Piscibacillus Halophilus sp. nov., a Moderately Halophilic Bacterium from a Hypersaline Iranian Lake

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published in INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY on 2009, available at: https://doi.org/10.1099/ijs.0.012013-0” 1 2 Piscibacillus halophilus sp. nov., a moderately halophilic bacterium from a 3 hypersaline Iranian lake 4 5 M. A. Amoozegar1, C. Sanchez-Porro2, R. Rohban1, M. Hajighasemi1 and A. Ventosa2 6 7 1Extremophiles Lab., Department of Microbiology, Faculty of Biology, College of 8 Science, University of Tehran, Tehran, Iran. 9 2Department of Microbiology and Parasitology, Faculty of Pharmacy, University of 10 Sevilla, 41012 Sevilla, Spain 11 12 13 Running title: Piscibacillus halophilus sp. nov. 14 Subject category: New taxa, Other Gram-positive bacteria 15 16 Author for correspondence: Antonio Ventosa, Department of Microbiology and 17 Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain. Tel. +34 18 95 455 6765; Fax +34 95 462 81 62. E-mail: [email protected] 19 20 The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of 21 strain HS224T is FM864227. 22 23 24 2 A Gram-positive, moderately halophilic bacterium, designated strain HS224T, was 25 isolated from the hypersaline lake Howz-Soltan in Iran. The strain was rod- 26 shaped, nonmotile and produced oval endospores. Phylogenetic analyses based on 27 16S rRNA gene sequences showed that strain HS224T is phylogenetically affiliated 28 to the genus Piscibacillus, exhibiting a sequence similarity of 98.5% with respect to 29 Piscibacillus salipiscarius; the other closely related species were Aquisalibacillus 30 elongatus (98.0%), Filobacillus milosensis (97.9%) and Tenuibacillus multivorans 31 (97.0%). Strain HS224T was able to grow at NaCl concentrations from 1 to 20% 32 (w/v), with optimum growth occurring at 10% (w/v) NaCl. The optimum 33 temperature and pH for growth were 35ºC and pH 7.5. Similarly to P. salipiscarius 34 the major polar lipids were phosphatidylglycerol and diphosphatidylglycerol, the 35 isoprenoid quinone was MK-7 and the peptidoglycan type was A1γ, with meso- 36 diaminopimelic acid as the diagnostic diamino acid. The major cellular fatty acids 37 were anteiso-C15:0, iso-C15:0, anteiso-C17:0 and iso-C16:0. The G+C content of the 38 DNA was 37.5 mol%. The DNA-DNA relatedness of the strain with respect to 39 Piscibacillus salipiscarius JCM 13188T was 30.8%. It is evident from the genotypic, 40 chemotaxonomic and phenotypc data that strain HS224T represents a new species 41 of the genus Piscibacillus, for which the name Piscibacillus halophilus sp. nov. is 42 proposed. The type strain is HS224T (=CCM 7596T =DSM 21633T =JCM 15721T 43 =LMG 24786T). 44 _______________________________________________________ 45 46 3 The genus Piscibacillus was proposed by Tanasupawat et al. (2007) for a moderately 47 halophilic bacterium isolated from fermented fish (pla-ra) in Thailand. The only species 48 of this genus is Piscibacillus salipiscarius, a Gam-positive, motile rod able to produce 49 oval endospores at cell-terminal position in swollen sporangia. It is a moderately 50 halophilic bacterium, able to grow from 2 to 30% (w/v) NaCl, showing optimal growth 51 at 10-20% (w/v) NaCl. This species contains meso-diaminopimelic acid in the cell-wall 52 peptidoglycan, menaquinone with seven isoprene units (MK-7) and 53 phosphatidylglycerol and diphosphatidylglycerol as polar lipids. Its major cellular fatty 54 acids are iso-C15:0 and anteiso-C15:0 and the DNA G+C content is 36.7 mol%. 55 Phylogenetically is a member of the family Bacillaceae, within a cluster including 56 species of the genera Filobacillus and Tenuibacillus (Tanasupawat et al., 2007). 57 In this paper we describe the isolation and phenotypic, chemotaxonomic, phylogenetic 58 and genotypic features of a new, moderately halophilic isolate, designated strain 59 HS224T, isolated from a hypersaline lake in Iran. 60 61 Strain HS224T was isolated from water of the hypersaline lake Howz-Soltan, which is 62 located near Qom city in the center of Iran. This lake covers an area of about 240 and 63 280 square kilometers in dry and wet seasons, respectively. The depth of salt layer 64 which covers almost all the surface of the playa varies between 20-46 meters and the pH 65 of the water, saline soil and salt sediments differs between 6.5 and 8.2. Major chemical 66 composition of the soil, brine, mud and salt consists of NaCl, KCl, MgSO4, MgCl2 and 67 Na2SO4. At the time of sampling, the temperature of the water was 32°C and the pH 7.6. 68 Strain HS224T was isolated during studies focused on the determination of the 69 biodiversity of the lake. It was isolated by diluting a water sample in sterile 10% (w/v) 70 4 salt solution, plating on 10% HM medium and incubating at 37ºC aerobically. The 10% 71 HM medium contained (g l-1): NaCl, 81; MgCl2, 7; MgSO4, 9.6; CaCl2, 0.36; KCl, 2; 72 NaHCO3, 0.06; NaBr, 0,026; proteose-peptone no. 3,5; yeast extract, 10, and glucose, 1. 73 (Ventosa et al., 1982). The pH of this medium was adjusted to pH 7.5. The strain was 74 subsequently purified three times by plating on the same medium and maintained on the 75 same 10% HM medium and at -80ºC on this medium without agar and supplemented 76 with 30% (v/v) glycerol. In our study, Piscibacillus salipiscarius JCM 13188T was used 77 as reference and was cultured on the same conditions than strain HS224T. 78 79 Cell morphology was examined by light microscopy (model CX 31; Olympus) using 80 cells from exponentially growing cultures. Gram staining was performed by the Burke 81 method (Murray et al., 1994) and the result was confirmed by the KOH test (Baron & 82 Finegold, 1990). The presence of endospores was investigated by using the Schaeffer- 83 Fulton staining method (Murray et al., 1994). Motility was analysed by the wet-mount 84 method (Murray et al., 1994). To determine the optimal temperature and pH for growth 85 of the strain, broth cultures were incubated at temperatures of 10–50 °C at intervals of 86 5°C and at pH 5–11 at intervals of 0.5 pH units. pH values below 6, pH 6–9 and pH 87 values above 9 were obtained using sodium acetate/acetic acid, Tris/HCl and 88 glycine/sodium hydroxide buffers, respectively. Growth at different salt contents (0, 2.5, 89 5, 7.5, 10, 15, 20, 25 and 30%, w/v) was tested on HM medium at pH 7.5. Growth was 90 monitored by turbidity at OD600 using a spectroscopic method (model UV-160 A; 91 Shimadzu). Catalase, oxidase and urease activities, nitrate reduction, hydrolysis of 92 aesculin, production of indole, methyl red and Voges–Proskauer tests were tested as 93 recommended by Smibert & Krieg (1994). Hydrolysis of Tween-80 was examined as 94 5 described by Harrigan & McCance (1976). Determination of acid production from 95 carbohydrates, as well as utilization of carbon and nitrogen sources, was performed as 96 recommended by Ventosa et al. (1982). Antibiotic susceptibility tests were performed 97 on Mueller–Hinton agar plus 10% (w/v) sea salts (Ventosa et al., 1982) seeded with a 98 bacterial suspension containing 1.5 ×106 c.f.u. ml-1 using discs (HiMedia) impregnated 99 with various antibiotics. The plates were incubated at 35°C for 48 h and the inhibition 100 zone was interpreted according the manufacturer’s manual. Other physiological and 101 biochemical tests were performed as described previously (Mata et al., 2002; Quesada 102 et al., 1984; Ventosa et al., 1982). 103 104 Strain HS224T was a Gram-positive, nonmotile rod able to produce oval endospores at 105 terminal position in swollen sporangia. When grown for 2 days at 35 ºC on 10% HM 106 medium, the colonies were circular, entire, smooth, cream and with a diameter of 2 mm. 107 This isolate was moderately halophilic, growing in media containing 1 to 20% (w/v) 108 NaCl and optimally in media containing 10% (w/v) NaCl. No growth was observed in 109 the absence of NaCl. Strain HS224T grew between pH 7.0 and 10.0, and optimally in 110 media with pH 7.5, and at temperatures ranging from 15 to 55 ºC, with optimal growth 111 at 35 ºC. Other phenotypic features are included in Table 1 and the species description. 112 113 Genomic DNA from strain HS224T was prepared using the method described by 114 Marmur (1961). The 16S rRNA gene was amplified by PCR with the forward primer 115 16F27 and the reverse primer 16R1488. Direct sequence determination of the PCR- 116 amplified DNA was carried out using an automated DNA sequencer model ABI 117 3130XL (Applied Biosystems). The 16S rRNA gene sequence analysis was performed 118 6 with the ARB software package (Ludwig et al., 2004). The 16S rRNA gene sequence 119 was aligned with the published sequences of the closely related bacteria and the 120 alignment was confirmed and checked against both primary and secondary structures of 121 the 16S rRNA molecule using the alignment tool of the ARB software package. 122 Phylogenetic trees were constructed using three different methods: maximum-likelihood 123 (Felsenstein, 1981), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou 124 & Nei, 1987), algorithms integrated in the ARB software for phylogenetic inference. 125 The 16S rRNA gene sequences used for phylogenetic comparisons were obtained from 126 the GenBank database and their strain designations and accession numbers are shown in 127 Figure 1. 128 129 Almost-complete 16S rRNA gene sequence (1472 bp) of strain HS224T was obtained 130 and used for initial BLAST searches in GenBank and phylogenetic analysis. The 131 identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene 132 sequence similarity were achieved using the EzTaxon server (http://www.eztaxon.org/; 133 Chun et al., 2007). The phylogenetic analysis, based on the maximum-parsimony 134 algorithm, revealed that strain HS224T formed a phyletic group with Piscibacillus 135 salipiscarius RBU1-1T, clustering at 98.5% similarity (Fig. 1). The other closely related 136 species were Aquisalibacillus elongatus (98.0% sequence similarity), Filobacillus 137 milosensis (97.9%) and Tenuibacillus multivorans (97.0%). Neighbour-joining and 138 maximum likelihood methods resulted in highly similar tree topologies and only 139 maximum-parsimony results are shown. 140 141 7 The G+C content of the genomic DNA was determined from the midpoint value of the 142 thermal denaturation profile (Marmur & Doty, 1962) using the equation of Owen & Hill 143 (1979), as previously described in detail (Ventosa et al., 1999). The G+C content of the 144 DNA of strain HS224T was 37.5 mol%. This value is very closely related to that 145 reported for Piscibacillus salipiscarius (Tanasupawat et al., 2007) as well as for other 146 closely related species (Table 1). 147 148 DNA-DNA hybridization studies were performed by using the competition procedure of 149 the membrane method (Johnson, 1994), described in detail by Arahal et al. (2001a, b). 150 The hybridization temperature used was 46.3 ºC, which is within the limit of validity for 151 the filter method (De Ley & Tijtgat, 1970) and the percentage of hybridization was 152 calculated according to Johnson (1994). The experiments were carried out in triplicate. 153 The level of DNA-DNA relatedness between strain HS224T and Piscibacillus 154 salipiscarius JCM 13188T was 30.8 %, low enough to classify strain HS224T as 155 representing a genotypically distinct species (Stackebrandt & Goebel, 1994; 156 Stackebrandt et al., 2002). 157 158 Analysis of polar lipids, quinones and peptidoglycan of the cell wall was carried out by 159 the Identification Service of the DSMZ (Braunschweig, Germany). The polar lipids 160 detected were phosphatidylglycerol and diphosphatidylglycerol; besides, two minor 161 phospholipids were detected. Strain HS224T contained MK-7 as the unique 162 menaquinone and a peptidoglycan cell wall type A1γ, with meso-diaminopimelic acid 163 as diagnostic diamino acid. All these features are the same as those found for 164 Piscibacillus salipiscarius (Tanasupawat et al., 2007). However, a key feature in order 165 8 to distinghish these two strains and species of other closely related genera is the 166 peptidoglycan type: the cell wall of Aquisalibacillus elongatus contains peptidoglycan 167 of the A4β type, with L-Orn as the diamino acid and D-Asp as the dicarboxylic amino 168 acid present in the cross-linkage (Márquez et al., 2008) and Filobacillus milosensis also 169 has peptidoglycan of the A4β type, but with D-Glu instead D-Asp in the cross-linkage 170 (Schlessner et al., 2001). 171 172 Fatty acids analysis was performed using the MIDI system (Microbial Identification 173 System). Cells were cultured on marine agar containing 10% (w/v) NaCl at 37ºC for 24 174 h. The extraction and the analysis were performed conform to the recommendations of 175 the MIDI system. This analysis was carried out by the Identification Service of the 176 Belgian Co-ordinated Collections of Microorganisms, Laboratory of Microbiology of 177 Gent (BCCM/LMG), Gent, Belgium. The cellular fatty acid profile of strain HS224T 178 was characterized by the fatty acids anteiso-C15:0 (35.3%), iso-C15:0 (26.3%), anteiso- 179 C17:0 (13.8%) and iso-C16:0 (9.4%) as the major fatty acids; other minor fatty acids 180 detected on strain HS224T were iso-C17:0 (5.8%), iso-C14:0 (2.6%), C16:1ω7c (2.5%) and 181 C16:0 (1.4%). This fatty acid composition is quite similar to that reported for 182 Piscibacillus salipiscarius, that also contained the four previously mentioned fatty acid 183 as major compounds (Tanasupawat et al., 2007). 184 185 The characteristics that differentiate strain HS224T from Piscibacillus salipiscarius and 186 other related species are summarized in Table 1. Our results show clearly that strain 187 HS224T is closely related to Piscibacillus salipiscarius; however, there are differences 188 in some phenotypic and chemotaxonomic features. Besides, DNA hybridization data 189 15 Table 1. Characteristics that distinguish strain HS224T from Piscibacillus salipiscarius 308 and other closely related species. 309 Taxa: 1, Piscibacillus halophilus HS224T (data from this study); 2, Piscibacillus 310 salipiscarius RBU1-1T (Tanasupawat et al., 2007); 3, Aquisalibacillus elongatus SH4sT 311 (Márquez et al., 2008); 4, Filobacillus milosensis SH 714T (Schlesner et al., 2001); 5, 312 Tenuibacillus multivorans 28-1T (Ren & Zhou, 2005). +, Positive; -, negative; W, 313 weakly positive; ND, no data available; meso-DAP, meso-diaminopimelic. 314 Characteristic 1 2 3 4 5 Cell size (µm) 0.5-0.7 X 2.5-4.0 0.4-0.5 x 1.5-4.0 0.5 x 2.0-10.0 0.3-0.4 x 3.0-7.0 0.3-0.5 x 2.0-6.0 Oxidase + + - - + Anaerobic growth + w - - - spore shape Oval, terminal at swollen-sporangia Oval, terminal at swollen-sporangia - Spherical, terminal in swollensporangia Spherical, terminal in swollen sporangia Motility - + - + - NaCl range (%, w/v) for growth (optimum) 1-20 (10) 2-30 (10-20) 3-20 (10) 2-23 (8-14) 1-20 (5) Temperature range (°C) (optimum) 15-55 (35) 15-48 (37) 20-55 (37) ND-42 (33-38) 21-42 (36-41) pH range for growth (optimum) 7.0-10.0 (7.5) 5.0-9.0 (7) 7.0-10.0 (7.5) 6.5-8.9 (7.3-7.8) 6.5-9.0 (7.0-8.0) Acid production from: Galactose - + ND - - D-glucose - + - - - D-fructose - + - - - Lactose - - - - Nitrate reduction - - + ND - Nitrite reduction - - + ND ND Hydrolysis of: Aesculin + ND - - + Starch - + - - - Casein + + - - + Gelatin + + - - + Tween 80 + - - ND - DNase - + ND + ND Peptidoglycan type/variant A1γ/meso-DAP A1γ/meso-DAP A4β/Orn-D-Asp A4β/Orn-D-Glu A1γ/meso-DAP DNA G+C content (mol%) 37.5 36.7 45.9 35.0 36.5-37 16 315 Legend to figure 316 Fig. 1. Maximum-parsimony phylogenetic tree, based on the 16S rRNA gene sequence 317 comparison, showing the relationship of strain HS224T with related species. The 318 accession numbers of the sequences used in this study are shown in parentheses after 319 the strain designation. Bacillus licheniformis DSM 13T was used as an outgroup. The 320 scale bar represents 0.01 substitutions per nucleotide position. 321 322 323 Halobacillus halophilus DSM 2266T (X62174) Halobacillus alkaliphilus FP5T (AM295006) Halobacillus campisalis ASL-17T (EF486356) Halobacillus yeomjeoni MSS-402T (AY881246) Halobacillus litoralis SL-4T (X94558) Halobacillus karajensis DSM 14948T (AJ486874) Halobacillus locisalis MSS-155T (AY190534) Salinibacillus aidingensis 25-7T (AY321436) Salinibacillus kushneri 8-2T (AY321434) Gracilibacillus halotolerans NNT (AF036922) Gracilibacillus dipsosauri DD1T (X82436) Paraliobacillus ryukyuensis 015-7T (AB087828) Virgibacillus proomii LMG 12370T (AJ012667) Virgibacillus pantothenticus NCDO 1765T (X60627) Virgibacillus marismortui 123T (AJ009793) Virgibacillus halodenitrificans ATCC 49067T (AB021186) Piscibacillus salipiscarius RBU1-1T (AB194046) Piscibacillus halophilus HS224T (FM864227) Aquisalibacillus elongatus SH4sT (AM911047) Filobacillus milosensis SH 714T (AJ238042) Tenuibacillus multivorans AS 1.3442T (AY319933) Alkalibacillus salilacus BH163T (AY671976) Alkalibacillus silvisoli BM2T (AB264528) Alkalibacillus haloalkaliphilus DSM 5271T (AJ238041) Alkalibacillus filiformis 4AGT (AJ493661) Bacillus licheniformis DSM 13T (X60623) 0.01 74 98 94 52 72 77 95 86 98 92 97 60 54