Fodinicurvata halophila sp. nov., a moderately halophilic bacterium from a marine saltern

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 01 March 2015, available at: https://doi.org/10.1099/ijs.0.000010 .” 1 Fodinicurvata halophila sp. nov., a moderately halophilic bacterium from a marine 1 saltern 2 3 Carmen Infante-Dominguez1, Paul A. Lawson2, Crystal N. Johnson2, Cristina Sánchez- 4 Porro1 and Antonio Ventosa1 5 6 1Department of Microbiology and Parasitology, Faculty of Pharmacy, University of 7 Sevilla, 41012 Sevilla, Spain. 8 2Department of Microbiology and Plant Biology, University of Oklahoma, Norman, 9 Oklahoma, 73019 USA. 10 11 Running title: Fodinicurvata halophila sp. nov. 12 Subject category: New taxa, Proteobacteria 13 14 Author for correspondence: Antonio Ventosa, Department of Microbiology and 15 Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain. E.-mail: 16 vent[email protected]s 17 18 The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of 19 strain BA45ALT is HG764424. 20 21 Two supplementary figures and a supplementary table are available with the online 22 version of this paper. 23 24 Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Fodinicurvata halophila-rev.doc 2 A Gram-stain-negative, rod-shaped, facultatively anaerobic, moderately halophilic 25 bacterium, designated strain BA45ALT, was isolated from water of a saltern of 26 Santa Pola, Alicante, Spain. Cells were motile, catalase and oxidase positive. Strain 27 BA45ALT grew at temperatures in the range 14-45 ºC (optimally at 37 ºC) and at 28 pH 5.0-9.0 (optimally at pH 7.5). It grew in media containing 5-20 % (w/v) salts 29 and optimally in media containing 10 % (w/v) salts. Phylogenetic analysis based 30 on the comparison of 16S rRNA gene sequences revealed that strain BA45ALT is a 31 member of the genus Fodinicurvata. The closest relatives to this strain were 32 Fodinicurvata fenggangensis YIM D812T and Fodinicurvata sediminis YIM D82T 33 with sequence similarities of 98.2 % and 97.4 %, respectively. DNA-DNA 34 hybridization between the novel isolate and these phylogenetically related species 35 revealed relatedness of 15 % and 30 %, respectively, with respect to the 36 aforementioned species. The major cellular fatty acids of strain BA45ALT were 37 C18:1ω7c, C16:0 and iso-C15:0. The G+C content of the genomic DNA of this strain 38 was 58.0 mol%. Its polar lipid pattern consisted of diphosphatidylglycerol, 39 phosphatidylmethylethanolamine and a number of unknown phospholipids and 40 lipids. Based on the phenotypic, chemotaxonomic and phylogenetic data presented 41 in this study strain BA45ALT constituted a novel species of the genus 42 Fodinicurvata, for which the name Fodinicurvata halophila sp. nov. is suggested. 43 The type strain of Fodinicurvata halophila is strain BA45ALT (= CCM 8504T = 44 CECT 8472T = JCM 19075T = LMG 27945T). 45 46 47 3 The genus Fodinicurvata belongs to the family Rhodospirillaceae, within the order 48 Rhodospirillales (Pfening & Trüper, 1971) of the class Alphaproteobacteria. This genus 49 was originally proposed by Wang et al. (2009) and to date it comprises two species, 50 Fodinicurvata sediminis (Wang et al., 2009) and Fodinicurvata fenggangensis (Wang et 51 al., 2009). Both species were isolated from a sediment sample collected from a salt 52 mine in Yunnan, south-west China. The species of the genus Fodinicurvata stain Gram- 53 negative, are facultatively anaerobic, non-motile, vibrioid or rod-shaped. Catalase and 54 oxidase are produced and polyhydroxybutyrate (PHB) granules are observed. The 55 predominant polar lipids consist of diphosphatidylglycerol, 56 phosphaitidylmethylethanolamine and phosphatidylcholine. Phospatidylinositol is 57 variable between species. Their DNA G+C content ranges from 61.5 – 62.3 mol% 58 (Wang et al., 2009). 59 Recent metagenomic studies have provided new insights into the microbial diversity 60 along the salinity gradient of salterns located in Santa Pola and Isla Cristina in Spain 61 (Ghai et al., 2011; Fernández et al., 2014 a,b; León et al., 2014; Ventosa et al., 2014). 62 During the course of a study to search for the most abundant microorganisms in these 63 salterns, a novel, moderately halophilic, yellow to cream pigmented, Gram-stain- 64 negative bacterium, strain BA45ALT, was isolated from a water sample. The pH of the 65 water was 7.4 and its salinity was 12 % (w/v) salts. At the time of sampling, the 66 temperature of the water was 28 ºC. In this paper we describe the taxonomic features of 67 this novel bacterium and propose it as a new species of the genus Fodinicurvata, as 68 Fodinicurvata halophila sp. nov. 69 Aliquots of the water sample were diluted with sterile salt solution, where 100 70 microliters was plated on saline media and incubated aerobically at 37º C. The isolation 71 4 medium used was prepared with a 15% (g l-1) salts mixture: NaCl, 117; MgCl2.6H2O, 72 19.5; MgSO4.7H2O, 30.5; CaCl2.2H2O, 0.3; KCl, 3; NaHCO3, 0.06; NaBr, 0.23 (Subow, 73 1931), supplemented with 0.25 % (w/v) yeast extract (Difco) and 1. 8 % (w/v) agar 74 (Difco). The pH of the medium was adjusted to 7.5 with 1 M KOH. A pure culture of 75 strain BA45ALT was obtained after several transfers on the same medium. For routine 76 growth, the strain was cultivated in the same medium but prepared with 10 % (w/v) 77 total salts. The culture was maintained at -80 ºC in the routine medium containing 50 % 78 (v/v) glycerol. Fodinicurvata fenggangensis DSM 21160T and Fodinicurvata sediminis 79 DSM 21159T obtained from the DSMZ (German Collection of Microorganisms and Cell 80 Cultures) were used as reference strains and were grown following the 81 recommendations of the culture collection. 82 Cell morphology and motility were examined using an Olympus CX41 microscope 83 equipped with phase-contrast optics. Cells were motile, both rod- and vibrioid-shaped 84 (Supplementary Fig. S1, available at IJSEM online). Colony morphology was observed 85 on routine medium under optimal growth conditions after incubation at 37º C for 5 86 days. Colonies of strain BA45ALT that formed on agar plates were circular, smooth, 87 entire, opaque, yellow-cream pigmented, with a size of 0.3-0.5 mm. The strain was able 88 to accumulate PHB, as was observed by using an Olympus CX41 microscope equipped 89 with phase-contrast optics. 90 Genomic DNA from strain BA45ALT was obtained using the method described by 91 Marmur (1961). The 16S rRNA gene of the isolate were amplified by PCR using 92 forward primer 16F27 and reverse primer 16R1488 (Mellado et al., 1995). An almost 93 complete 16S rRNA gene sequence (1392 nt) of strain BA45ALT was obtained. The 94 identification of the closest phylogenetic neighbours and calculation of pairwise 16S 95 5 rRNA gene sequence similarity were achieved using the EzTaxon-e tool (Kim et al., 96 2012). The 16S rRNA gene sequence similarities between BA45ALT and the most 97 closely related Fodinicurvata type strains, Fodinicurvata fenggangensis YIM D812T 98 and Fodinicurvata sediminis YIM D82T were 98.2 and 97.4 %, respectively. The 16S 99 rRNA gene sequence was aligned and checked against both primary and secondary 100 structures of the 16S rRNA molecule using the alignment tool of the ARB software 101 package. Phylogenetic trees were constructed using three different methods, namely the 102 maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Fitch, 1971) and 103 neighbour-joining (Saitou & Nei, 1987) algorithms integrated in the ARB software. The 104 maximum-likelihood phylogenetic tree is presented in Fig. 1. Similar tree topologies 105 were obtained using other treeing methods: neighbour-joining and maximum-parsimony 106 (Fig. S2) 107 Although no precise correlation exists between the percentage of 16S rRNA sequence 108 divergence and species delineation, it is generally recognized that divergence values of 109 1.3% or more are significant (Stackebrandt & Goebel, 1994). However, due to the high 110 sequence similarity values to its close relatives, DNA-DNA hybridization studies were 111 performed. These studies were carried out using the competition procedure of the 112 membrane method (Johnson, 1994) as described by Ventosa et al. (2004). The levels of 113 DNA-DNA relatedness for strain BA45ALT with respect to Fodinicurvata 114 fenggangensis DSM 21160T and Fodinicurvata sediminis DSM 21159T were 30 and 15 115 %, respectively. These data confirmed that strain BA45ALT represents a novel species 116 of the genus Fodinicurvata, having DNA-DNA hybridization values <70 % with respect 117 to the type strains of the most closely related species of the genus Fodinicurvata (Waine 118 et al., 1987; Stackebrandt & Goebel, 1994). 119 6 Optimal conditions for growth and range were determined by growing the strain in the 120 routine medium prepared at the following salinity values: 0.5, 3, 5, 7, 7.5, 8, 10, 12, 15, 121 20 and 25% (w/v) total salts and at temperatures from 5 to 45 ºC (in increments of 5 ºC) 122 and from 35 to 40 ºC in increments of one unit. The pH range for isolate was 123 determined in the routine medium at optimal salinity adjusted to pH values ranging 124 from 4 to 10 (in increments of one pH unit). The growth was determined by monitoring 125 the optical density using a spectrophotometer at 600 nm. 126 Strain BA45ALT grew in media containing 5-20 % (w/v) salts and optimally in media 127 containing 10 % (w/v) salts. No growth was observed in the absence of NaCl. It grew at 128 temperatures in the range 14-45 ºC (optimally at 37 ºC) and at pH 5.0-9.0 (optimally at 129 pH 7.5). The strain was able to grow anaerobically, determined after incubation in an 130 anaerobic chamber (GasPak Anaerobic system, BBL). 131 All phenotypic features were carried out growing the strain in the routine medium 132 prepared at 10% (w/v) total salts, pH 7.5 and at 37 ºC. The production of acid from 133 different carbohydrates was tested in a medium with 0.5 % (w/v) yeast extract and 134 supplemented with 1% (w/v) of the carbohydrate tested (sterilized separately) (Ventosa 135 et al., 1982). To determine the utilization of different organic substrates as carbon and 136 energy sources, a medium containing 0.05 % (w/v) yeast extract and supplemented with 137 1 % (w/v) of the tested substrate, was used (Ventosa et al., 1982). The results for the 138 utilization of different substrates are included in the species description. 139 Catalase activity was determined by adding a 1% (w/v) H2O2 solution to colonies on 140 solid medium. The oxidase test was performed using a DrySlide assay (Difco). Strain 141 BA45ALT was catalase and oxidase positive. The following tests were conducted as 142 described by Cowan and Steel (1977): hydrolysis of aesculin, casein, gelatin, Tween 80, 143 7 and starch; the urease, methyl red, and indole production, Vogues-Proskauer test; nitrate 144 and nitrite reduction, and Simmons citrate. The following tests were negative: methyl 145 red, Voges-Proskauer, indole production from tryptone and Simmons’s citrate, casein 146 and starch hydrolysis. The strain was positive for nitrate reduction but nitrite was not 147 reduced. Both gelatin and aesculin were hydrolyzed and the urease test was positive. 148 Other phenotypic characteristics of strain BA45ALT are included in the species 149 description. 150 Antimicrobial compounds sensitivity tests were performed by spreading the culture 151 suspension on solid medium plates and applying discs impregnated with antimicrobial 152 compounds following the technique of Bauer-Kirby (Bauer et al., 1966). Susceptibility 153 to antimicrobial compounds was determined on agar plates using antimicrobial agent 154 discs with the following concentrations: ampicillin (10 µg), chloramphenicol (30 µg), 155 erythromycin (15 µg), gentamicin (10 µg), nalidixic acid (30 µg), neomycin (10 µg), 156 novobiocin (30 µg), penicillin G (10 U) and rifampicin (30 µg). Strain BA45ALT was 157 resistant to ampicillin, erythromycin, gentamicin, nalidixic acid, neomycin, novobiocin, 158 penicillin G, and rifampicin but susceptible to chloramphenicol. 159 Several phenotypic differences were observed between strain BA45ALT and the two 160 closely related Fodinicurvata species, including those concerning motility, 161 physiological and cultural features, nitrate reduction, hydrolysis of gelatin and aesculin, 162 H2S production, and utilization of several carbohydrates (Table 1). 163 The G+C content of the genomic DNA of strain BA45ALT was determined from the 164 mid-point value (Tm) of the thermal denaturation profile (Marmur & Doty, 1962), 165 obtained with a Perkin-Elmer UV-Vis Lambda 20 spectrophotometer at 260 nm. The 166 8 DNA G+C content of strain BA45ALT was 58.0 mol%, a value close to those reported 167 for species of the genus Fodinicurvata (Wang et al., 2009). 168 Cell biomass for fatty acid, polar lipid analyses and quinone composition of strain 169 BA45ALT, Fodinicurvata sediminis DSM 21159T and Fodinicurvata fenggangensis 170 DSM 21160T was obtained by cultivation in nutrient agar (NA) supplemented with 5 % 171 NaCl, incubated at 28 ºC for 5 days. The whole-cell fatty acid composition of strain 172 BA45ALT and the reference strains was determined on cultures reaching the late- 173 exponential stage of growth according to the four quadrants streak method (Sasser, 174 1990). Fatty acid methyl esters were prepared and analysed according to the standard 175 protocol of the Microbial Identification System (MIDI, Version 6.1; Identification 176 Library TSBA40 4.1; Microbial ID). Extracts were analyzed using a Hewlett Packard 177 model HP6890A gas chromatograph equipped with a flame-ionization detector as 178 described by Kämpfer & Kroppenstedt (1996). The fatty acid profiles of strain 179 BA45ALT included C18:1 ω7c (35.6 %), iso-C16:0 (16.3 %) and iso-C15:0 (10.1 %) as the 180 major fatty acids, followed by iso-C17:1 ω9c (5.8 %), C17:0 (5.5 %), C16:0 (3.3 %), C18:1 181 2OH (3 %). The fatty acid profile of strain BA45ALT was clearly different from those of 182 the related taxa. Although all three species produce C18:1 ω7c as a major product, the 183 novel organism also produces major amounts of iso-C16:0 and iso-C15:0 which are lacking 184 in Fodinicurvata sediminis DSM 21159T and Fodinicurvata fenggangensis DSM 185 21160T (Table S1). 186 The polar lipids of strain BA45ALT and the reference strains were analyzed as described 187 by Groth et al. (1996). The polar lipids detected in strain BA45ALT were 188 diphosphatidylglycerol, phosphatidylmethylethanolamine and a number of unknown 189 phospholipids and lipids (Supplementary Figure S2). It is important to note that in our 190 15 Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. J. 312 (1997). The clustral X windows interface: Flexible strategies for multiple sequence 313 alignment aided by quality analysis tools. Nucleic Acids Res 24, 4876–4882. 314 Tindall, B. J., Rosselló-Móra, R., Busse, H.-J., Ludwig, W. & Kämpfer, P. (2010). 315 Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst 316 Evol Microbiol 60, 249–266. 317 Tindall, B. J., Sikorski, J., Smibert, R. A. & Krieg, N. R. (2007). Phenotypic 318 characterization and the principles of comparative systematics. In Methods for General 319 and Molecular Microbiology, pp. 330-393. Edited by C. A. Reddy, T. J. Beveridge, J. 320 A. Breznak, G. A. Marzluf, T. M. Schmidt and R. 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Extremophiles 18, 811-824. 335 Wang, Y. X., Liu, J.-H., Zhang, X. X., Chen, Y.-G., Wang, Z. G., Chen, Y., Li, Q. 336 Y., Peng, Q. & Cui, X. L. (2009). Fodinicurvata sediminis gen. nov., sp. nov. and 337 Fodinicurvata fenggangensis sp. nov., poly-ß-hydroxybutyrate-producing bacteria in 338 the family Rhodospirillaceae. Int J Syst Evol Microbiol 59, 2575–2581. 339 Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., 340 Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E., Stackebrandt, 341 E., Starr, M. P. & Trüper, H. G. (1987). Report of the ad hoc committee on 342 reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463-464. 343 17 Table 1. Characteristics that differentiate strain BA45ALT from related species of the 344 genus Fodinicurvata. 345 Taxa: 1, strain BA45ALT; 2, Fodinicurvata fenggangensis DSM 21160T; 3, 346 Fodinicurvata sediminis DSM 21159T. All data are from the present study, except 347 where indicated. +, Positive reaction; -, negative reaction. 348 Characteristic 1 2 3 Colony pigmentation Yellow-cream Cream-white Cream-white Cell size (width x length; µm) 0.05-0.1 x 0.2-1.5 0.2-04 x 0.5-1.3 0.3-0.5 x 0.7-1.5 Motility + - - Temperature range (ºC) 14-45 15-42 15-42 NaCl range (%, w/v) 5-20 1.5-20 1.5-20 pH range 5-9 6.5-8.5 6.5-8.5 Nitrate reduction + - - Hydrolysis of: Aesculin Gelatin + + - - + - Utilization of carbon sources: Fructose Lactose Fumarate Hippurate L-ornithine + + + + + - - - - - - - - - - Simmons citrate - + + H2S production + - - Major fatty acids C18:1 ω7c, iso-C16:0 and iso- C15:0 C18:1 ω7c, 2-OH-C18:1 and C16:0 C18:1 ω7c, 2-OH-C18:1 and C16:0 Polar lipids Diphosphatidylglycerol, phosphatidylmethylethanola mine and unknown phospholipids and lipids Diphosphatidylglycerol, phosphatidylmethylethanola mine and four unknown phospholipids and two unknown lipids Diphosphatidylglycerol, phosphatidylmethylethanol a mine and four unknown phospholipids and two unknown lipids DNA G+C content (mol%) 58.0 62.3 61.5 Source Salt water Deposit of salt mine* Deposit of salt mine* *Data from Wang et al. (2009). 349 350 351 18 352 Legend to figure 353 354 Fig. 1. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences, 355 showing the relationships between strain BA45ALT and representatives of the family 356 Rhodospirillaceae. Bootstrap percentages (based on 1000 replications) ≥ 70 % are 357 shown at branch points. Bar represents 0.1 substitutions per nucleotide position. 358 Azospirillum melinis TMCY 0552T (DQ022958) Azospirillum largimobile ACM 2041T (X90759) Azospirillum oryzae COC8T (AB185396) Azospirillum zeae N7T (DQ682470) Azospirillum lipoferum ATCC 29707T (M59061) Azospirillum thiophilum BV-sT ( EU678791) Azospirillum canadense DS2T (DQ393891) Azospirillum rugosum IMMIB AFH-6T (AM419042) Azospirillum brasilense NCIMB 11860T (Z29617) Azospirillum halopraeferens Au4T (X79731) Rhodocista centenaria ATCC 43720T (D12701) Rhodocista pekingensis 3-pT (AF523824) Skermanella parooensis ACM 2042T (X90760) Inquilinus ginsengisoli Gsoil 080T (AB245352) Inquilinus limosus AU476T (AY043374) Thalassobaculum litoreum CL-GR58T (EF203900) Phaeospirillum molischianum ATCC 14031T (M59067) Phaeospirillum fulvum NCIMB 11762T (D14433) Phaeospirillum chandramohanii JA145T (AM779061) Magnetospirillum gryphiswaldense MSR-1T (Y10109) Magnetospirillum magnetotacticum DSM 3856T (Y10110) Telmatospirillum siberiense 26-4b1T (AF524863) Defluviicoccus vanus Ben 114T (AF179678) Tistrella mobilis IAM 14872T (AB071665) Fodinicurvata sediminis YIM D82T (FJ357426) Fodinicurvata fenggangensis YIM D812T (FJ357427) Rhodovibrio salinarum NCIMB 2243T (D14432) Rhodovibrio sodomensis DS1T (M59072) Caenispirillum bisanense K92T (EF100694) Roseospirillum parvum 930IT (AJ011919) Roseospira mediosalina BN 280T (AJ000989) Roseospira marina CE2105T (AJ298879) Roseospira goensis JA135T (AM283537) Roseospira visakhapatnamensis JA131T (AM282560) Rhodospira trueperi ATCC 70022T (AJ001276) Rhodospirillum photometricum DSM 122T (AJ222662) Rhodospirillum rubrum ATCC 11170T (CP000230) Thalassospira lucentensis QMT2T (AF358664) Escherichia coli ATCC 11775T (X80725) 0.10 Fodinicurvata halophila BA45ALT (HG764424) 97 100 97 100 100 100 99 96 98 100 99 100 93 100 96 100 100 77 78 88 72 81 78 Figure 1 Click here to download Figure: Fig. 1.ppt Supplementary Material File Click here to download Supplementary Material Files: Suppl Material Fodinicurvata halophila.pdf