Labeled glucuronosyldiglyceride was formed from UDP-[ 14 C]glucuronic acid and added diglycerides in a cell-free preparation. KCl and NaCl were inhibitory, with 0.5 M causing more than 80% inhibition (318). (iv) “Pseudomonas halosaccharolytica.” The major phospholipids of “P. halosaccharolytica” are PE, PG, CL, and an unidentified phosphoglycolipid—a glucosyl derivative of PG containing phosphate, glycerol, fatty acids, and glucose in the ratio 1:2:2:1. When grown in the lower-salinity range (0.5 to 1 M NaCl), PE was twice as abundant as PG, while at high salinity (3 M or more), the amounts of PE and PG were approximately equal (121, 122, 230, 231). At a combination of high temperature and high salt concentration, acidic phospholipids were especially abundant (73.6 mol%), due to an increase in CL and glucosyl-PG levels with a decrease in the PE level (112). Upon entering the stationary growth phase, the content of CL increased at the expense of PG. The cytoplasmic membrane contained a higher proportion of acidic polar lipids than did the outer membrane (50 and 40% respectively, in cells grown in 2 M NaCl). When 1% glucose was added to the medium, the phosphoglycolipid content increased to 25% of the total lipid. Thus, the phospholipid pattern resembled that of other moderate halophiles discussed above, with the phosphoglycolipid being possibly identical to the a-glucosyl-PG identified in H. israelensis (230). As in H. israelensis, UDP-glucose served as the glucosyl donor for its biosynthesis (229). The glucuronosyldiglyceride of H. israelensis was not detected in “P. halosaccharolytica” (230). The outer membrane proteins of “P. halosaccharolytica” 528 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from the vector pHSG575 were screened on minimal medium with elevated salinity; one clone, carrying the recombinant plasmid pOSM1, was able to grow at 5% NaCl thanks to ectoine production. Sequencing of a 4.4-kb fragment of pOSM1 revealed three open reading frames involved in ectoine synthesis: ectA, encoding L-diaminobutyric acid acetyltransferase; ectB, encoding L-diaminobutyric acid transaminase; and ectC, encoding ectoine synthase. The M. halophilus ectC gene had 47% identity to the corresponding gene of Halomonas sp. strain KS-3 (208). The genes were osmoregulated in E. coli, as demonstrated by an increasing cytoplasmic ectoine concentration in response to salinity (180). To identify the ectoine biosynthetic genes of H. elongata DSM 3043, Ca´novas et al. (24) isolated mutants blocked in its synthesis. Two of these Tn1732-induced mutants accumulated ectoine precursors, as shown by 13 C-NMR and mass spectroscopy. Mutant CHR62 accumulated low levels of diaminobutyric acid, while mutant CHR63 contained high concentrations of N-g-acetyldiaminobutyric acid, suggesting that strain CHR62 could be defective in the diaminobutyric acid acetyltransferase gene and strain CHR63 could be defective in the ectoine synthase gene. The genes were isolated by colony hybridization, using a 370-bp chromosomal fragment flanking the Tn1732 insertion in mutant CHR63 as a probe against a genomic library of H. elongata. One cosmid clone thus identified was able to complement the mutations in both CHR62 and CHR63, confirming that it carried the wild-type genes encoding the enzymes for the synthesis of N-g-acetyldiaminobutyric acid and ectoine. Preliminary data on the sequence of the isolated region revealed that the predicted protein encoded by the ectoine synthase gene of H. elongata is highly homologous to the corresponding protein sequences of Halomonas sp. strain KS-3 and M. halophilus (22). The organization of the ectoine synthesis genes in H. elongata, most probably contained in one operon, seems to differ from that in M. halophilus (22, 24). The genes encoding the transport system for glycine betaine in H. elongata DSM 3043 have recently been isolated (22). For this purpose, a gene bank of this organism was transferred by conjugation to an E. coli strain unable to take up glycine betaine and transconjugants were selected for growth in minimal medium containing 0.75 M NaCl and 1 mM betaine. A number of cosmid clones carrying the genes for glycine betaine transport were obtained. To isolate the genes of the choline-betaine pathway of H. elongata, the same gene bank was used to complement E. coli MKH13 harboring plasmid pJB004 (carrying the E. coli choline transport betT gene). Three cosmid clones were isolated that conferred to MKH13(pJB004) the ability to oxidize choline to glycine betaine, as shown by thin-layer chromatography of cytoplasmic extracts of the transconjugants. The isolated region has been sequenced, showing that it contains three genes highly homologous to the betI,betB, and betA genes of E. coli (22). At least in some bacteria, halotolerance may be linked to the presence of certain plasmids. Preliminary studies have indicated that when H. elongata was cured of its single plasmid, it lost its ability to grow in 3.4 M NaCl (351). Similarly, a euryhaline strain designated “Spirillum luteum” was able to grow at up to about 1.2 M salt. However, after plasmid curing by acriflavine treatment, growth could be obtained at up to about 0.7 M only. It was concluded that salt resistance in this organism is probably controlled by a plasmid (216, 217). It is to be regretted that these studies were not pursued in further depth. BIOTECHNOLOGICAL APPLICATIONS Moderately halophilic bacteria have the potential for exciting and promising applications. Not only do many of them produce compounds of industrial interest (enzymes, polymers, and osmoprotectans), but also they possess useful physiological properties which can facilitate their exploitation for commercial purposes. First, most of them can grow at high salt concentrations (352), minimizing the risk of contamination. Second, they are easy to grow, and their nutritional requirements are simple: the majority can use a large range of compounds as their sole carbon and energy source (176). Moreover, as discussed above, many of the genetic tools developed for the nonhalophilic bacteria can be applied to the moderate halophiles, and hence their genetic manipulation seems relatively simple. In spite of all this, the moderately halophilic bacteria have not yet been used extensively for biotechnological purposes. In this section, we discuss some of the current industrial applications of these halophiles and emphasize some expected future developments. Fermented Foods Tetragenococcus strains are involved in the fermentation of soy sauce. In soy sauce manufacture, ground wheat and soy grains are suspended in water with about 19% NaCl and incubated for up to 9 months in the dark (172). Halophilic lactococci (Tetragenococcus halophilus) are used as starters for the fermentation and typically develop densities of up to 10 8 CFU/ ml in soy sauce mash with about 3 M NaCl (289, 331). Tetragenococcus muriatianus is involved in the preparation of fermented liver sauce (304). In the preparation of Thai fish sauce (nam pla, a food condiment widely used in Southeast Asia), moderate halophiles and halotolerant bacteria are used (Bacillus spp., coryneform bacteria, and, more rarely, pseudomonads, most of these tolerating up to 20 to 30% salt). Extremely halophilic red archaea are also found during the process (326). Enzymes As described above, a number of extra- and intracellular enzymes from moderately halophilic bacteria have been isolated and characterized. These include hydrolases (amylases, nucleases, phosphatases, and proteases), which are currently of commercial interest. Nuclease H produced by “M. varians subsp. halophilus” may be useful for the industrial production of the flavoring agent 59-inosinic acid and 59-guanylic acid (140) by using a bioreactor system with a column of flocculated cells (241). Esterases with high affinity for short-chain esters are made by a strain of H. elongata growing in Danish bacon curing brines (118), and the isolation of novel Bacillus species such as B. salexigens, which produces an extracellular nuclease in saline medium (89), may also lead to the development of industrially promising processes. Compatible Solutes Moderate halophiles accumulate high cytoplasmatic concentrations of low-molecular-weight organic compounds to cope with the osmotic stress and to maintain positive turgor pressure. The ability to produce and accumulate high concentrations of these compounds makes moderate halophiles useful for the biotechnological production of these osmolytes. Some compatible solutes, especially glycine betaine and ectoines, have gained considerable attention in recent years, because they may be used as stress protectants (against high salinity, VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 535 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from thermal denaturation, desiccation, and freezing) and stabilizers of enzymes, nucleic acids, membranes and whole cells (80, 82, 181). The industrial use of these compounds in enzyme technology (biosensor technologies, PCR, etc.) and for pharmaceuticals and cosmetics is a most promising field (84–86, 346). Ectoine and hydroxyectoine exert a remarkable protective effect on a number of labile enzymes such as lactate dehydrogenase and phosphofructokinase. In a study with several compatible solutes (glycine betaine, trehalose, glycerol, proline, ectoines, and sugars), hydroxyectoine showed the highest efficiency of protection of lactate dehydrogenase against freezethaw treatment and heat stress whereas ectoine was the most effective freeze-stabilizing agent for phosphofructokinase (80, 181). Ectoine and hydroxyectoine can as yet be produced only biologically. The production of ectoine for industrial purposes by using H. halodenitrificans has recently been established (325). Cells were grown in an anaerobic fed-batch fermentation process, in a synthetic medium with glycerol at high concentration as the carbon source. Once a high cell density was reached, ectoine was extracted by “bacterial milking”, a process previously developed with H. elongata, in which an osmotic down-shock from 10 to 2% NaCl is employed, resulting in excretion of about 80% of the intracellular ectoine to the surrounding medium (88, 305). Subsequent exposure of the cells to a hyperosmotic shock (from 2 to 10% NaCl) restored the original level of ectoine in a period of 10 h. Thus, a yield of 2 g ectoine per liter of medium per day was obtained. Ectoine and hydroxyectoine can also be produced by the gram-positive moderate halophile Marinococcus strain M52. A maximum yield of 35.3 g per liter cell dry weight was reached (77). The use of a dialysis reactor, in which cells were grown in an inner chamber fed with fish peptone and glucose, resulted in a dramatic increase in yield: up to 132 g of cell dry weight with about 20% hydroxyectoine was obtained (164). Another organism used for the production of ectoine is the actinomycete Nocardiopsis lucentensis A5-1, isolated from a saline soil near Alicante, Spain (179, 371). Advances in fermentation technology and genetic engineering of moderate halophiles will allow large amounts of these compatible solutes to become available. As stated above, the genes involved in the synthesis of ectoine and its regulation in halophilic bacteria have recently been cloned (24, 179, 207), opening the way to its overproduction. It may also enable the transfer of genes for salt and drought tolerance from moderate halophiles to agricultural crops, such as wheat, rice, and barley, enabling them to grow in more saline soils. Polymers Bacterial polysaccharides are of great value as enhancers of oil recovery because of their surfactant activity and bioemulsifying properties. Since the conditions in oil deposits are often saline, the use of salt-resistant surfactants may be advantageous. Pfiffner et al. (260) isolated more than 200 bacterial strains capable of producing extracellular polysaccharides from oilwells and oil well-associated environments. The predominant type was a facultative anaerobic, as yet unidentified Bacillus species, growing at up to 12% NaCl. It produced a polysaccharide with pseudoplastic behavior, resistant to shear and thermal degradation, and showing higher viscosities at dilute concentrations and elevated temperatures than commercial polymers such as xanthan gum. The polymer is a charged heteropolysaccharide containing glucose, arabinose, mannose, ribose, and small amounts of allose and glucosamine. Production was optimal under anaerobic conditions in media with 4 to 10% NaCl. H. eurihalina F2-7 produces large amounts of an extracellular polyanionic polysaccharide (263). The polymer, consisting of 42% carbohydrates (mostly hexoses) and 15% protein, is a potent emulsifying agent, exhibiting pseudoplastic behavior. It forms gels of high viscosity at acid pH. In view of these properties, the polysaccharide (EPS V2-7) may find broad applications in pharmaceuticals, in the food industry, and in biodegradation processes (21). Having a high content in sulfate groups, this polysaccharide was shown to have remarkable in vitro immunomodulating activity, enhancing the proliferative effect of human lymphocytes as a response to the presence of the anti-CD3 monoclonal antibody in blood (258). Degradation of Toxic Compounds Hypersaline waters and soils are often contaminated with heavy metals or other toxic compounds from anthropogenic sources. Hypersaline wastewaters are generated during the manufacture of chemicals such as pesticides, pharmaceuticals, and herbicides and during oil and gas recovery processes. Conventional microbiological treatment processes do not function at high salt concentrations, and therefore the use of moderately halophilic bacteria should be considered (250, 251). Hydrocarbon-degrading moderate halophiles have been isolated from a variety of environments, including the Great Salt Lake (360) and Antarctic saline lakes (196). Woolard and Irvine (366) reported the utilization of a biofilm of a moderately halophilic bacterium isolated from a saltern at the Great Salt Lake, Utah, for the treatment of hypersaline wastewaters containing phenol. By using a batch biofilm reactor, more than 99% of the phenol was removed from a waste containing 15% salt. Benzoate and other aromatic compounds can be degraded by H. halodurans by cleavage of aromatic rings (291). A moderately halophilic bacterium isolated from a hypersaline spring in Utah degrades highly toxic organophosphorus compounds. The microorganism (strain JD6.5) grew at 2 to 24% salt and was tentatively identified as an Alteromonas strain (38). The enzyme responsible, organophosphorus acid anhydrase, was purified and characterized. Five additional halophilic bacteria, showing hydrolytic activity against several organophosphorus compounds and related chemicals, were isolated (37). Such enzymes may have considerable potential for the decontamination and mineralization of chemical warfare agents. Moderate halophiles belonging to the family Halomonadaceae have been recently isolated from highly saline sites contaminated with the herbicide 2,4-dichlorophenoxyacetic acid; they were able to utilize chloroaromatic compounds as sources of carbon and energy. One of the isolates, strain I-18, showed high activities of catechol 1,2-dioxygenase, muconate cycloisomerase, and dienelactone hydrolase at about 1.0 M NaCl and pH 8.4 to 9.4 (185). This strain was also able to utilize other aromatic compounds including benzoic acid, 3-chloro- benzoic acid, and 4-chlorophenol. Other Potential Applications Moderately halophilic bacteria may have numerous additional potential uses in biotechnology, yet to be exploited. (i) It has been claimed that moderate halophiles may be used to remove phosphate from saline environments, as a cheaper alternative to chemical approaches (275). (ii) Moderate halophiles could be used in the recovery of hypersaline waste brines derived from the olive oil industry and leather- or fur-curing processes. (iii) They may be screened for the production of bioactive compounds such as antibiotics. (iv) Biological surfac- 536 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from tants derived from moderate halophiles may have a variety of applications in industry. Thus, Yakimov et al. (369) recently isolated a moderate halophile which synthesizes a novel glycolipid belonging to a powerful novel class of biosurfactants. The bacterium (strain MM1, a member of the Halomonadaceae) was isolated from saline water and sediment samples by enrichment on n-hexadecane. The purified surfactant is a powerful surface-active agent, showing a strong positive effect on chlorinated biphenyl degradation, probably due to the solubilization of these compounds in water. (v) New restriction endonucleases and other enzymes for molecular biological studies are expected to be present in microorganisms from hypersaline habitats and will be discovered and exploited. (vi) Many moderate halophiles produce orange or pink colonies, probably due to the production of carotenoids as a protective mechanisms against photooxidation processes. Carotenoids have major applications in the food industry as food-coloring agents and as additives in health food products. Therefore, investigations of the utilization of moderate halophiles as producers of carotenoids could be of great interest. Finally, we may note that in the last few years, considerable economic effort has been made to facilitate the exploitation of the potentialities of extremophilic microorganisms such as moderate halophiles. Thus, in Europe, a consortium of 39 teams funded by the BIOTECH-Programme of the European Commission has recently finished a 3-year project aimed at isolating and/or selecting the extremophiles with the best potential for industry. In early 1997, a new project was initiated to utilize these microbes as cell factories for the production of commercially interesting compounds. It may be expected that the moderately halophilic bacteria will shortly be used routinely in biotechnology as their astonishing properties are increasingly exploited. CONCLUSION AND FUTURE DIRECTIONS Studies carried out, specially during the last decade, have increased our current knowledge about different aspects of moderately halophilic bacteria, such as their physiology, ecology, taxonomy or phylogenetic relationships with other microorganisms, and, to a lesser extent, their genetics. Besides, there are several fields in which their industrial applications are more promising, and, as in the case of other extremophilic microorganisms, they have an important biotechnological potential as a source of compatible solutes, enzymes, and other compounds of industrial interest. Moreover, moderately halophilic bacteria constitute an excellent model for the molecular study of the osmoregulatory mechanisms that permit them to grow over a wide range of salt concentrations. This aspect has very exciting potentialities, such as, for instance, their possible application in agriculture to construct salt-resistant plants carrying procaryotic genes encoding enzymes for the synthesis of osmoprotective compounds. For all these studies, the availability of genetic tools and an in-depth knowledge of their molecular genetics will be necessary, and thus, this field could be identified currently as one of the most attractive for researchers. Systematic and phylogenetic studies have defined a large number of species to be included within the moderately halophilic bacteria, distributed over at least half of the major phylogenetic branches of the bacteria. Molecular ecology techniques available nowadays should be used to determine in more detail the ecological distribution of these halophilic microorganisms and the roles they play in hypersaline environments, as well as their contribution to microbial transformation processes. The use of such techniques would enable the elucidation of the biodiversity of moderately halophilic bacteria and the identification of species that constitute the predominant populations in these extreme habitats. ACKNOWLEDGMENTS We thank Carol D. Litchfield (Fairfax, Va.), Erwin A. Galinski (Mu¨nster), and Carmen Vargas (Seville) for critical reading of parts of the manuscript and invaluable comments. C. Vargas prepared some figures. Work in the laboratory of A. Ventosa and J. J. Nieto was supported by grants from the Biotech Programme of the European Commission (Generic Projects “Biotechnology of Extremophiles” [grant BIO2- CT93-0273] and “Extremophiles as Cell Factories” [grant BIO4-CT96- 0488]), the Ministerio de Educacio´n y Ciencia, Spain (grants PB93- 0920 and BIO97-1876-CE), and Junta de Andalucı´a. A. Oren acknowledges the United States—Israel Binational Science Foundation (BSF, Jerusalem, Israel; grant 95-00027). REFERENCES 1. Adams, R., J. Bygraves, M. Kogut, and N. J. Russell. 1987. The role of osmotic effects in haloadaptation of Vibrio costicola. J. Gen. Microbiol. 133: 1861–1870. 2. Adams, R. L., M. Kogut, and N. J. Russell. 1990. The effect of salinity on growth and lipid composition of a moderately halophilic Gram-negative bacterium HX. Biochem. Cell Biol. 68:249–254. 3. Adams, R. L., and N. J. Russell. 1992. Interactive effects of salt concentration and temperature on growth and lipid composition in the moderately halophilic bacterium Vibrio costicola. Can. J. Microbiol. 38:823–827. 4. Adkins, J. P., M. T. Madigan, L. Mandelco, C. R. Woese, and R. S. Tanner. 1993. Arhodomonas aquaeolei gen. nov., sp. nov., an aerobic halophilic bacterium isolated from a subterranean brine. Int. J. Syt. Bacteriol. 43:514– 520. 5. Ahonkhai, I., M. Kamekura, and D. J. Kushner. 1989. Effects of salts on the aspartate transcarbamylase of a halophilic eubacterium, Vibrio costicola. Biochem. Cell Biol. 67:666–669. 6. Al-Tai, A. M., and J.-S. Ruan. 1994. Nocardiopsis halophila sp. nov., a new halophilic actinomycete isolated from soil. Int. J. Syst. Bacteriol. 44:474– 478. 7. Arvantis, N., C. Vargas, G. Tegos, A. Perysinakis, J. J. Nieto, A. Ventosa, and C. Drainas. 1995. Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae. Appl. Environ. Microbiol. 61:3821–3825. 8. Azachi, M., A. Oren, P. Gurevich, S. Sarig, and Y. Henis. 1995. Transformation of formaldehyde by a Halomonas sp. Can. J. Microbiol. 41:548–553. 9. Baxter, R. M., and N. E. Gibbons. 1954. The glycerol dehydrogenases of Pseudomonas salinaria,Vibrio costicolus, and Escherichia coli in relation to bacterial halophilism. Can. J. Biochem. Physiol. 32:206–217. 10. Baxter, R. M., and N. E. Gibbons. 1956. Effects of sodium and potassium chloride on certain enzymes of Micrococcus halodenitrificans and Pseudomonas salinaria. Can. J. Microbiol. 2:599–606. 11. Bengis-Garber, C., and D. J. Kushner. 1981. Purification and properties of 59-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium. J. Bacteriol. 146:24–32. 12. Bengis-Garber, C., and D. J. Kushner. 1982. Role of membrane-bound 59-nucleotidase in nucleotide uptake by the halophile Vibrio costicola.J. Bacteriol. 149:808–815. 13. Bennloch, S., S. G. Acinas, A. J. Martı´nez-Murcia, and F. Rodrı´guez- Valera. 1996. Description of prokaryotic biodiversity along the salinity gradient of a multipond solar saltern by direct PCR amplification of 16S rDNA. Hydrobiologia 329:19–31. 14. Bennloch, S., A. J. Martı´nez-Murcia, and F. Rodrı´guez-Valera. 1995. Sequencing of bacterial and archaeal 16S rRNA genes directly amplified from a hypersaline environment. Syst. Appl. Microbiol. 18:574–581. 15. Bernard, T., M. Jebbar, Y. Rassouli, S. Himdi-Kabbab, J. Hamelin, and C. Blanco. 1993. Ectoine accumulation and osmotic regulation in Brevibacterium linens. J. Gen. Microbiol. 139:129–136. 16. Brisou, J., D. Courtois, and F. Denis. 1974. Microbiological study of a hypersaline lake in French Somaliland. Appl. Microbiol. 27:819–822. 17. Brown, A. D. 1976. Microbial water stress. Bacteriol. Rev. 40:803–846. 18. Bylund, J. E., J. K. Dyer, D. E. Feely, and E. L. Martin. 1990. Alkaline and acid phosphatases from the extensively halotolerant bacterium Halomonas elongata. Curr. Microbiol. 20:125–131. 19. Bylund, J. E., J. K. Dyer, T. L. Thompson, and E. L. Martin. 1991. Alanine dehydrogenase activity of the extensively halotolerant eubacterium Halomonas elongata. Microbios 66:45–53. 20. Calvo, C., A. G. de la Paz, V. Bejar, E. Quesada, and A. Ramos-Cor- menzana. 1988. Isolation and characterization of phage F9-11 from a lysogenic Deleya halophila strain. Curr. Microbiol. 17:49–53. VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 537 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from 21. Calvo, C., M. R. Ferrer, F. Martı´nez-Chewca, V. Bejar, and E. Quesada. 1995. Some rheological properties of the extracellular polysaccharide produced by Volcaniella eurihalina F2-7. Appl. Biochem. Biotechnol. 55:45–54. 22. Ca´novas, D. Unpublished results. 23. Ca´novas, D., C. Vargas, L. N. Csonka, A. Ventosa, and J. J. Nieto. 1996. Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway. J. Bacteriol. 178:7221–7226. 24. Ca´novas, D., C. Vargas, F. Iglesias-Guerra, L. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto. 1997. Isolation and characterization of salt-sensi- tive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes. J. Biol. Chem. 272:25794–25801. 25. Ca´novas, D., C. Vargas, A. Ventosa, and J. J. Nieto. 1997. Salt sensitive and auxotrophic mutants of Halomonas elongata and H. meridiana by use of hydroxylamine mutagenesis. Curr. Microbiol. 34:85–90. 26. Cazzulo, J. J. 1978. The adenosine triphosphatase from the moderate halophile, Vibrio costicola, p. 447–451. In S. R. Caplan and M. Ginzburg (ed.), Energetics and structure of halophilic microorganisms. Elsevier/ North-Holland Biomedical Press, Amsterdam, The Netherlands. 27. Cazzulo, J. J. 1979. Las bacterias halofilas moderadas. Rev. Argent. Microbiol. 11:118–128. 28. Chan, K., O. C. Leung, and L. H. Lee. 1979. Influence of temperature on ionic sparing effect and cell-associated cations in the moderate halophile, Micrococcus varians var. halophilus. Microbios 24:81–91. 29. Choquet, C. G., I. Ahonkhai, M. Klein, and D. J. Kushner. 1991. Formation and role of glycine betaine in the moderate halophile Vibrio costicola. Arch. Microbiol. 155:153–158. 30. Choquet, C. G., M. Kamekura, and D. J. Kushner. 1989. In vitro protein synthesis by the moderate halophile Vibrio costicola: site of action of Cl 2 ions. J. Bacteriol. 171:880–886. 31. Christian, J. H. B., and J. A. Waltho. 1962. Solute concentrations within cells of halophilic and non-halophilic bacteria. Biochim. Biophys. Acta 65: 506–508. 32. Ciulla, R. A., M. R. Diaz, B. F. Taylor, and M. F. Roberts. 1997. Organic osmolytes in aerobic bacteria from Mono Lake, an alkaline, moderately hypersaline environment. Appl. Environ. Microbiol. 63:220–226. 33. Claus, D., F. Fahmay, H. J. Rolf, and N. Torunoglu. 1983. Sporosarcina halophila sp. nov., an obligate, slightly halophilic bacterium from salt marsh soils. Syst. Appl. Bacteriol. 4:496–506. 34. Coronado, M.-J., C. Vargas, H. J. Kunte, E. A. Galinski, A. Ventosa, and J. J. Nieto. 1995. Influence of salt concentration on the susceptibility of moderately halophilic bacteria to antimicrobials and its potential use for genetic transfer studies. Curr. Microbiol. 31:365–371. 35. Cummings, S. P., and D. J. Gilmour. 1995. The effect of NaCl on the growth of Halomonas species: accumulation and utilization of compatible solutes. Microbiology 141:1413–1418. 36. Cummings, S. P., M. P. Williamson, and D. J. Gilmour. 1993. Turgor regulation in a novel Halomonas species. Arch. Microbiol. 160:319–323. 37. DeFrank, J. J., W. T. Beaudry, T. C. Cheng, S. P. Harvey, A. N. Stroup, and L. L. Szafraniec. 1993. Screening of halophilic bacteria and Alteromonas species for organophosphorus hydrolyzing enzyme activity. Chem. Biol. Interact. 87:141–148. 38. DeFrank, J. J., and T. Cheng. 1991. Purification and properties of an organophosphorus acid anhydrase from a halophilic bacterial isolate. J. Bacteriol. 173:1938–1943. 39. Del Moral, A., B. Prado, E. Quesada, T. Garcı´a, R. Ferrer, and A. Ramos- Cormenzana. 1988. Numerical taxonomy of moderately halophilic Gramnegative rods from an inland saltern. J. Gen. Microbiol. 134:733–741. 40. Del Moral, A., E. Quesada, V. Bejar, and A. Ramos-Cormenzana. 1987. Evolution of bacterial flora from a subterranean saline well by gradual salinity changes in enrichment media. J. Appl. Bacteriol. 62:465–471. 41. Del Moral, A., E. Quesada, and A. Ramos-Cormenzana. 1987. Distribution and types of bacteria isolated from an inland saltern. Ann. Microbiol. 138: 59–66. 42. Del Moral, A., E. Roldan, J. Navarro, M. Monteoliva-Sanchez, and A. Ramos-Cormenzana. 1987. Formation of calcium carbonate crystals by moderately halophilic bacteria. Geomicrobiol. J. 5:79–87. 43. Del Moral, A., J. Severin, A. Ramos-Cormenzana, H. G. Tru¨per, and E. A. Galinski. 1994. Compatible solutes in new moderately halophilic isolates. FEMS Microbiol. Lett. 122:165–172. 44. Del Moral, A., M. J. Valderrama, M. R. Ferrer, F. Pera`n, E. Quesada, and A. Ramos-Cormenzana. 1991. Effect of external salinity changes on cellular composition of some ions and amino acids in Deleya halophila. Res. Microbiol. 142:103–107. 45. De Me´dicis, E. 1986. Magnesium, manganese and mutual depletion systems in halophilic bacteria. FEMS Microbiol. Rev. 37:137–143. 46. De Me´dicis, E., J. Paquette, J.-J. Gauthier, and D. Shapcott. 1986. Magnesium and manganese content of halophilic bacteria. Appl. Environ. Microbiol. 52:567–573. 47. De Me´dicis, E., and B. Rossignol. 1977. Pyruvate kinase from the moderate halophile, Vibrio costicola. Can. J. Biochem. 55:825–833. 48. De Me´dicis, E., and B. Rossignol. 1979. The halophilic properties of pyruvate kinase from Vibrio costicola, a moderate halophile. Experientia 35: 1546–1547. 49. Denariaz, G., W. J. Payne, and J. Le Gall. 1989. A halophilic denitrifier, Bacillus halodenitrificans sp. nov. Int. J. Syst. Bacteriol. 39:145–151. 50. Deutch, C. E. 1994. Characterization of a novel salt-tolerant Bacillus sp. from the nasal cavities of desert iguanas. FEMS Microbiol. Lett. 121:55–60. 51. Diaz, M. R., and B. F. Taylor. 1996. Metabolism of methylated osmolytes by aerobic bacteria from Mono Lake, a moderately alkaline environment. FEMS Microbiol. Ecol. 19:239–247. 52. Dobson, S. J., R. R. Colwell, T. A. McMeekin, and P. D. Franzmann. 1993. Direct sequencing of the polymerase chain reaction-amplified 16S rRNA gene of Flavobacterium gondwanense sp. nov. and Flavobacterium salegens sp. nov., two new species from a hypersaline Antarctic lake. Int. J. Syst. Bacteriol. 43:77–83. 53. Dobson, S. J., and P. D. Franzmann. 1996. Unification of the genera Deleya (Baumann et al. 1983), Halomonas (Vreeland et al. 1980), and Halovibrio (Fendrich 1988) and the species Paracoccus halodenitrificans (Robinson and Gibbons 1952) into a single genus, Halomonas, and placement of the genus Zymobacter in the family Halomonadaceae. Int. J. Syst. Bacteriol. 46: 550–558. 54. Dobson, S. J., S. R. James, P. D. Franzmann, and T. A. McMeekin. 1990. Emended description of Halomonas halmophila (NCBM 1971 T ). Int. J. Syst. Bacteriol. 40:462–463. 55. Dobson, S. J., S. R. James, P. D. Franzmann, and T. A. McMeekin. 1991. A numerical taxonomic study of some pigmented bacteria isolated from Organic Lake, an Antarctic hypersaline lake. Arch. Microbiol. 156:56–61. 56. Dobson, S. J., T. A. McMeekin, and P. D. Franzmann. 1993. Phylogenetic relationships between some members of the genera Deleya,Halomonas, and Halovibrio. Int. J. Syst. Bacteriol. 43:665–673. 57. Doronina, N. V., and Y. A. Trotsenko. 1997. Aerobic methylotrophic bacterial communities of hypersaline ecosystems. Microbiology 66:130–136. 58. Duckworth, A. W., W. D. Grant, B. E. Jones, and R. van Steenbergen. 1996. Phylogenetic diversity of solar lake alkaliphiles. FEMS Microbiol. Ecol. 19: 181–191. 59. Economou, A., A. Roussis, D. Milioni, and P. Katinakis. 1989. Patterns of protein synthesis in the moderately halophilic bacterium Deleya halophila in response to sudden changes in external salinity. FEMS Microbiol. Ecol. 62: 103–110. 60. Elazari-Volcani, B. 1940. Ph.D. thesis. The Hebrew University of Jerusalem, Jerusalem. (In Hebrew.) 61. Falkenberg, P., A. T. Matheson, and C. F. Rollin. 1976. The properties of ribosomal proteins from a moderate halophile. Biochim. Biophys. Acta 434: 474–482. 62. Falkenberg, P., M. Yaguchi, C. F. Rollin, A. T. Matheson, and R. Wydro. 1979. The N-terminal sequence of the ribosomal ‘A’ protein from two moderate halophiles, Vibrio costicola and an unidentified moderate (NRCC 41227). Biochim. Biophys. Acta 578:207–215. 63. Falkenberg, P., M. Yaguchi, C. Roy, M. Zucker, and A. T. Matheson. 1986. The primary structure of the ribosomal A-protein (L12) from the moderate halophile NRCC 41227. Biochem. Cell Biol. 64:675–680. 64. Farrow, J. A. E., C. Ash, S. Wallbanks, and M. D. Collins. 1992. Phylogenetic analysis of the genera Planococcus,Marinococcus and Sporosarcina and their relationships to members of the genus Bacillus. FEMS Microbiol. Lett. 93:167–172. 65. Fendrich, C. 1988. Halovibrio variabilis gen. nov. sp. nov., Pseudomonas halophila sp. nov. and a new halophilic aerobic coccoid eubacterium from Great Salt Lake, Utah. Syst. Appl. Microbiol. 11:36–43. 66. Fendrich, C., and B. Schink. 1988. Degradation of glucose, glycerol and acetate by aerobic bacteria in surface water of Great Salt Lake, Utah, USA. Syst. Appl. Microbiol. 11:94–96. 67. Fernandez-Castillo, R., C. Vargas, J. J. Nieto, A. Ventosa, and F. Ruiz- Berraquero. 1992. Characterization of a plasmid from moderately halophilic eubacteria. J. Gen. Microbiol. 138:1133–1137. 68. Ferrer, M. R., J. Quevedo-Sarmiento, V. Bejar, R. Delgado, A. Ramos- Cormenzana, M. A. Rivadeneyra. 1988. Calcium carbonate formation by Deleya halophila: effect of salt concentration and incubation temperature. Geomicrobiol. J. 6:49–57. 69. Ferrer, M. R., J. Quevedo-Sarmiento, M. A. Rivadeneyra, V. Bejar, R. Delgado, and A. Ramos-Cormenzana. 1988. Calcium carbonate precipitation by two groups of moderately halophilic microorganisms at different temperatures and salt concentrations. Curr. Microbiol. 17:221–227. 70. Flannery, W. L., and D. M. Kennedy. 1962. The nutrition of Vibrio costicola. I. A simplified synthetic medium. Can. J. Microbiol. 8:923–928. 71. Forsyth, M. P., and D. J. Kushner. 1970. Nutrition and distribution of salt response in populations of moderately halophilic bacteria. Can. J. Microbiol. 16:253–261. 72. Forsyth, M. P., D. B. Shindler, M. B. Gochnauer, and D. J. Kushner. 1971. Salt tolerance of intertidal marine bacteria. Can. J. Microbiol. 17:825–828. 73. Franzmann, P. D. 1991. The microbiota of saline lakes of the Vestfold Hills, Antarctica, p. 9–14. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 74. Franzmann, P. D., H. R. Burton, and T. A. McMeekin. 1987. Halomonas 538 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from subglaciescola, a new species of halotolerant bacteria isolated from Antarctica. Int. J. Syst. Bacteriol. 37:27–34. 75. Franzmann, P. D., and B. J. Tindall. 1990. A chemotaxonomic study of members of the family Halomonadaceae. Syst. Appl. Microbiol. 13:142–147. 76. Franzmann, P. D., U. Wehmeyer, and E. Stackebrandt. 1988. Halomonadaceae fam. nov., a new family of the class Proteobacteria to accommodate the genera Halomonas and Deleya. Syst. Appl. Microbiol. 11:16–19. 77. Frings, E., T. Sauer, and E. A. Galinski. 1995. Production of hydroxyectoine: high cell-density cultivation and osmotic downshock of Marinococcus strain M52. J. Biotechnol. 43:53–61. 78. Fritze, D. 1996. Bacillus haloalkaliphilus sp. nov. Int. J. Syst. Bacteriol. 46: 98–101. 79. Galinski, E. A. 1989. The potential use of halophilic bacteria for the production of organic chemicals and enzyme protective agents, p. 375–379. In M. S. Da Costa, J. C. Duarte, and R. A. D. Williams (ed.), Microbiology of extreme environments and its potential for biotechnology. Elsevier Applied Science, London, United Kingdom. 80. Galinski, E. A. 1993. Compatible solutes of halophilic eubacteria: molecular principles, water-solute interaction, stress protection. Experientia 49:487– 496. 81. Galinski, E. A. 1994. Halophile and halotolerante Eubakterien, p. 89–112. In K. Hausmann and B. P. Kremer (ed.), Extremophile Mikroorganismen in ausgefallenen Lebensra¨umen. VHC, Weinheim, Germany. 82. Galinski, E. A. 1995. Osmoadaptation in bacteria. Adv. Microb. Physiol. 37: 273–328. 83. Galinski, E. A. Personal communication. 84. Galinski, E. A., and K. Lippert. 1991. Novel compatible solutes and their potential application as stabilizers in enzyme technology, p. 351–358. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 85. Galinski, E. A., and P. Louis. 1998. Compatible solutes: ectoine production and gene expression, p. 187–202. In A. Oren (ed.), Microbiology and biogeochemistry of hypersaline environments. CRC Press, Inc., Boca Raton, Fla. 86. Galinski, E. A., and B. J. Tindall. 1992. Biotechnological prospects for halophiles and halotolerant micro-organisms, p. 76–114. In R. A. Herbert and R. J. Sharp (ed.), Molecular biology and biotechnology of extremophiles. Blackie, Glasgow, United Kingdom. 87. Galinski, E. A., and H. G. Tru¨per. 1994. Microbial behaviour in saltstressed ecosystems. FEMS Microbiol. Rev. 15:95–108. 88. Galinski, E. A., H. G. Tru¨per, and T. Sauer. 1993. European patent application EP93/03687 (CI,C12P1/00). 89. Garabito, M. J., D. R. Arahal, E. Mellado, M. C. Ma´rquez, and A. Ventosa. 1997. Bacillus salexigens sp. nov., a new moderately halophilic Bacillus species. Int. J. Syst. Bacteriol. 47:735–741. 90. Garcia, M. T., J. J. Nieto, A. Ventosa, and F. Ruiz-Berraquero. 1987. The susceptibility of the moderate halophile Vibrio costicola to heavy metals. J. Appl. Bacteriol. 63:63–66. 91. Garcia, M. T., A. Ventosa, F. Ruiz-Berraquero, and M. Kocur. 1987. Taxonomic study and amended description of Vibrio costicola. Int. J. Syst. Bacteriol. 37:251–256. 92. Garcia de la Paz, A. M., A. Perez Martinez, C. Calvo Sainz, and A. Ramos Cormenzana. 1989. Isolation and characterisation of bacteriophages active on moderately halophilic microorganisms, p. 425. In M. S. Da Costa, J. C. Duarte, and R. A. D. Williams (ed.), Microbiology of extreme environments and its potential for biotechnology. Elsevier Applied Science, London, United Kingdom. 93. Ghandbhir, M., I. Rasched, P. Marlie`re, and R. Mutzel. 1995. Convergent evolution of amino acid usage in archaebacterial and eubacterial lineages adapted to high salt. Res. Microbiol. 146:113–120. 94. Gilboa, H., M. Kogut, S. Chalamish, R. Regev, Y. Avi-Dor, and N. J. Russell. 1991. Use of 23 Na nuclear magnetic resonance spectroscopy to determine the true intracellular concentration of free sodium in a halophilic eubacterium. J. Bacteriol. 173:7021–7023. 95. Gochnauer, M. B., G. G. Leppard, P. Komaratat, M. Kates, T. Novitsky, and D. J. Kushner. 1975. Isolation and characterization of Actinopolyspora halophila, gen. et sp. nov., an extremely halophilic actinomycete. Can. J. Microbiol. 21:1500–1511. 96. Goel, U., T. Kauri, H.-W. Ackermann, and D. J. Kushner. 1996. A moderately halophilic Vibrio from a Spanish saltern and its lytic bacteriophage. Can. J. Microbiol. 42:1015–1023. 97. Goldberg, M., and H. Gilboa. 1978. Sodium exchange between two sites. The binding of sodium to halotolerant bacteria. Biochim. Biophys. Acta 538:268–283. 98. Goldberg, M., M. Rizk, and H. Gilboa. 1983. Lithium nuclear magnetic resonance measurements in halotolerant bacterium B a1 . Biochim. Biophys. Acta 763:35–40. 99. Grant, M. A., S. E. Cronin, and L. I. Hochstein. 1984. Solubilization and resolution of the membrane-bound nitrite reductase from Paracoccus halodenitrificans into nitrite and nitric oxide reductases. Arch. Microbiol. 140: 183–186. 100. Grant, M. A., and L. I. Hochstein. 1984. A dissimilatory nitrite reductase in Paracoccus halodenitrificans. Arch. Microbiol. 137:79–84. 101. Grant, W. D. 1991. General view of halophiles, p. 15–37. In K. Horikoshi and W. D. Grant (ed.), Superbugs. Microorganisms in extreme environments. Japan Scientific Societies Press, Tokyo, Japan. 102. Grant, W. D., and W. E. Mwatha. 1989. Bacteria from alkaline, saline environments, p. 64–67. In T. Hattori, Y. Ishida, Y. Maruyama, R. Y. Morita, and A. Uchida (ed.), Recent advances in microbial ecology. Japan Scientific Societies Press, Tokyo, Japan. 103. Greenberg, E. P., and E. Canale-Parola. 1976. Spirochaeta halophila sp. nov., a fermentative anaerobic bacterium from a high-salinity pond. Arch. Microbiol. 110:185–194. 104. Guixa-Boixareu, N., J. I. Caldero´n-Paz, M. Heldal, G. Bratbak, and C. Pedro´s-Alio´. 1996. Viral lysis and bacterivory as prokaryotic loss factors along a salinity gradient. Aquat. Microb. Ecol. 11:215–227. 105. Hamaide, F., D. J. Kushner, and G. D. Sprott. 1983. Proton motive force and Na 1 /H 1 antiport in a moderate halophile. J. Bacteriol. 156:537–544. 106. Hamaide, F., D. J. Kushner, and G. D. Sprott. 1985. Proton circulation in Vibrio costicola. J. Bacteriol. 161:681–686. 107. Hamaide, F., G. D. Sprott, and D. J. Kushner. 1984. Energetics of sodiumdependent a-aminoisobutyric acid transport in the moderate halophile Vibrio costicola. Biochim. Biophys. Acta 766:77–87. 108. Hamaide, F., G. D. Sprott, and D. J. Kushner. 1984. Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium, Vibrio costicola. Arch. Microbiol. 140:231–235. 109. Hamana, K. 1997. Polyamine distribution patterns within the families Aeromonadaceae, Vibrionaceae, Pasteurellaceae, and Halomonadaceae, and related genera of the gamma subclass of the Proteobacteria. J. Gen. Appl. Microbiol. 43:49–59. 110. Hanna, K., C. Bengis-Garber, D. J. Kushner, M. Kogut, and M. Kates. 1984. The effect of salt concentration on the phospholipid and fatty acid composition of the moderate halophile Vibrio costicola. Can. J. Microbiol. 30:669–675. 111. Hao, M. V., M. Kocur, and K. Komagata. 1984. Marinococcus gen. nov., a new genus for motile cocci with meso-diaminopimelic acid in the cell walls; and Marinococcus albus sp. nov., and Marinococcus halophilus (Novitsky and Kushner) comb. nov. J. Gen. Appl. Microbiol. 30:449–459. 112. Hara, H., A. Hyono, S. Kuriyama, I. Yano, and M. Masui. 1980. ESR studies on the membrane properties of a moderately halophilic bacterium. II. Effect of extreme growth conditions on liposome properties. J. Biochem. 88:1275–1282. 113. Hara, H., and M. Masui. 1985. Effect of NaCl concentration on the synthesis of membrane phospholipid in a halophilic bacterium. FEMS Microbiol. Ecol. 31:279–282. 114. Hart, D. J., and R. H. Vreeland. 1988. Changes in the hydrophobic-hydro- philic cell surface character of Halomonas elongata in response to NaCl. J. Bacteriol. 170:132–135. 115. Hebert, A. M., and R. H. Vreeland. 1987. Phenotypic comparison of halotolerant bacteria: Halomonas halodurans sp. nov., nom. rev., comb. nov. Int. J. Syst. Bacteriol. 37:347–350. 116. Henis, Y., and J. Eren. 1963. Preliminary studies on the microflora of a highly saline soil. Can. J. Microbiol. 9:902–904. 117. Higa, A. I., and J. J. Cazzulo. 1978. The adenosine triphosphatase from the moderate halophile, Vibrio costicola. FEMS Microbiol. Lett. 3:157–160. 118. Hinrichsen, L. L., M. C. Montel, and R. Talon. 1994. Proteolytic and lipolytic activities of Micrococcus roseus,Halomonas elongata and Vibrio sp. isolated from Danish bacon curing brines. Int. J. Food Microbiol. 22:115– 126. 119. Hipkiss, A. P., D. W. Armstrong, and D. J. Kushner. 1980. Protein turnover in a moderately halophilic bacterium. Can. J. Microbiol. 26:196–203. 120. Hiramatsu, T., Y. Ohno, H. Hara, S. Toriyama, I. Yano, and M. Masui. 1978. Salt-dependent change of cell envelope components of a moderately halophilic bacterium, Pseudomonas halosaccharolytica, p. 515–520. In S. R. Caplan and M. Ginzburg (ed.), Energetics and structure of halophilic microorganisms. Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands. 121. Hiramatsu, T., Y. Ohno, H. Hara, I. Yano, and M. Masui. 1980. Effects of NaCl concentration on the envelope components in a moderately halophilic bacterium, Pseudomonas halosaccharolytica, p. 189–200. In H. Morishita and M. Masui (ed.), Saline environments. Proceedings of the Japanese Conference on Halophilic Microbiology. Nakanishi Printing Co., Kyoto, Japan. 122. Hiramatsu, T., I. Yano, and M. Masui. 1980. Effect of NaCl concentration on the protein species and phospholipid composition of the outer membrane in a moderately halophilic bacterium. FEMS Microbiol. Lett. 7:289– 292. 123. Hirsch, P. 1980. Distribution and pure culture studies of morphologically distinct Solar Lake microorganisms, p. 41–60. In A. Nissenbaum (ed.), Hypersaline brines and evaporitic environments. Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands. 124. Hirsch, P., and B. Hoffmann. 1989. Dichotomicrobium thermohalophilum, gen. nov., spec. nov., budding prosthecate bacteria from the Solar Lake (Sinai) and some related strains. Syst. Appl. Microbiol. 11:291–301. VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 539 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from 125. Hochman, A., A. Nissany, and M. Amizur. 1988. Nitrate reduction and assimilation by a moderately halophilic, halotolerant bacterium Ba 1 . Biochim. Biophys. Acta 965:82–89. 126. Hochstein, L. I., and G. A. Tomlinson. 1984. The growth of Paracoccus halodenitrificans in a defined medium. Can. J. Microbiol. 30:837–840. 127. Hof, T. 1935. Investigations concerning bacterial life in strong brines. Rev. Trav. Bot. Neerl. 32:92–171. 128. Huval, J. H., R. Latta, R. Wallace, D. J. Kushner, and R. H. Vreeland. 1995. Description of two new species of Halomonas:Halomonas israelensis sp. nov. and Halomonas canadensis sp. nov. Can. J. Microbiol. 41:1124–1131. 129. Hyono, A., S. Kuriyama, H. Hara, I. Yano, and M. Masui. 1979. Thick viscous structures in the lipid membranes of a moderately halophilic Gramnegative bacterium. FEBS Lett. 103:192–196. 130. Hyono, A., S. Kuriyama, H. Hara, I. Yano, and M. Masui. 1980. ESR studies on the membrane properties of a moderately halophilic bacterium. I. Physical properties of lipid bilayers in whole cells. J. Biochem. 88:1267– 1274. 131. Imhoff, J., and F. Rodriguez-Valera. 1984. Betaine is the main compatible solute of halophilic eubacteria. J. Bacteriol. 160:478–479. 132. Imhoff, J. F. 1986. Osmoregulation and compatible solutes in eubacteria. FEMS Microbiol. Rev. 39:57–66. 133. Imhoff, J. F. 1993. Osmotic adaptation in halophilic and halotolerant microorganisms, p. 211–253. In R. H. Vreeland and L. I. Hochstein (ed.), The biology of halophilic bacteria. CRC Press, Inc., Boca Raton, Fla. 134. Inbar, L., and A. Lapidot. 1988. The structure and biosynthesis of new tetrahydropyrimidine derivatives in actinomycin D producer Streptomyces parvulus. Use of 13 C and 15 N-labeled L-glutamate and 13 C and 15 N NMR spectroscopy. J. Biol. Chem. 263:16014–16022. 135. James, S. R., S. J. Dobson, P. D. Franzmann, and T. A. McMeekin. 1990. Halomonas meridiana, a new species of extremely halotolerant bacteria isolated from Antarctic saline lakes. Syst. Appl. Microbiol. 13:270–278. 136. Javor, B. J. 1989. Hypersaline environments. Microbiology and biogeochemistry. Springer-Verlag KG, Berlin, Germany. 137. Johnson, K. G., P. H. Lanthier, and M. B. Gochnauer. 1986. Studies of two strains of Actinopolyspora halophila, an extremely halophilic actinomycete. Arch. Microbiol. 143:370–378. 138. Kamekura, M. 1986. Production and function of enzymes of eubacterial halophiles. FEMS Microbiol. Rev. 39:145–150. 139. Kamekura, M., S. Bardo´cz, P. Anderson, R. Wallace, and D. J. Kushner. 1986. Polyamines in moderately and extremely halophilic bacteria. Biochim. Biophys. Acta 880:204–208. 140. Kamekura, M., T. Hamakawa, and H. Onishi. 1982. Application of halophilic nuclease H of Micrococcus varians subsp. halophilus to commercial production of flavoring agent 59-GMP. Appl. Environ. Microbiol. 44:994– 995. 141. Kamekura, M., and D. J. Kushner. 1984. Effect of chloride and glutamate ions on in vitro protein synthesis by the moderate halophile Vibrio costicola. J. Bacteriol. 160:385–390. 142. Kamekura, M., and H. Onishi. 1974. Halophilic nuclease from a moderately halophilic Micrococcus varians. J. Bacteriol. 119:339–344. 143. Kamekura, M., and H. Onishi. 1976. Effect of magnesium and some nutrients on the growth and nuclease formation of a moderate halophile, Micrococcus varians var. halophilus. Can. J. Microbiol. 22:1567–1576. 144. Kamekura, M., and H. Onishi. 1978. Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. halophilus. Can. J. Microbiol. 24:703–709. 145. Kamekura, M., and H. Onishi. 1978. Properties of the halophilic nuclease of a moderate halophile, Micrococcus varians subsp. halophilus. J. Bacteriol. 133:59–65. 146. Kamekura, M., and H. Onishi. 1982. Cell-associated cations of the moderate halophile Micrococcus varians ssp. halophilus grown in media of high concentrations of LiCl, NaCl, KCl, RbCl, or CsCl. Can. J. Microbiol. 28: 155–161. 147. Kamekura, M., and H. Onishi. 1983. Inactivation of nuclease H of the moderate halophile Micrococcus varians ssp. halophilus during cultivation in the presence of salting-in type salts. Can. J. Microbiol. 29:46–51. 148. Kamekura, M., R. Wallace, A. R. Hipkiss, and D. J. Kushner. 1985. Growth of Vibrio costicola and other moderate halophiles in a chemically defined minimal medium. Can. J. Microbiol. 31:870–872. 149. Karamanou, S., and P. Katinakis. 1988. Heat shock proteins in the moderately halophilic bacterium Deleya halophila: protective effect of high salt concentration against thermal shock. Ann. Microbiol. 139:505–514. 150. Katinakis, P. 1989. The pattern of protein synthesis induced by heat-shock of the moderately halophilic bacterium Chromobacterium marismortui: protective effect of high salt concentration against the thermal shock. Microbiologica 12:61–67. 151. Kauri, T., H.-W. Ackermann, U. Goel, and D. J. Kushner. 1991. A bacteriophage of a moderately halophilic bacterium. Arch. Microbiol. 156:435– 438. 152. Ken-Dror, S., and Y. Avi-Dor. 1985. Regulation of respiration by Na 1 and K 1 in the halotolerant bacterium Ba 1 . Arch. Biochem. Biophys. 234:238– 245. 153. Ken-Dror, S., J. K. Lanyi, B. Schobert, B. Silver, and Y. Avi-Dor. 1986. An NADH:quinone oxidoreductase of the halotolerant bacterium Ba 1 is specifically dependent on sodium ions. Arch. Biochem. Biophys. 244:766–772. 154. Ken-Dror, S., R. Preger, and Y. Avi-Dor. 1986. Functional characterization of the uncoupler-insensitive Na 1 pump of the halotolerant bacterium, Ba 1 . Arch. Biochem. Biophys. 244:122–127. 155. Ken-Dror, S., R. Preger, and Y. Avi-Dor. 1986. Role of betaine in the control of respiration and osmoregulation of a halotolerant bacterium. FEMS Microbiol. Rev. 39:115–120. 156. Ken-Dror, S., R. Shnaiderman, and Y. Avi-Dor. 1984. Uncoupler-stimu- lated Na 1 pump and its possible role in the halotolerant bacterium, Ba 1 . Arch. Biochem. Biophys. 229:640–649. 157. Khire, J. M. 1994. Production of moderately halophilic amylase by newly isolated Micrococcus sp. 4 from a salt pan. Lett. Appl. Microbiol. 19:210– 212. 158. Kobayashi, T., M. Kamekura, W. Kanlayakrit, and H. Onishi. 1986. Production, purification, and characterization of an amylase from the moderate halophile, Micrococcus varians subspecies halophilus. Microbios 46:165– 177. 159. Kocur, M. 1984. Genus Paracoccus Davis 1969, 384 al , p. 399–402. In N. R. Krieg and J. G. Holt (ed.), Bergey’s manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore, Md. 160. Kogut, M. 1991. The “true” intracellular environment of moderately halophilic eubacteria, p. 217–224. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 161. Kogut, M., and W. M. Madira. 1978. Dihydrostreptomycin as a probe to study the effects of salt concentration during growth on cell constituents in a moderate halophile, p. 521–527. In S. R. Caplan and M. Ginzburg (ed.), Energetics and structure of halophilic microorganisms. Elsevier/North Holland Biomedical Press, Amsterdam, The Netherlands. 162. Kogut, M., J. R. Mason, and N. J. Russell. 1992. Isolation of salt-sensitive mutants of the moderately halophilic eubacterium Vibrio costicola. Curr. Microbiol. 24:325–328. 163. Kogut, M., and N. J. Russell. 1984. The growth and phospholipid composition of a moderately halophilic bacterium during adaptation to changes in salinity. Curr. Microbiol. 10:95–98. 164. Krahe, M., G. Antranikian, and H. Ma¨rkl. 1996. Fermentation of extremophilic microorganisms. FEMS Microbiol. Rev. 18:271–285. 165. Kuchta, T., and N. J. Russell. 1994. Glycinebetaine stimulates, but NaCl inhibits, fatty acid biosynthesis in the moderately halophilic eubacterium HX. Arch. Microbiol. 161:234–238. 166. Kunte, H. J., and E. A. Galinski. 1995. Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5and Tn1732 in Halomonas elongata. FEMS Microbiol. Lett. 128:293–299. 167. Kuriyama, S., H. Hara, T. Hiramatsu, A. Hyono, I. Yano, and M. Masui. 1982. ESR studies on the lipid bilayers of separated outer and cytoplasmic membranes of a moderately halophilic bacterium. Can. J. Microbiol. 60: 830–837. 168. Kushner, D. J. 1978. Life in high salt and solute concentrations: halophilic bacteria, p. 317–368. In D. J. Kushner (ed.), Microbial life in extreme environments. Academic Press, Ltd., London, United Kingdom. 169. Kushner, D. J. 1986. Molecular adaptation of enzymes, metabolic systems and transport systems in halophilic bacteria. FEMS Microbiol. Rev. 39:121– 127. 170. Kushner, D. J. 1988. What is the “true” internal environment of halophilic and other bacteria? Can. J. Microbiol. 34:482–486. 171. Kushner, D. J. 1989. Halophilic bacteria: life in and out of salt, p. 60–64. In T. Hattori, Y. Ishida, Y. Maruyama, R. Y. Morita, and A. Uchida (ed.), Recent advances in microbial ecology. Japan Scientific Societies Press, Tokyo, Japan. 172. Kushner, D. J. 1989. Halophilic bacteria: their life in and out of salt, p. 280– 288. In M. S. Da Costa, J. C. Duarte, and R. A. D. Williams (ed.), Microbiology of extreme environments and its potential for biotechnology. Elsevier Applied Science, London, United Kingdom. 173. Kushner, D. J. 1991. Halophiles of all kinds: what are they up to now and where do they come from? p. 63–71. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 174. Kushner, D. J. 1993. Growth and nutrition of halophilic bacteria, p. 87–103. In R. H. Vreeland and L. I. Hochstein (ed.), The biology of halophilic bacteria. CRC Press, Inc., Boca Raton, Fla. 175. Kushner, D. J., F. Hamaide, and R. A. MacLeod. 1983. Development of salt-resistant active transport in a moderately halophilic bacterium. J. Bacteriol. 153:1163–1171. 176. Kushner, D. J., and M. Kamekura. 1988. Physiology of halophilic eubacteria, p. 109–138. In F. Rodriguez-Valera (ed.), Halophilic bacteria, vol. I. CRC Press, Inc., Boca Raton, Fla. 177. Lanyi, J. K. 1974. Salt-dependent properties of proteins from extremely halophilic bacteria. Bacteriol. Rev. 38:272–290. 178. LeFevre, E., and L. A. Round. 1919. A preliminary report upon some halophilic bacteria. J. Bacteriol. 4:177–182. 540 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from 179. Lippert, K., and E. A. Galinski. 1992. Enzyme stabilization by ectoine-type compatible solutes: protection against heating, freezing and drying. Appl. Microbiol. Biotechnol. 37:61–65. 180. Louis, P., and E. A. Galinski. 1997. Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. Microbiology 143:1141– 1149. 181. Louis, P., H. G. Tru¨per, and E. A. Galinski. 1994. Survival of Escherichia coli during drying and storage in the presence of compatible solutes. Appl. Microbiol. Biotechnol. 41:648–688. 182. Lowe, S. E., M. K. Jain, and J. G. Zeikus. 1993. Biology, ecology, and biotechnological applications of anaerobic bacteria adjusted to environmental stresses in temperature, pH, salinity, or substrates. Microbiol. Rev. 57:451–509. 183. MacLeod, R. A. 1986. Salt requirements for membrane transport and solute retention in some moderate halophiles. FEMS Microbiol. Rev. 39:109–113. 184. MacLeod, R. A. 1991. Is the Na 1 -activated NADH-quinone-acceptor oxidoreductase in marine bacteria and moderate halophiles a primary electrogenic Na 1 pump? p. 97–106. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 185. Maltseva, O., C. McGowan, R. Fulthorpe, and P. Oriel. 1996. Degradation of 2,4-dichlorophenoxyacetic acid by haloalkaliphilic bacteria. Microbiology 142:1115–1122. 186. Mancinelli, R. L., S. Cronin, and L. I. Hochstein. 1986. The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans. Arch. Microbiol. 145:202–208. 187. Marquez, M. C., A. Ventosa, and F. Ruiz-Berraquero. 1987. A taxonomic study of heterotrophic halophilic and non-halophilic bacteria from a solar saltern. J. Gen. Microbiol. 133:45–56. 188. Marquez, M. C., A. Ventosa, and F. Ruiz-Berraquero. 1990. Marinococcus hispanicus, a new species of moderately halophilic gram-positive cocci. Int. J. Syst. Bacteriol. 40:165–169. 189. Ma´rquez, M. C., A. Ventosa, and F. Ruiz-Berraquero. 1992. Phenotypic and chemotaxonomic characterization of Marinococcus halophilus. Syst. Appl. Microbiol. 15:63–69. 190. Martin, E. L., T. Duryea-Rice, R. H. Vreeland, L. Hilsabeck, and C. Davis. 1983. Effects of NaCl on the uptake of a-[ 14 C]aminoisobutyric acid by the halotolerant bacterium Halomonas elongata. Can. J. Microbiol. 29:1424– 1429. 191. Martı´nez-Murcia, A. J., S. G. Acinas, and F. Rodriguez-Valera. 1995. Evaluation of prokaryotic diversity by restrictase digestion of 16S rDNA directly amplified from hypersaline environments. FEMS Microbiol. Ecol. 17:247– 256. 192. Masui, M., and S. Wada. 1973. Intracellular concentrations of Na 1 ,K 1 and Cl 2 of a moderately halophilic bacterium. Can. J. Microbiol. 19:1181–1186. 193. Matheson, A. T., G. D. Sprott, I. J. McDonald, and H. Tessier. 1976. Some properties of an unidentified halophile: growth characteristics, internal salt concentration, and morphology. Can. J. Microbiol. 22:780–786. 194. Matheson, A. T., M. Yaguchi, R. N. Nazar, L. P. Visentin, and G. E. Willick. 1978. The structure of ribosomes from moderate and extreme halophilic bacteria, p. 481–500. In S. R. Caplan and M. Ginzburg (ed.), Energetics and structure of halophilic microorganisms. Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands. 195. McMeekin, T. A., and P. D. Franzmann. 1988. Effect of temperature on the growth rates of halotolerant and halophilic bacteria isolated from Antarctic saline lakes. Polar Biol. 8:281–285. 196. McMeekin, T. A., P. D. Nichols, S. D. Nichols, A. Juhasz, and P. D. Franzmann. 1993. Biology and biotechnological potential of halotolerant bacteria from Antarctic saline lakes. Experientia 49:1042–1046. 197. Mellado, E. Unpublished results. 198. Mellado, E., J. A. Asturias, J. J. Nieto, K. N. Timmis, and A. Ventosa. 1995. Characterization of the basic replicon of pCM1, a narrow-host-range plasmid from the moderate halophile Chromohalobacter marismortui. J. Bacteriol. 177:3433–3450. 199. Mellado, E., M. T. Garcia, J. J. Nieto, S. Kaplan, and A. Ventosa. 1997. Analysis of the genome of Vibrio costicola: pulsed-field gel electrophoretic analysis of genome size and plasmid-content. Syst. Appl. Microbiol. 20:20– 26. 200. Mellado, E., E. R. B. Moore, J. J. Nieto, and A. Ventosa. 1995. Phylogenetic inferences and taxonomic consequences of 16S ribosomal DNA sequence comparison of Chromohalobacter marismortui,Volcaniella eurihalina, and Deleya salina and reclassification of V. eurihalina as Halomonas eurihalina comb. nov. Int. J. Syst. Bacteriol. 45:712–716. 201. Mellado, E., E. R. B. Moore, J. J. Nieto, and A. Ventosa. 1996. Analysis of 16S rRNA gene sequences of Vibrio costicola strains: description of Salinivibrio costicola gen. nov., comb. nov. Int. J. Syst. Bacteriol. 46:817–821. 202. Mellado, E., J. J. Nieto, and A. Ventosa. 1995. Construction of novel shuttle vectors for use between moderately halophilic bacteria and Escherichia coli. Plasmid 34:157–164. 203. Miguelez, E., and D. J. Gilmour. 1994. Regulation of cell volume in the salt tolerant bacterium Halomonas elongata. Lett. Appl. Microbiol. 19:363–365. 204. Miller, J. M., S. J. Dobson, P. D. Franzmann, and T. A. McMeekin. 1994. Reevaluating the classification of Paracoccus halodenitrificans with sequence comparisons of 16S ribosomal DNA. Int. J. Syst. Bacteriol. 44:360– 361. 205. Miller, K. J. 1985. Effects of temperature and sodium chloride concentrations on the phospholipid and fatty acid composition of a halotolerant Planococcus sp. J. Bacteriol. 162:263–270. 206. Miller, K. J. 1986. Effects of monovalent and divalent salts on the phospholipid and fatty acid compositions of a halotolerant Planococcus sp. Appl. Environ. Microbiol. 52:580–582. 207. Miller, K. J., and S. Leschine. 1984. A halotolerant Planococcus from Antarctic dry valleys. Curr. Microbiol. 11:205–210. 208. Min-Yu, L., H. Ono, and M. Takano. 1993. Gene cloning of ectoine synthase from Halomonas sp. Annu. Rep. Int. Cent. Coop. Res. Biotechnol. Jpn. 16:193–200. 209. Mojica, F. J. M., E. Cisneros, C. Ferrer, F. Rodriguez-Valera, and G. Juez. 1997. Osmotically induced response in representatives of halophilic prokaryotes: the bacterium Halomonas elongata and the archaeon Haloferax volcanii. J. Bacteriol. 179:5471–5481. 210. Monteoliva-Sanchez, M., M. R. Ferrer, A. Ramos-Cormenzana, E. Quesada, and M. Monteoliva. 1988. Cellular fatty acid composition of Deleya halophila: effect of growth temperature and salt concentration. J. Gen. Microbiol. 134:199–203. 211. Monteoliva-Sanchez, M., and A. Ramos-Cormenzana. 1986. Effect of growth temperature and salt concentration on the fatty acid composition of Flavobacterium halmephilum CCM2831. FEMS Microbiol. Lett. 33:51–54. 212. Monteoliva-Sanchez, M., and A. Ramos-Cormenzana. 1987. Cellular fatty acid composition in moderately halophilic Gram-negative rods. J. Appl. Bacteriol. 62:361–366. 213. Monteoliva-Sanchez, M., and A. Ramos-Cormenzana. 1987. Cellular fatty acid composition of Planococcus halophilus NRCC 14033 as affected by growth temperature and salt concentration. Curr. Microbiol. 15:133–136. 214. Monteoliva-Sanchez, M., A. Ramos-Cormenzana, and N. J. Russell. 1993. The effect of salinity and compatible solutes on the biosynthesis of cyclopropane fatty acids in Pseudomonas halosaccharolytica. J. Gen. Microbiol. 139:1877–1884. 215. Monteoliva-Sanchez, M., A. Ventosa, and A. Ramos-Cormenzana. 1989. Cellular fatty acid composition of moderately halophilic cocci. Syst. Appl. Microbiol. 12:141–144. 216. Morishita, H. 1978. Control by episome on salt-resistance in bacteria, p. 431–439. In H. Noda (ed.), Origin of life. Japan Scientific Societies Press, Tokyo, Japan. 217. Morishita, H. 1978. Genetic regulation on salt resistance in halophilic bacteria, p. 599–606. In S. R. Caplan and M. Ginzburg (ed.), Energetics and structure of halophilic microorganisms. Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands. 218. Mota, R. R., M. C. Ma´rquez, D. A. Arahal, E. Mellado, and A. Ventosa. 1997. Polyphasic taxonomy of Nesterenkonia halobia. Int. J. Syst. Bacteriol. 47:1231–1235. 219. Mullakhanbhai, M. F., and H. Larsen. 1975. Halobacterium volcanii spec. nov., a Dead Sea halobacterium with a moderate salt requirement. Arch. Microbiol. 104:207–214. 220. Mylona, P., and P. Katinakis. 1992. Oxidative stress in the moderately halophilic bacterium Deleya halophila: effect of NaCl concentration. Experientia 48:54–57. 221. Nicolaus, B., F. Marsiglia, E. Esposito, L. Lama, A. Trincone, G. di Prisco, A. Gambacorta, M. J. Valderrama, and W. D. Grant. 1992. Isolation of extremely halotolerant cocci from Antarctica. FEMS Microbiol. Lett. 99: 145–150. 222. Nieto, J. J. 1991. The response of halophilic bacteria to heavy metals, p. 173–179. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 223. Nieto, J. J., R. Fernandez-Castillo, M. T. Garcia, E. Mellado, and A. Ventosa. 1993. Survey of antimicrobial susceptibility of moderately halophilic eubacteria and extremely halophilic aerobic archaeobacteria: utilization of antimicrobial resistance as a genetic marker. Syst. Appl. Microbiol. 16:352–360. 224. Nieto, J. J., R. Ferna´ndez-Castillo, M. C. Ma´rquez, A. Ventosa, E. Quesada, and F. Ruiz-Berraquero. 1989. Survey of metal tolerance in moderately halophilic eubacteria. Appl. Environ. Microbiol. 55:2385–2390. 225. Nikolayev, Y. A., and N. I. Matveyeva. 1990. Energization of alanine transport compared in the moderate halophilic bacterium Vibrio costicola and the halotolerant bacterium Micrococcus varians at different pH. Mikrobiologiya 59:933–937. 226. Nikolayev, Y. A., N. I. Matveyeva, and V. K. Plakunov. 1990. The properties of amino acid transport systems in some weak and temperate halophiles as well as in halotolerant bacteria. Mikrobiologiya 59:213–221. 227. Novitsky, T. J., and D. J. Kushner. 1975. Influence of temperature and salt concentration on the growth of a facultatively halophilic “Micrococcus” sp. Can. J. Microbiol. 21:107–110. 228. Novitsky, T. J., and D. J. Kushner. 1976. Planococcus halophilus sp. nov., a VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 541 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from facultatively halophilic coccus. Int. J. Syst. Bacteriol. 26:53–57. 229. Ohno, Y., H. Hara, S. Toriyama, I. Yano, and M. Masui. 1980. Biosynthesis of glucophospholipids in Pseudomonas halosaccharolytica, p. 181–187. In H. Morishita and M. Masui (ed.), Saline environments. Proceedings of the Japanese Conference on Halophilic Microbiology. Nakanishi Printing Co., Kyoto, Japan. 230. Ohno, Y., I. Yano, T. Hiramatsu, and M. Masui. 1976. Lipids and fatty acids of a moderately halophilic bacterium, no. 101. Biochim. Biophys. Acta 424: 337–350. 231. Ohno, Y., I. Yano, and M. Masui. 1979. Effect of NaCl concentration and temperature on phospholipid and fatty acid composition of a moderately halophilic bacterium Pseudomonas halosaccharolytica. J. Biochem. 85:413– 421. 232. Ollivier, B., P. Caumette, J.-L. Garcia, and R. A. Mah. 1994. Anaerobic bacteria from hypersaline environments. Microbiol. Rev. 58:27–38. 233. Onishi, H. 1972. Halophilic amylase from a moderately halophilic Micrococcus. J. Bacteriol. 109:570–574. 234. Onishi, H. 1972. Salt response of amylase produced in media of different NaCl or KCl concentrations by a moderately halophilic Micrococcus. Can. J. Microbiol. 18:1617–1620. 235. Onishi, H., H. Fuchi, K. Konomi, O. Hidaka, and M. Kamekura. 1980. Isolation and distribution of a variety of halophilic bacteria and their classification by salt-response. Agric. Biol. Chem. 44:1253–1258. 236. Onishi, H., and O. Hidaka. 1978. Purification and properties of amylase produced by a moderately halophilic Acinetobacter sp. Can. J. Microbiol. 24: 1017–1023. 237. Onishi, H., and M. Kamekura. 1972. Micrococcus halobius sp. nov. Int. J. Syst. Bacteriol. 22:233–236. 238. Onishi, H., T. Kobayashi, N. Morita, and M. Baba. 1984. Effect of salt concentration on the cadmium tolerance of a moderately halophilic cadmium tolerant Pseudomonas sp. Agric. Biol. Chem. 48:2441–2448. 239. Onishi, H., T. Mori, S. Takeuchi, K. Tani, T. Kobayashi, and M. Kamekura. 1983. Halophilic nuclease of a moderately halophilic Bacillus sp.: production, purification and characteristics. Appl. Environ. Microbiol. 45: 24–30. 240. Onishi, H., and K. Sonoda. 1979. Purification and some properties of an extracellular amylase from a moderate halophile, Micrococcus halobius. Appl. Environ. Microbiol. 38:616–620. 241. Onishi, H., H. Yokoi, and M. Kamekura. 1991. An application of a bioreactor with flocculated cells of halophilic Micrococcus varians subsp. halophilus which preferentially adsorbed halophilic nuclease H to 59-nucleotide production, p. 341–349. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 242. Oren, A. 1988. The microbial ecology of the Dead Sea. Adv. Microb. Ecol. 10:193–229. 243. Oren, A. 1990. Estimation of the contribution of halobacteria to the bacterial biomass and activity in a solar saltern by the use of bile salts. FEMS Microbiol. Ecol. 73:41–48. 244. Oren, A. 1990. The use of protein synthesis inhibitors in the estimation of the contribution of halophilic archaebacteria to bacterial activity in hypersaline environments. FEMS Microbiol. Ecol. 73:187–192. 245. Oren, A. 1990. Thymidine incorporation in saltern ponds of different salinities: estimation of in situ growth rates of halophilic archaeobacteria and eubacteria. Microb. Ecol. 19:43–51. 246. Oren, A. 1991. Estimation of the contribution of archaebacteria and eubacteria to the bacterial biomass and activity in hypersaline ecosystems: novel approaches, p. 25–31. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic bacteria. Plenum Press, New York, N.Y. 247. Oren, A. 1993. Ecology of extremely halophilic microorganisms, p. 25–53. In R. H. Vreeland and L. I. Hochstein (ed.), The biology of halophilic bacteria. CRC Press, Inc., Boca Raton, Fla. 248. Oren, A. 1995. Comment on Convergent evolution of amino acid usage in archaebacterial and eubacterial lineages adapted to high salt (M. Ghandbhir et al., Res. Microbiol. 146:113–120, 1995). Res. Microbiol. 146:805– 806. 249. Oren, A., G. Bratbak, and M. Heldal. 1997. Occurrence of virus-like particles in the Dead Sea. Extremophiles 1:143–149. 250. Oren, A., P. Gurevich, M. Azachi, and Y. Henis. 1992. Microbial degradation of pollutants at high salt concentrations. Biodegradation 3:387–398. 251. Oren, A., P. Gurevich, M. Azachi, and Y. Henis. 1993. Microbial degradation of pollutants at high salt concentrations, p. 263–274. In E. Rosenberg (ed.), Microorganisms to combat pollution. Kluwer Academic Publishers, Dordrecht, The Netherlands. 252. Oren, A., M. Heldal, and S. Norland. 1997. X-ray microanalysis of intracellular ions in the anaerobic halophilic eubacterium Haloanaerobium praevalens. Can. J. Microbiol. 43:588–592. 253. Paster, B. J., F. E. Dewhirst, W. G. Weisburg, L. A. Tordoff, G. J. Fraser, R. B. Hespell, T. B. Stanton, L. Zablen, L. Mandelco, and C. R. Woese. 1991. Phylogenetic analysis of the spirochetes. J. Bacteriol. 173:6101–6109. 254. Peerbaye, Y., and D. J. Kushner. 1993. Effects of penicillin on a moderately halophilic bacterium, Vibrio costicola. Curr. Microbiol. 26:229–232. 255. Peleg, E., and A. Tietz. 1971. Glycolipids of a halotolerant moderately halophilic bacterium. FEMS Microbiol. Lett. 15:309–312. 256. Peleg, E., and A. Tietz. 1973. Phospholipids of a moderately halophilic halotolerant bacterium. Isolation and identification of glucosylphosphatidylglycerol. Biochim. Biophys. Acta 306:368–379. 257. Peleg, E., A. Tietz, and I. Friedberg. 1980. Effects of salts and ionophores on proline transport in a moderately halotolerant bacterium. Biochim. Biophys. Acta 595:118–128. 258. Pe´rez, M. E., M. J. Montes, V. Be´jar, E. Quesada, and C. Ruiz. 1997. Efecto del polisaca´rido V2-7 de Halomonas eurihalina cepa F2-7 sobre linfocitos humanos, abstr. 222. In Abstracts del XVI Congreso de la Sociedad Espan˜ola de Microbiologia. 259. Peters, P., E. A. Galinski, and H. G. Tru¨per. 1990. The biosynthesis of ectoine. FEMS Microbiol. Lett. 71:157–162. 260. Pfiffner, S. M., M. J. McInerney, G. E. Jenneman, and R. M. Knapp. 1986. Isolation of halotolerant, thermotolerant, facultative polymer-producing bacteria and characterization of the exopolymer. Appl. Environ. Microbiol. 51:1224–1229. 261. Post, J. F. 1977. The microbial ecology of the Great Salt Lake. Microb. Ecol. 3:143–165. 262. Prado, B., A. Del Moral, E. Quesada, R. Rı´os, M. Monteoliva-Sanchez, V. Campos, and A. Ramos-Cormenzana. 1991. Numerical taxonomy of moderately halophilic Gram-negative rods isolated from the Salar de Atacama, Chile. Syst. Appl. Microbiol. 14:275–281. 263. Quesada, E., V. Bejar, and C. Calvo. 1993. Exopolysaccharide production by Volcaniella eurihalina. Experientia 49:1037–1041. 264. Quesada, E., V. Bejar, M. J. Valderrama, and A. Ramos-Cormenzana. 1987. Growth characteristics and salt requirement of Deleya halophila in a defined medium. Curr. Microbiol. 16:21–25. 265. Quesada, E., V. Bejar, M. J. Valderrama, A. Ventosa, and A. Ramos- Cormenzana. 1985. Isolation and characterization of moderately halophilic nonmotile rods from different saline habitats. Microbiologia SEM 1:89–96. 266. Quesada, E., M. J. Valderrama, V. Bejar, A. Ventosa, M. C. Gutierrez, F. Ruiz-Berraquero, and A. Ramos-Cormenzana. 1990. Volcaniella eurihalina gen. nov., sp. nov., a moderately halophilic nonmotile gram-negative rod. Int. J. Syst. Bacteriol. 40:261–267. 267. Quesada, E., A. Ventosa, F. Rodriguez-Valera, L. Megias, and A. Ramos- Cormenzana. 1983. Numerical taxonomy of moderately halophilic gramnegative bacteria from hypersaline soils. J. Gen. Microbiol. 129:2649–2657. 268. Quesada, E., A. Ventosa, F. Rodriguez-Valera, and A. Ramos-Cormenzana. 1982. Types and properties of some bacteria isolated from hypersaline soils. J. Appl. Microbiol. 53:155–161. 269. Quesada, E., A. Ventosa, F. Ruiz-Berraquero, and A. Ramos-Cormenzana. 1984. Deleya halophila, a new species of moderately halophilic bacteria. Int. J. Syst. Bacteriol. 34:287–292. 270. Quevedo-Sarmiento, J., A. Del Moral, M. R. Ferrer, and A. Ramos-Cor- menzana. 1988. Antibiotic-resistant moderately halophilic Gram negative rods from hypersaline waters. Chemosphere 17:2233–2242. 271. Quevedo-Sarmiento, J., A. Del Moral, M. R. Ferrer, and A. Ramos-Cor- menzana. 1989. Antibiotic-resistant moderately halophilic Gram negative motile rods from hypersaline waters, p. 423. In M. S. Da Costa, J. C. Duarte, and R. A. D. Williams (ed.), Microbiology of extreme environments and its potential for biotechnology. Elsevier Applied Science, London, United Kingdom. 272. Rafaeli-Eshkol, D. 1968. Studies on halotolerance in a moderately halophilic bacterium. Effect of growth conditions on salt resistance of the respiratory system. Biochem. J. 109:679–685. 273. Rafaeli-Eshkol, D., and Y. Avi-Dor. 1968. Studies on halotolerance in a moderately halophilic bacterium. Effect of betaine on salt resistance of the respiratory system. Biochem. J. 109:687–691. 274. Ramos-Cormenzana, A. 1989. Ecological distribution and biotechnological potential of halophilic microorganisms, p. 289–309. In M. S. Da Costa, J. C. Duarte, and R. A. D. Williams (ed.), Microbiology of extreme environments and its potential for biotechnology. Elsevier Applied Science, London, United Kingdom. 275. Ramos-Cormenzana, A. 1991. Halophilic organisms and the environment, p. 15–24. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 276. Ramos-Cormenzana, A. 1993. Ecology of moderately halophilic bacteria, p. 55–86. In R. H. Vreeland and L. I. Hochstein (ed.), The biology of halophilic bacteria. CRC Press, Inc., Boca Raton, Fla. 277. Regev, R., I. Peri, H. Gilboa, and Y. Avi-Dor. 1990. 13 C NMR study of the interrelation between synthesis and uptake of compatible solutes in two moderately halophilic eubacteria. Arch. Biochem. Biophys. 278:106–112. 278. Rı´os, M. Unpublished results. 279. Rivadeneyra, M. A., R. Delgado, A. Del Moral, M. R. Ferrer, and A. Ramos-Cormenzana. 1994. Precipitation of calcium carbonate by Vibrio spp. from an inland saltern. FEMS Microbiol. Ecol. 13:197–204. 280. Rivadeneyra, M. A., R. Delgado, E. Quesada, and A. Ramos-Cormenzana. 1989. Does the high Mg 21 content inhibit the CaCO 3 precipitation by Deleya halophila? p. 418. In M. S. Da Costa, J. C. Duarte, and R. A. D. Williams (ed.), Microbiology of extreme environments and its potential for biotechnology. Elsevier Applied Science, London, United Kingdom. 542 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from 281. Rivadeneyra, M. A., R. Delgado, E. Quesada, and A. Ramos-Cormenzana. 1991. Precipitation of calcium carbonate by Deleya halophila in media containing NaCl as sole salt. Curr. Microbiol. 22:185–190. 282. Rivadeneyra, M. A., A. Ramos-Cormenzana, G. Delgado, and R. Delgado. 1996. Process of carbonate precipitation by Deleya halophila. Curr. Microbiol. 32:308–313. 283. Robinson, J. 1952. The effects of salts on the nitratase and lactic dehydrogenase of Micrococcus halodenitrificans. Can. J. Bot. 30:155–163. 284. Rodriguez-Valera, F. 1986. The ecology and taxonomy of aerobic chemoorganotrophic halophilic eubacteria. FEMS Microbiol. Rev. 39:17–22. 285. Rodriguez-Valera, F. 1988. Characteristics and microbial ecology of hypersaline environments, p. 3–30. In F. Rodriguez-Valera (ed.), Halophilic bacteria, vol. 1. CRC Press, Inc., Boca Raton, Fla. 286. Rodriguez-Valera, F., A. Ventosa, G. Juez, and J. F. Imhoff. 1985. Variation of environmental features and microbial populations with salt concentrations in a multi-pond saltern. Microb. Ecol. 11:107–115. 287. Rodriguez-Valera, F., F. Ruiz-Berraquero, and A. Ramos-Cormenzana. 1980. Behaviour of mixed populations of halophilic bacteria in continuous cultures. Can. J. Microbiol. 26:1259–1263. 288. Rodriguez-Valera, F., F. Ruiz-Berraquero, and A. Ramos-Cormenzana. 1981. Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations. Microb. Ecol. 7:235– 243. 289. Ro¨lling, W. F. M., and H. van Verseveld. 1996. Characterization of Tetragenococcus halophila populations in Indonesian soy mash (kecap) fermentation. Appl. Environ. Microbiol. 62:1203–1207. 290. Romano, I., B. Nicolaus, L. Lama, M. C. Manca, and A. Gambacorta. 1996. Characterization of a haloalkalophilic strictly aerobic bacterium, isolated from Pantelleria island. Syst. Appl. Microbiol. 19:326–333. 291. Rosenberg, A. 1983. Pseudomonas halodurans sp. nov., a halotolerant bacterium. Arch. Microbiol. 136:117–123. 292. Ruan, J.-S., A. M. Al-Tai, Z.-H. Zhou, and L.-H. Qu. 1994. Actinopolyspora iraquiensis sp. nov., a new halophilic actinomycete isolated from soil. Int. J. Syst. Bacteriol. 44:759–763. 293. Russell, N. J. 1989. Adaptive modifications in membranes of halotolerant and halophilic microorganisms. J. Bioenerg. Biomembr. 21:93–113. 294. Russell, N. J. 1993. Lipids of halophilic and halotolerant microorganisms, p. 163–210. In R. H. Vreeland and L. I. Hochstein (ed.), The biology of halophilic bacteria. CRC Press, Boca Raton, Fla. 295. Russell, N. J., R. Adams, J. Bygraves, and M. Kogut. 1986. Cell envelope phospholipid changes in a moderate halophile during phenotypic adaptation to altered salinity and osmotic stress. FEMS Microbiol. Rev. 39:103– 107. 296. Russell, N. J., and M. Kogut. 1985. Haloadaptation: salt sensing and cellenvelope changes. Microbiol. Sci. 2:345–350. 297. Russell, N. J., M. Kogut, and M. Kates. 1985. Phospholipid biosynthesis in the moderately halophilic bacterium Vibrio costicola during adaptation to changing salt concentrations. J. Gen. Microbiol. 131:781–789. 298. Sadler, M., M. McAninch, R. Alico, and L. I. Hochstein. 1980. The intracellular Na 1 and K 1 composition of the moderately halophilic bacterium, Paracoccus halodenitrificans. Can. J. Microbiol. 26:496–502. 299. Sakhnini, A., and H. Gilboa. 1993. Double quantum sodium NMR studies of the halotolerant Ba 1 bacterium. Biophys. Chem. 46:21–25. 300. Salvarrey, M. S., J. J. B. Cannata, and J. J. Cazzulo. 1989. Phosphoenolpyruvate carboxykinase from the moderate halophile Vibrio costicola. Purification, physicochemical properties and the effect of univalent-cation salts. Biochem. J. 260:221–230. 301. Salvarrey, M. S., and J. J. Cazzulo. 1980. Some properties of the NADP- specific malic enzyme from the moderate halophile Vibrio costicola. Can. J. Microbiol. 26:50–57. 302. Salvarrey, M. S., J. J. Cazzulo, and J. J. B. Cannata. 1995. Effects of divalent cations and nucleotides on the 14 CO 2 -oxaloacetate exchange catalyzed by the phosphoenol pyruvate carboxykinase from the moderate halophile, Vibrio costicola. Biochem. Mol. Biol. Int. 36:1225–1234. 303. Sa´nchez Amat, A., and F. Torrella. 1989. Isolation and characterization of marine and salt pond halophylic bdellovibrios. Can. J. Microbiol. 35:771– 778. 304. Satomi, M., B. Kimura, M. Mizoi, T. Sato, and T. Fujii. 1997. Tetragenococcus muriaticus sp. nov., a new moderately halophilic lactic acid bacterium isolated from fermented squid liver sauce. Int. J. Syst. Bacteriol. 47: 832–836. 305. Sauer, T., and E. A. Galinski. Bacterial milking: a novel bioprocess for production of compatible solutes. Biotechnol. Bioeng., in press. 306. Severin, J., A. Wohlfarth, and E. A. Galinski. 1992. The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria. J. Gen. Microbiol. 138:1629–1638. 307. Shah, V. H., and J. D. H. De Sa. 1964. Studies on halotolerant and halophilic bacteria. I. Isolation and salt response. Indian J. Exp. Biol. 2:181–184. 308. Shindler, D. B., R. M. Wydro, and D. J. Kushner. 1977. Cell-bound cations of the moderately halophilic bacterium Vibrio costicola. J. Bacteriol. 130: 698–703. 309. Shkedy-Vinkler, C., and Y. Avi-Dor. 1975. Betaine-induced stimulation of respiration at high osmolarities in a halotolerant bacterium. Biochem. J. 150:219–226. 310. Shnaiderman, R., and Y. Avi-Dor. 1982. The uptake and extrusion of salts by the halotolerant bacterium, Ba 1 . Arch. Biochem. Biophys. 213:177–185. 311. Simon, R. D., A. Abeliovich, and S. Belkin. 1994. A novel terrestrial halophilic environment: the phylloplane of Atriplex halinus, a salt-excreting plant. FEMS Microbiol. Ecol. 14:99–110. 312. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath (ed.). 1980. Approved list of bacterial names. Int. J. Syst. Bacteriol. 30:225–420. 313. Skerratt, J. H., P. D. Nichols, C. A. Mancuso, S. R. James, S. J. Dobson, T. A. McMeekin, and H. Burton. 1991. The phospholipid ester-linked fatty acid composition of members of the family Halomonadaceae and genus Flavobacterium: a chemotaxonomic guide. Syst. Appl. Microbiol. 14:8–13. 314. Smith, F. B. 1938. An investigation of a taint in rib bones of bacon. The determination of halophilic vibrios (n. spp.). Proc. R. Soc. Queensl. 49:29– 52. 315. Spring, S., W. Ludwig, M. C. Marquez, A. Ventosa, and K.-H. Schleifer. 1996. Halobacillus gen nov., with descriptions of Halobacillus litoralis sp. nov. and Halobacillus trueperi sp. nov., and transfer of Sporosarcina halophila to Halobacillus halophilus comb. nov. Int. J. Syst. Bacteriol. 46:492– 496. 316. Stackebrandt, E., C. Koch, O. Gvozdiak, and P. Schumann. 1995. Taxonomic dissection of the genus Micrococcus:Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov. and Micrococcus Cohn 1987 gen. emend. Int. J. Syst. Bacteriol. 45:682–692. 317. Stern, N., and A. Tietz. 1973. Glycolipids of a halotolerant, moderately halophilic bacterium. I. The effect of growth medium and age of culture on lipid composition. Biochim. Biophys. Acta 296:130–135. 318. Stern, N., and A. Tietz. 1973. Glycolipids of a halotolerant, moderately halophilic bacterium. II. Biosynthesis of glucuronosyldiglyceride by cell-free particles. Biochim. Biophys. Acta 296:136–144. 319. Stern, N., and A. Tietz. 1978. Glycolipids of a halotolerant, moderately halophilic bacterium. Biosynthesis of glucosylphosphatidylglycerol by cellfree particles. Biochim. Biophys. Acta 530:357–366. 320. Sutton, G. C., P. J. Quinn, and N. J. Russell. 1990. The effect of salinity on the composition of fatty acid double bond isomers and sn-1/sn-2 positional distribution in membrane phospholipids of a moderately halophilic eubacterium. Curr. Microbiol. 20:43–46. 321. Sutton, G. C., N. J. Russell, and P. J. Quinn. 1990. The effect of salinity on the phase behaviour of purified phosphatidylethanolamine and phosphatidylglycerol isolated from a moderately halophilic eubacterium. Chem. Phys. Lipids 56:135–147. 322. Sutton, G. C., N. J. Russell, and P. J. Quinn. 1991. The effect of salinity on the phase behaviour of total lipid extracts and binary mixtures of the major phospholipids isolated from a moderately halophilic eubacterium. Biochim. Biophys. Acta 1061:235–246. 323. Tao, T., N. Yasuda, H. Ono, A. Shinmyo, and M. Takano. 1992. Purification and characterization of 2,4-diaminobutyric acid transaminase from Halomonas sp. Annu. Rep. Int. Centre Coop. Res. Biotechnol. Jpn. 15:187–199. 324. Tegos, G., C. Vargas, G. Vartholomatos, A. Perysinakis, J. J. Nieto, A. Ventosa, and C. Drainas. 1997. Identification of a promoter region on the Halomonas elongata cryptic plasmid pHE1 employing the inaZ reporter gene of Pseudomonas syringae. FEMS Microbiol. Lett. 154:45–51. 325. Thomas, T., and E. A. Galinski. 1996. Anaerobic high-cell-density (HCD) fermentation and cyclic production of compatible solutes with halophilic, denitrifying bacteria, abstr. 192. In Abstracts of the First International Congress on Extremophiles. 326. Thongthai, C., and P. Suntinanalert. 1991. Halophiles in Thai fish sauce (nam pla), p. 381–388. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 327. Tindall, B. J. 1988. Prokaryotic life in the alkaline, saline, athalassic environment, p. 31–67. In F. Rodriguez-Valera (ed.) Halophilic bacteria, vol. I. CRC Press, Inc., Boca Raton, Fla. 328. Tokuda, H., and T. Unemoto. 1983. Growth of a marine Vibrio alginolyticus and moderately halophilic V. costicola becomes uncoupler resistant when the respiration-dependent Na 1 pump functions. J. Bacteriol. 156:636–643. 329. Tru¨per, H. G., and E. A. Galinski. 1986. Concentrated brines as habitats for microorganisms. Experientia 42:1182–1187. 330. Tru¨per, H. G., J. Severin, A. Wohlfarth, E. Mu¨ller, and E. A. Galinski. 1991. Halophily, taxonomy, phylogeny and nomenclature, p. 3–7. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 331. Uchida, K., and C. Kanbe. 1993. Occurrence of bacteriophages lytic for Pediococcus halophilus, a halophilic lactic-acid bacterium, in soy sauce fermentation. J. Gen. Appl. Microbiol. 39:429–437. 332. Udagawa, T., T. Unemoto, and H. Tokuda. 1986. Generation of Na 1 electrochemical potential by the Na 1 -motive NADH oxidase and Na 1 /H 1 antiport system of a moderately halophilic Vibrio costicola. J. Biol. Chem. 261:2616–2622. 333. Unemoto, T., A. Akagawa, and M. Hayashi. 1993. Correlation between the respiration-driven Na 1 pump and Na 1 -dependent amino acid transport in moderately halophilic bacteria. J. Gen. Microbiol. 139:2779–2782. VOL. 62, 1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 543 on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from 334. Unemoto, T., A. Akagawa, M. Mizugaki, and M. Hayashi. 1992. Distribution of Na 1 -dependent respiration and a respiration-driven electrogenic Na 1 pump in moderately halophilic bacteria. J. Gen. Microbiol. 138:1999– 2005. 335. Unemoto, T., M. Hayashi, and K. Terao. 1977. Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus by chaotropic anions. Biochim. Biophys. Acta 500:425–431. 336. Valderrama, M. J., E. Quesada, V. Bejar, A. Ventosa, M. C. Gutierrez, F. Ruiz-Berraquero, and A. Ramos-Cormenzana. 1991. Deleya salina sp. nov., a moderately halophilic gram-negative bacterium. Int. J. Syst. Bacteriol. 41: 377–384. 337. Van Qua, D., U. Simidu, and N. Taga. 1981. Purification and some properties of halophilic protease produced by a moderately halophilic marine Pseudomonas sp. Can. J. Microbiol. 27:505–510. 338. Vargas, C., M. J. Coronado, A. Ventosa, and J. J. Nieto. 1997. Host range, stability and compatibility of broad host-range-plasmids and a shuttle vector in moderately halophilic bacteria. Evidence of intrageneric and intergeneric conjugation in moderate halophiles. Syst. Appl. Microbiol. 20:173–181. 339. Vargas, C., R. Ferna´ndez-Castillo, D. Ca´novas, A. Ventosa, and J. J. Nieto. 1995. Isolation of cryptic plasmids from moderately halophilic eubacteria of the genus Halomonas. Characterization of a small plasmid from H. elongata and its use for shuttle vector construction. Mol. Gen. Genet. 246:411–418. 340. Ventosa, A. 1988. Taxonomy of moderately halophilic heterotrophic eubacteria, p. 71–84. In F. Rodriguez-Valera (ed.), Halophilic bacteria, vol. I. CRC Press, Inc., Boca Raton, Fla. 341. Ventosa, A. 1994. Taxonomy and phylogeny of moderately halophilic bacteria, p. 231–242. In F. G. Priest (ed.), Bacterial diversity and systematics. Plenum Press, New York, N.Y. 342. Ventosa, A., M. T. Garcı´a, M. Kamekura, H. Onishi, and F. Ruiz-Ber- raquero. 1989. Bacillus halophilus sp. nov., a moderately halophilic Bacillus species. Syst. Appl. Microbiol. 12:162–166. 343. Ventosa, A., M. C. Gutierrez, M. T. Garcia, and F. Ruiz-Berraquero. 1989. Classification of “Chromobacterium marismortui” in a new genus, Chromohalobacter gen. nov., as Chromohalobacter marismortui comb. nov., nom. rev. Int. J. Syst. Bacteriol. 39:382–386. 344. Ventosa, A., M. C. Marquez, F. Ruiz-Berraquero, and M. Kocur. 1990. Salinicoccus roseus gen. nov., sp. nov., a new moderately halophilic Grampositive coccus. Syst. Appl. Microbiol. 13:29–33. 345. Ventosa, A., M. C. Ma´rquez, N. Weiss, and B. J. Tindall. 1992. Transfer of Marinococcus hispanicus to the genus Salinicoccus as Salinicoccus hispanicus comb. nov. Syst. Appl. Microbiol. 15:530–534. 346. Ventosa, A., and J. J. Nieto. 1995. Biotechnological applications and potentialities of halophilic microorganisms. World J. Microbiol. Biotechnol. 11:85–94. 347. Ventosa, A., F. Rodriguez-Valera, J. S. Poindexter, and W. S. Reznikoff. 1984. Selection for moderately halophilic bacteria by gradual salinity increases. Can. J. Microbiol. 30:1279–1282. 348. Vilhelmsson, O., H. Hafsteinsson, and J. K. Kristja´nsson. 1996. Isolation and characterization of moderately halophilic bacteria from fully cured salted cod (bachalao). J. Appl. Bacteriol. 81:95–103. 349. Villar, M., A. P. de Ruiz Holgado, J. J. Sanchez, R. E. Trucco, and G. Oliver. 1985. Isolation and characterization of Pediococcus halophilus from salted anchovies (Engraulis anchoita). Appl. Environ. Microbiol. 49:664– 666. 350. Volcani, B. E. 1944. The microorganisms of the Dead Sea, p. 71–85. In Papers collected to commemorate the 70th anniversary of Dr. Chaim Weizmann. Collective volume. The Daniel Sieff Research Institute, Rehovoth, Israel. 351. Vreeland, R. H. 1987. Mechanisms of halotolerance in microorganisms. Crit. Rev. Microbiol. 14:311–356. 352. Vreeland, R. H. 1992. The family Halomonadaceae, p. 3181–3188. In A. Balows, H. G. Tru¨per, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.), The prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications. 2nd ed., vol. IV. Springer-Verlag, New York, N.Y. 353. Vreeland, R. H. 1993. Taxonomy of halophilic bacteria, p. 105–134. In R. H. Vreeland and L. I. Hochstein (ed.), The biology of halophilic bacteria. CRC Press, Inc., Boca Raton, Fla. 354. Vreeland, R. H., R. Anderson, and R. G. E. Murray. 1984. Cell wall and phospholipid composition and their contribution to the salt tolerance of Halomonas elongata. J. Bacteriol. 160:879–883. 355. Vreeland, R. H., S. L. Daigle, S. T. Fields, D. J. Hart, and E. L. Martin. 1991. Physiology of Halomonas elongata in different NaCl concentrations, p. 233–241. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 356. Vreeland, R. H., and J. H. Huval. 1991. Phenotypic characterization of halophilic bacteria from ground water sources in the United States, p. 53– 60. In F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y. 357. Vreeland, R. H., C. D. Litchfield, E. L. Martin, and E. Elliot. 1980. Halomonas elongata, a new genus and species of extremely salt-tolerant bacteria. Int. J. Syst. Bacteriol. 30:485–495. 358. Vreeland, R. H., and E. L. Martin. 1980. Growth characteristics, effects of temperature, and ion specificity of the halotolerant bacterium Halomonas elongata. Can. J. Microbiol. 26:746–752. 359. Vreeland, R. H., B. D. Mierau, C. D. Litchfield, and E. L. Martin. 1983. Relationship of the internal solute composition to the salt tolerance of Halomonas elongata. Can. J. Microbiol. 29:407–414. 360. Ward, D. M., and T. D. Brock. 1978. Hydrocarbon biodegradation in hypersaline environments. Appl. Environ. Microbiol. 35:353–359. 361. Weisser, J., and H. G. Tru¨per. 1985. Osmoregulation in a new haloalkaliphilic Bacillus from the Wadi Natrun (Egypt). Syst. Appl. Microbiol. 6:7–11. 362. Wilkansky, B. 1936. Life in the Dead Sea. Nature 138:467. 363. Wohlfarth, A., J. Severin, and E. A. Galinski. 1990. The spectrum of compatible solutes in heterotrophic halophilic eubacteria of the family Halomonadaceae. J. Gen. Microbiol. 136:705–712. 364. Wohlfarth, A., J. Severin, and E. A. Galinski. 1993. Identification of N d - acetylornithine as a novel osmolyte in some Gram-positive halophilic eubacteria. Appl. Microbiol. Biotechnol. 39:568–573. 365. Wood, A. P., and D. P. Kelly. 1991. Isolation and characterisation of Thiobacillus halophilus sp. nov., a sulphur-oxidising autotrophic eubacterium from a Western Australian hypersaline lake. Arch. Microbiol. 156:277–280. 366. Woolard, C. R., and R. L. Irvine. 1992. Biological treatment of hypersaline wastewater by a biofilm of halophilic bacteria, abstr. 14. In Abstracts of the Annual Water Environmental Federation Conference. 367. Wydro, R., M. Kogut, and D. J. Kushner. 1975. Salt response of ribosomes of a moderately halophilic bacterium. FEBS Lett. 60:210–215. 368. Wydro, R. M., W. Madira, T. Hiramatsu, M. Kogut, and D. J. Kushner. 1977. Salt-sensitive in vitro protein synthesis by a moderately halophilic bacterium. Nature 269:824–825. 369. Yakimov, M. M., P. N. Golyshin, S. Lang, F. Wagner, E. Moore, W. R. Abraham, and K. N. Timmis. 1996. New moderate halophilic marine strain MM1 produces novel class of biosurfactants, abstr. 182. In Abstracts of the First International Congress on Extremophiles. 370. Yamada, T., I. Shiio, and F. Egami. 1954. On the halophilic alkaline phosphomonoesterase. Proc. Jpn. Acad. Sci. 30:113–115. 371. Yassin, A. F., E. A. Galinski, A. Wohlfarth, K.-D. Jahnke, K. P. Schaal, and H. G. Tru¨per. 1993. A new actinomycete species, Nocardiopsis lucentensis sp. nov. Int. J. Syst. Bacteriol. 43:266–271. 372. Yokoi, H., and H. Onishi. 1989. Effects of several metal ions, especially zinc ions, on RNA degradation by halophilic nuclease H in solution, or adsorbed on flocculated cells of halophilic Micrococcus. Agric. Biol. Chem. Tokyo 53: 1817. 373. Yorkovsky, Y., and B. L. Silver. 1997. Mn-superoxide dismutase from the halophilic halotolerant bacterium Ba 1 —isolation and active site spectroscopic studies. J. Inorg. Biochem. 65:35–43. 374. Yoshida, M., K. Mastubara, T. Kudo, and K. Horikoshi. 1991. Actinopolyspora mortivallis sp. nov., a moderately halophilic actinomycete. Int. J. Syst. Bacteriol. 41:15–20. 544 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV. on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://mmbr.asm.org/Downloaded from