Spiribacter roseus sp. nov., a moderately halophilic species of the genus Spiribacter from salterns

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 October 2016, available at: https://doi.org/10.1099/ijsem.0.001338 .” 1 1 Spiribacter roseus sp. nov., a new moderately halophilic species of the genus 2 Spiribacter from salterns 3 4 5 María José León, Blanca Vera-Gargallo, Cristina Sánchez-Porro and Antonio Ventosa* 6 7 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of 8 Sevilla, 41012 Sevilla, Spain 9 10 11 Running title: Spiribacter roseus sp. nov. 12 13 Subject category: New Taxa - Proteobacteria 14 15 *Corresponding author. Tel: +34 954556765; fax: +34 954628162. 16 E-mail address: [email protected] (A. Ventosa). 17 18 19 20 21 The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of 22 Spiribacter roseus SSL50T is LT578364. 23 24 Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document) Spiribacter roseus sp nov revised.docx 2 Four pink-pigmented, non-motile, Gram-staining-negative and moderately 25 halophilic curved rods, designated strains SSL50T, SSL25, SSL97 and SSL4, were 26 isolated from a saltern located in Isla Cristina, Huelva, South West Spain. 27 Phylogenetic analyses based on 16S rRNA gene sequences showed that they were 28 members of the genus Spiribacter, most closely related to Spiribacter curvatus UAH- 29 SP71T (99.3-99.5 % sequence similarity) and Spiribacter salinus M19-40T (96.5- 30 96.7 %). Other related species were Alkalilimnicola ehrlichii MLHE-1T (95.1- 31 95.3 %), Arhodomonas recens RS91T (95.1-95.2 %) and Arhodomonas aquaeolei 32 ATCC 49307T (95.0-95.1 %), all members of the family Ectothiorhodospiraceae. The 33 major fatty acids were C18:1 ω6c and/or C18:1 ω7c, C16:0, and C12:0. The DNA G+C 34 range was 64.0-66.3 mol%. The DNA-DNA hybridization between strains SSL50T, 35 SSL25, SSL97, SSL4, and Spiribacter curvatus UAH-SP71T were 37-49 %. The 36 average nucleotide identity (ANIb) values between the strain SSL50T genome and 37 the two other Spiribacter species, S. curvatus UAH-SP71T and S. salinus M19-40T 38 were 82.4 % and 79.1 %, respectively, supporting the proposal of a new species of 39 the genus Spiribacter. On the basis of the polyphasic analysis, the four new isolates 40 are considered to represent a novel species of Spiribacter, for which the name 41 Spiribacter roseus sp. nov. is proposed. The type strain is SSL50T (= CECT 9117T = 42 IBRC-M 11076T). 43 44 3 Hypersaline environments are extreme habitats in which the main life limiting factor is 45 their high salt concentration. In this regard, multi-pond solar salterns constitute excellent 46 models for studies focused on the microbial diversity and ecology over a wide range of 47 salt concentrations. The microbial community of crystallizer ponds has been extensively 48 studied and their most abundant archaeal and bacterial inhabitants have been obtained in 49 pure culture, whereas intermediate salinity ponds have received much less attention 50 (Ventosa et al., 2014). Moreover, studies using culture-independent techniques for the 51 analysis of the microbial diversity dwelling in intermediate salinity saltern ponds have 52 shown that the most abundant microbes have not been retrieved in axenic cultures yet, 53 possibly due to their slow growing time and particular requirement of nutrients being very 54 different to the ones commonly used in laboratories studies (Ghai et al., 2011; Fernández 55 et al., 2014a; 2014b). As a consequence, culture-based studies conducted on these habitats 56 have led to the isolation of microorganisms detected in negligible abundance by culture- 57 independent techniques, which provide a more realistic view of their diversity. This 58 prevents scientific community from studying and understanding the primary relationships 59 and functions of microbes in these natural environments. 60 Recently, León et al. (2014) described the genus Spiribacter, an abundant moderate 61 halophile thriving in intermediate salinity saltern ponds as ascertained by metagenomic 62 assemblies and recruitment analysis. It belongs to the family Ectothiorhodospiraceae, 63 within the order Chromatiales, class Gammaproteobacteria in the phylum 64 Proteobacteria. To date, this genus includes two species isolated from salterns, 65 Spiribacter salinus (León et al. 2014; Oren and Garrity, 2014) and Spiribacter curvatus 66 (León et al. 2015), which genomes were sequenced and assembled into a single contig 67 (López-Pérez et al., 2013). Spiribacter cells are characterized by a peculiar morphology, 68 4 adopting short and thin curved rods morphology on young cultures and spiral shapes at 69 the stationary phase. 70 Metagenomic studies carried out from concentrator saltern ponds with 19 % and 21 % 71 NaCl located in Santa Pola (East coast of Spain) and Isla Cristina (Southwest of Spain), 72 respectively, showed that a high number of the metagenomics contigs assembled 73 belonged to Spiribacter (Fernández et al. 2014b; León et al., 2014), which indicates that 74 this genus is very abundant in these environment. However, a closer look at these contigs 75 revealed that some of them had low similarity to S. salinus and S. curvatus genomes, 76 while still being syntenic to them. This suggested that there may be other very abundant 77 species in these environments. On the basis of these data, four strains closely related to 78 Spiribacter, were obtained from the water of ponds of Isla Cristina saltern in Spain. In 79 this study we combine a genomic analysis as well as a polyphasic approach with the aim 80 of defining the taxonomic position of these new strains. Based on the obtained data we 81 propose the placement of the four strains as a new species within the genus Spiribacter, 82 for which we propose the name Spiribacter roseus sp. nov. 83 84 Taking into consideration the culture conditions already used for the cultivation of the 85 previously described species of Spiribacter, the growth characteristics and the peculiar 86 morphology adopted by the cells of this genus, we carried out a sampling aimed to 87 exclusively obtain other bacterial strains phylogenetically related to the genus 88 Spiribacter. Four strains, SSL50T, SSL25, SSL97 and SSL4, were isolated from water 89 samples from a pond of a marine saltern located in Isla Cristina, Huelva, South West 90 Spain. Samples were collected in sterile containers and transported within 4-5 h of its 91 collection to the laboratory and plated on a modified SM medium, previously described 92 by León et al. (2014), designated as SMM medium. The composition of the SMM 93 5 medium was (g l-1): NaCl, 46.8; MgCl2·6H2O, 19.5; MgSO4·7H2O, 30.5; CaCl2, 0.5; KCl, 94 3.0; NaHCO3, 0.1; NaBr, 0.35; casein digest, 5.0 and sodium pyruvate, 1.1. The pH was 95 adjusted to 7.5 with KOH 4 M. Strains were routinely grown in this medium at 37 ºC for 96 10 days. These cultures were maintained at -80 ºC in SMM medium containing 50 % (v/v) 97 glycerol. The type strains Spiribacter salinus M19-40T and Spiribacter curvatus UAH- 98 SP71T were used as reference strains for comparative purposes. They were cultivated 99 under the same conditions as the four new isolates. 100 101 For the determination of the cellular morphology and motility, cultures grown on SMM 102 medium were examined under a phase-contrast microscope (Olympus CX41). Cellular 103 morphology was also examined by electron microscopy after growth on SMM medium 104 at 37 ºC on a shaking incubator at 200 rpm until they reached the exponential-stationary 105 phase. The cells were then collected by centrifugation and fixed in 1.6 % glutaraldehyde 106 buffered with 0.1 M cacodilate (pH 7.4) for one hour at room temperature and 24 hours 107 at 4 ºC. They were then postfixed in a 1 % (w/v) osmium tetroxide solution for one hour 108 at 4 ºC. The dehydration was performed in an increasing series of acetone (50-100 %) and 109 the samples were embedded in Spurr resin and stained with 2 % (w/v) aqueous uranyl 110 acetate for 2 hours. Blocks were obtained by polymerization at 70 ºC for 8 hours. After 111 hardening, ultrathin sections were cut with a diamond knife and then counterstained with 112 uranyl acetate and lead citrate using an automatic counterstaining machine (Leica AC20). 113 The thin sections were observed using a Zeiss Libra 120 at the CITIUS Center of the 114 University of Sevilla, Spain. As previously reported for species of Spiribacter, the cells 115 of strains SSL50T, SSL25, SSL97 and SSL4 have small sizes, forming thin curved rods 116 on young cultures and spirals at the late exponential and stationary phase. These spiral 117 cells are not as long as those reported for Spiribacter salinus M19-40T. At the beginning 118 6 of the exponential phase, the cells look like curved rods-nearly closed rings-with a size 119 of 0.2-0.3 µm wide by 1.6-1.8 µm long (Fig 1). When they reach the late exponential 120 phase cells have spiral shapes, 2.0-3.0 µm long. Polyalkanoate inclusion bodies are 121 observed. By both, optical and electron microscopy, we also observe round structures, 122 with a prolongation in many cases, along all the growth phases. These structures were 123 also present in Spiribacter salinus M19-40T (León et al., 2014), but they are more evident 124 in the four new isolates. 125 126 The morphology of colonies, their size and pigmentation were observed on SMM solid 127 medium after 10 days of incubation at 37 ºC. Optimal conditions for growth were 128 determined by growing the strains in SMM liquid medium adjusted to 0, 0.2, 0.4, 0.6, 0.8, 129 1.0, 1.3, 1.6, 1.8, 2.0 and 2.2 M NaCl, and at temperatures of 15, 20, 28, 30, 37, 40 and 130 45 °C, respectively. Growth rates were determined by monitoring the increase in the 131 optical density at 600 nm. The pH range for the isolates was tested in SMM medium 132 adjusted to pH 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 9.0 and 10.0 with the addition of the appropriate 133 buffering capacity to each medium (Sánchez-Porro et al., 2009). All biochemical tests 134 were carried out in SMM medium at 37 C, unless otherwise stated. Growth under 135 anaerobic conditions (with H2/CO2) was determined by incubating strains in an anaerobic 136 chamber in SMM solid medium. Catalase activity was determined by adding a 1 % (w/v) 137 H2O2 solution to colonies on SMM agar medium. Oxidase test was performed using the 138 Dry Slide Assay (Difco). The following tests were determined as described by Cowan & 139 Steel (1965) growing the cells on SMM medium: hydrolysis of aesculin, casein, gelatin, 140 starch or Tween 80, Voges-Proskauer and methyl red tests, production of indole, 141 phosphatase, urease, nitrate and nitrite reduction. Citrate utilization was determined on 142 Simon’s citrate medium supplemented with 10 % (w/v) salts. For determining the range 143 7 of substrates used as carbon and energy sources or as carbon, nitrogen and energy sources, 144 the classical medium of Koser (1923) as modified by Ventosa et al. (1982) was used. 145 Substrates were added as filter-sterilized solutions to give a final concentration of 1 g l-1, 146 except for carbohydrates, which were used at 2 g l-1. 147 Cells of the new isolates were Gram-stain-negative, non-motile, and strictly aerobic. The 148 four strains showed catalase and oxidase activities. They were moderately halophilic 149 bacteria, able to grow in SMM medium with 8 to 20 % total salts, with optimal growth at 150 10 % salts; they were not able to grow in the absence of NaCl. The cells were able to 151 grow from 15 to 40 ºC, being the optimal growth at 37 ºC. The pH range for growth was 152 7-9 with the optimal growth at pH 7.5-8.0. Urease and phosphatase were positive for all 153 strains (in the species Spiribacter curvatus UAH-SP71T urease was negative, but 154 phosphatase was positive). Nitrate and nitrite were not reduced. Hydrolysis of aesculin, 155 starch, casein, gelatine and Tween 80 were negative. The available genomic sequence of 156 the type strain SSL50T has been of considerable importance in the phenotypic 157 characterization of this new bacterium because it has allowed us to contrast the 158 experimental data obtained in the laboratory with those obtained from the genomic 159 analysis. The analysis of the complete genome of the strain SSL50T showed a simplified 160 metabolic versatility for this bacterium, as occurs with the other two Spiribacter species. 161 Strains SSL50T, SSL25, SSL97 and SSL4 had very reduced ability to utilize organic 162 compounds as sole carbon and energy sources (Table 1). 163 164 Genomic DNA from strains SSL50T, SSL25, SSL97 and SSL4 was prepared using the G- 165 spinTM Total extraction kit (Intron Biotechnology) according to the manufacturer’s 166 instructions. The 16S rRNA gene sequences of strains SSL50T, SSL25, SSL97 and SSL4 167 were obtained from the complete genome sequence. The almost-complete 16S rRNA 168 8 gene sequence of strains SSL50T (1548 bp), SSL25 (1548 bp), SSL97 (1549 bp) and SSL4 169 (1548 bp) were obtained, deposited in GenBank, and used for initial BLAST searches in 170 GenBank and for phylogenetic analysis. The identification of phylogenetic neighbours 171 and the calculation of pairwise 16S rRNA gene sequence similarities were achieved using 172 the EzTaxon-e tool (Kim et al., 2012). The 16S rRNA gene sequence analysis showed 173 that strains SSL50T, SSL25, SSL97 and SSL4 were members of the genus Spiribacter. 174 The 16S rRNA gene sequence similarities between strains SSL50T, SSL25, SSL97 and 175 SSL4 were 99.9 % and between these four strains and the most closely related species of 176 Spiribacter, Spiribacter curvatus UAH-SP71T were 99.3 %, 99.3 %, 99.5 % and 99.4 %, 177 respectively, and with Spiribacter salinus M19-40T were 96.5 %, 96.5 %, 96.7 % and 178 96.5 % sequence similarities, respectively. Other related species were Alkalilimnicola 179 ehrlichii MLHE-1T (95.1-95.3 %), Arhodomonas recens RS91T (95.1-95.2 %) and 180 Arhodomonas aquaeolei ATCC 49307T (95.0-95.1 %), all members of the family 181 Ectothiorhodospiraceae. Those high similarity values might indicate that the new strains 182 could constitute novel strains of the species Spiribacter curvatus UAH-SP71T within the 183 genus Spiribacter. However, some significant differences in the cultures of these new 184 strains, such as their growth rates, colonies pigmentation and the optical density reached, 185 with respect to the species Spiribacter curvatus UAH-SP71T were noticed and led us to 186 further investigate their taxonomic affiliation. 187 The 16S rRNA gene sequences were aligned with the published sequences from closely 188 related bacteria and the alignments were confirmed and checked against both primary and 189 secondary structures of the 16S rRNA molecule using the alignment tool of the ARB 190 software package version 5.5 (Ludwig et al., 2004). Phylogenetic trees were constructed 191 using three different methods: maximum-parsimony (Fitch, 1971), neighbour-joining 192 (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1981) algorithms integrated 193 15 16s rRNA gene sequence database with phylotypes that represent uncultured species. Int 340 J SystEvol Microbiol 62, 716-721. 341 Konstantinidis, K. 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(1982). Numerical taxonomy of moderately halophilic Gram-negative 394 rods. J Gen Microbiol 128, 1959-1968. 395 Ventosa A. (2008). Aquisalibacillus elongatus gen. nov., sp. nov., a moderately 396 halophilic bacterium of the family Bacillaceae isolated from a saline lake. Int J Syst Evol 397 Microbiol 58, 1922-1926. 398 Versalovic, J., Schneider, M., de Bruijn, F. J. & Lupski, J. R. (1994). Genomic 399 fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. 400 Methods Mol Cell Biol 5, 25–40. 401 402 18 Legend to figures 403 404 Fig. 1. Maximum-parsimony phylogenetic tree based on the complete 16S rRNA gene 405 sequences showing the relationships between strains SSL50T, SSL25, SSL97, SSL4 and 406 the most closely related species of the genus Spiribacter and other members of the family 407 Ectothiorhodospiraceae. Sequence accession numbers used are shown in parentheses. 408 Bootstrap values higher than 70 % are indicated at branch-points. Thermus thermophilus 409 HB8T (GenBank accession no. X07998) was used as an outgroup. The 410 GenBank/EMBL/DDBJ accession number of each sequence is shown in parentheses. Bar, 411 0.01 nucleotide changes per position. 412 413 Fig. 2. Transmission electron microphotographs (A-D) of thin sections of cells of 414 Spiribacter roseus SSL50T grown on SMM liquid medium at late exponential phase. Bars, 415 0.2 µm (A), 1 µm (B); 1 µm (C); 0.5 µm (D). 416 417 19 Table 1. Differential characteristics of strains SSL50T, SSL25, SSL97, SSL4 and the type 418 strain of the two closely related species of the genus Spiribacter. 419 Strains: 1, SSL50T; 2, SSL25; 3, SSL97; 4, SSL4; 5, Spiribacter curvatus UAH-SP71T; 420 6, Spiribacter salinus M19-40T. 421 +, Positive; -, negative. 422 423 a Data from the Tm method; b data from the genome sequence; c data (Tm and genome 424 sequence, respectively) from León et al. (2015). 425 426 Characteristic 1 2 3 4 5 6 Cell morphology Curved rodsnearly closed rings, short spiral cells Curved rods-nearly closed rings, short spiral cells Curved rodsnearly closed rings, short spiral cells Curved rodsnearly closed rings, short spiral cells Thin curved rods, some spiral cells Thin curved rods, long spiral cells Colony pigmentation Pink Pale pink Pale pink Pink Dark pink Pink Cell size (µm) 0.2-0.3 x 1.6-1.8 0.2-0.3 x 1.6-1.8 0.2-0.3 x 1.6- 1.8 0.2-0.3 x 1.6- 1.8 0.5-1.5 0.3 x 0.8-1.8 NaCl range (M) 0.8-2.2 0.6-2.2 0.6-2.2 0.6-2.2 0.8-3.4 0.6-2.4 NaCl optimum (M) 0.8-1.0 1.3-1.6 1.0-1.3 1.3-1.6 1.7 0.8-1.0 Temperature range for growth (ºC) 15-40 15-40 15-40 15-40 5-40 15-40 pH range 7.0-9.0 7.0-9.0 7.0-9.0 7.0-9.0 5.0-10.0 6.0-9.0 pH optimum 7.5-8.0 7.5-8.0 7.5-8.0 7.5-8.0 8.0 7.5-8.0 Urease + + + + - + Phosphatase + + + + + - DNA G+C content (%) 64.2a (66.0b) 66.3a 64.0a 64.0a 60.4 (63.9)c 60.0 (62.7)c 20 Table 2. Cellular fatty acids composition of strain SSL50T and the most closely related 427 species of the genus Spiribacter. All data were obtained under the same growth 428 conditions. Values are percentages of the total cellular fatty acids. Strains: 1, strain 429 SSL50T; 2, Spiribacter salinus M19-40T (León et al., 2014) and 3, Spiribacter curvatus 430 UAH-SP71T (León et al., 2015). Values lower than 0.5 % are not shown. -, Not detected. 431 432 Fatty acid 1 2 3 C9:0 - 0.6 - C10:0 0.7 0.6 1.2 C10:0 3-OH 3.3 4.0 6.4 C12:0 6.7 3.5 5.7 C12:0 3-OH 3.1 1.3 1.2 C14:1 ω5c - 0.8 - Summed feature 2* - - 2.0 Summed feature 3* 2.5 8.6 3.6 C14:0 0.6 0.8 0.8 C16:0 28.3 9.3 13.4 Summed feature 8* 50.0 60.6 60.6 C17:0 - 1.8 - C17:1 ω8c - 2.1 - C17:1 ω6c - 1.0 - C18:0 2.1 1.2 1.6 C18:1 ω9c 0.8 - - Summed feature 7* 3.0 1.9 0.6 C19:0 cyclo ω8c 3.9 1.0 2.2 * Summed features are groups of two or three fatty acids that could not be separated by 433 GC with the MIDI system. Summed feature 2 comprised C14:0 3OH, iso-C16:1 and/or C12:0 434 aldehyde, summed feature 3 comprised C16:1 ω6c and/or C16:1 ω7c, summed feature 7 435 21 comprised C19:1 ω6c and/or C19:0 cyclo ω10c and summed feature 8 comprised C18:1 ω6c 436 and/or C18:1 ω7c. 437 438 Figure 1 Click here to download Figure Figure 1.jpg Figure 2A Click here to download Figure Fig2A.tif Figure 2B Click here to download Figure Fig2B.tif