From Metagenomics to Pure Culture: Isolation and Characterization of the Moderately Halophilic Bacterium Spiribacter salinus gen. nov., sp. nov. María José León,a Ana B. Fernández,a Rohit Ghai,b Cristina Sánchez-Porro,a Francisco Rodriguez-Valera,b Antonio Ventosaa Downloaded from Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spaina; Evolutionary Genomics Group, Division of Microbiology, Miguel Hernández University, San Juan, Alicante, Spainb Recent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dom- inated by both Euryarchaeota and halophilic bacteria, with a gammaproteobacterium closely related to the genera Alkalilimni- cola and Arhodomonas being one of the most predominant microorganisms, making up to 15% of the total prokaryotic popula- tion. Here we used several strategies and culture media in order to isolate this organism in pure culture. We report the isolation and taxonomic characterization of this new, never before cultured microorganism, designated M19-40T, isolated from a saltern http://aem.asm.org/ located in Isla Cristina, Spain, using a medium with a mixture of 15% salts, yeast extract, and pyruvic acid as the carbon source. Morphologically small curved cells (young cultures) with a tendency to form long spiral cells in older cultures were observed in pure cultures. The organism is a Gram-negative, nonmotile bacterium that is strictly aerobic, non-endospore forming, hetero- trophic, and moderately halophilic, and it is able to grow at 10 to 25% (wt/vol) NaCl, with optimal growth occurring at 15% (wt/ vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that strain M19-40T has a low similarity with other previously described bacteria and shows the closest phylogenetic similarity with species of the genera Alkalilimnicola (94.9 to 94.5%), Alkalispirillum (94.3%), and Arhodomonas (93.9%) within the family Ectothiorhodospiraceae. The phenotypic, genotypic, and chemotaxonomic features of this new bacterium showed that it constitutes a new genus and species, for which the on February 8, 2016 by USE/BCTA.GEN UNIVERSITARIA LMG ؍ IBRC-M 10768T ؍ CECT 8282T ؍) name Spiribacter salinus gen. nov., sp. nov., is proposed, with strain M19-40T 27464T) being the type strain. ypersaline environments are widely distributed habitats rep- respectively) and a crystallizer pond (with 37% salts) by deep shot- Hresented by a variety of aquatic and terrestrial systems (1). gun sequencing of environmental DNA. These studies have per- Most microbiological studies in these extreme environments have mitted the determination of the microbial diversity of these ponds been carried out on aquatic habitats, especially saline lakes and with intermediate salinities in comparison with that of the crys- marine salterns, which are used for the commercial production of tallizer pond (15, 19). Comparison of the 16S rRNA reads with salt by evaporation of seawater (1, 2). One of the salterns that has sequences in databases indicated that the major groups at the in- been most extensively studied during the last 30 years is located in termediate-salinity ponds were the Euryarchaeota (which was the Santa Pola, Alicante, Spain; these studies have been based on cul- predominant group at the crystallizer pond), as well as the phyla ture-dependent (3–5) as well as culture-independent (6–11) tech- Proteobacteria and Bacteroidetes. At the genus level, several halo- niques. The studies carried out in this hypersaline habitat have archaea (members of the genera Halorubrum, Haloquadratum, permitted the in-depth determination of the microbial diversity of and Natronomonas) were still very abundant in ponds with 19 or the ponds of the saltern, especially that of the crystallizers (ponds 33% salts, but a large diversity composed of members of many in which the NaCl is precipitated). The crystallizers are dominated different bacterial genera was also evident (with representatives by the square-shaped haloarchaeal species Haloquadratum wals- related to the genera Salinibacter, Alkalilimnicola, Arhodomonas, byi, the Gram-negative rod Salinibacter ruber, a member of the Roseovarius, and Oceanicola)(15, 19). phylum Bacteroidetes, and the recently described Nanohaloar- The assembly of the metagenomic data sets corresponding to chaeota (6, 7, 12–16). the intermediate-salinity saltern ponds permitted us to determine In contrast to the most concentrated ponds, few studies have that several organisms that had not been previously isolated or been carried out on the ponds of intermediate salinity designated characterized were abundant in these habitats, including two concentrators (4, 5, 10, 17, 18). Early studies based on culture- dependent techniques indicated that the most frequently isolated halophilic bacteria were members of the genera Pseudomonas-Al- Received 8 February 2014 Accepted 12 April 2014 teromonas-Alcaligenes (probably the current members of the Ha- Published ahead of print 18 April 2014 lomonadaceae) and Salinivibrio, as well as haloarchaeal members Editor: S.-J. Liu of the genera Haloferax and Haloarcula (4). However, later molec- Address correspondence to Antonio Ventosa, [email protected]. ular studies based on PCR amplification of the 16S rRNA gene and Supplemental material for this article may be found at http://dx.doi.org/10.1128 denaturing gradient gel electrophoresis showed that these were /AEM.00430-14. not the dominant groups (10, 18). Copyright © 2014, American Society for Microbiology. All Rights Reserved. Very recently, we have carried out metagenomic studies on doi:10.1128/AEM.00430-14 three intermediate-salinity ponds (with 13, 19, and 33% total salts, 3850 aem.asm.org Applied and Environmental Microbiology p. 3850–3857 July 2014 Volume 80 Number 13 Spiribacter salinus, a New Halophilic Bacterium Downloaded from http://aem.asm.org/ FIG 1 Phylogenetic tree of 16S rRNA gene sequences based on the maximum parsimony algorithm showing the position of Spiribacter salinus M19-40T and closely related species. GenBank sequence accession numbers are shown in parentheses. Bootstrap values higher than 50% are indicated at branch points. Chromatium okenii DSM 169T was used as the outgroup. Filled circles indicate that the corresponding nodes were also obtained in the trees generated with the maximum likelihood and neighbor-joining algorithms. Bar, 0.01 substitution per nucleotide position. groups consisting of Euryarchaeota with high GϩC contents and carried out using an automated DNA sequencer (model ABI 3130XL; Euryarchaeota with low GϩC contents (different from Halo- Applied Biosystems). The identification of phylogenetic relatives and cal- on February 8, 2016 by USE/BCTA.GEN UNIVERSITARIA quadratum walsbyi), a group of Actinobacteria with low GϩC con- culation of pairwise 16S rRNA gene sequence similarities were achieved tents, and interestingly, a group belonging to the class Gammapro- using the EzTaxon-e server (23). The 16S rRNA gene sequence analysis teobacteria which may account for up to 15% of the total was performed with the ARB software package (24). The 16S rRNA gene prokaryotic population of the intermediate-salinity ponds. This sequences were aligned with the published sequences of the closely related abundant group of microbes was phylogenetically distantly re- bacteria, and the alignment was confirmed and checked against both the primary and secondary structures of the 16S rRNA molecule using the lated to the genera Alkalilimnicola, Arhodomonas, and Nitrococcus. alignment tool of the ARB software package. Phylogenetic trees were con- The pI of the predicted proteins was very similar to that of Alka- structed using three different methods, the maximum likelihood (25), lilimnicola ehrlichii and indicated that this microorganism is a maximum parsimony (26), and neighbor-joining (27) methods, and the salt-out strategist (15, 19). algorithms integrated in the ARB software for phylogenetic inference. A In this paper, we describe the isolation and characterization of bootstrap test (28) was performed by calculating 1,000 replicate trees in the most abundant bacterium, according to the previous meta- order to assess the robustness of the topology. The 16S rRNA gene se- genomic studies. This bacterium is a member of the Ectothiorho- quences used for phylogenetic comparisons were obtained from the dospiraceae, with Alkalilimnicola and Arhodomonas being the most GenBank database, and the strain designations and accession numbers are closely related genera. We have characterized this new isolate tax- shown in Fig. 1. onomically and describe it as a new genus and species. Genome recruitment. The recruitment of the complete genome of strain M19-40T (29) from metagenomic data sets recently obtained from MATERIALS AND METHODS hypersaline habitats with different salt concentrations (15, 30, 31) was Isolation and culture conditions. Strain M19-40T was isolated from a carried out via BLASTN analysis. A restrictive cutoff of 95% identity in at water sample obtained in October 2011 from a pond of a marine saltern least 50 bp was established to guarantee that only similarities at the level of located in Isla Cristina, Huelva, southwest Spain. The isolation medium nearly identical microbes were counted. used was designed on the basis of the nutritional requirements described Morphological, physiological, and biochemical characteristics. For for Alkalilimnicola ehrlichii (20), after incubation at 37°C for 6 weeks.