From Metagenomics to Pure Culture: Isolation and Characterization of the Moderately Halophilic Bacterium Spiribacter salinus gen. nov., sp. nov

Abstract

bRecent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dominated by both Euryarchaeota and halophilic bacteria, with a gammaproteobacterium closely related to the genera Alkalilimnicola and Arhodomonas being one of the most predominant microorganisms, making up to 15% of the total prokaryotic population. 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 M19-40 T , isolated from a saltern 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, heterotrophic, 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 M19-40 T 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 name Spiribacter salinus gen. nov., sp. nov., is proposed, with strain M19-40 T ‫؍(‬ CECT 8282 T ‫؍‬ IBRC-M 10768 T ‫؍‬ LMG 27464 T ) being the type strain.

Microbial diversity of hypersaline environments: a metagenomic approach

“…Number related species or genera within the family Ectothiorhodospiraceae are present and await classification [16 ].…”

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

“…This has prevented the scientific community from studying and understanding the primary relationships and functions of microbes in these natural environments. León et al, 2014), which indicated that this genus is very abundant in these environments. However, a closer look at these contigs revealed that some of them had low similarity to the genomes of S. salinus and S. curvatus, while still being syntenic to them.…”

“…Moreover, studies using culture-independent techniques for the analysis of the microbial diversity dwelling in saltern ponds of intermediate salinity have shown that the most abundant microbes have not been retrieved in axenic cultures yet, possibly due to their slow growing time and particular requirement of nutrients being very different to the ones commonly used in laboratory studies (Ghai et al, 2011;Fern andez et al, 2014a, b). This has prevented the scientific community from studying and understanding the primary relationships and functions of microbes in these natural environments.Recently, León et al (2014) described the genus Spiribacter to accommodate an abundant moderate halophile thriving in intermediate-salinity saltern ponds as ascertained by metagenomic assemblies and recruitment analysis. It belongs to the family Ectothiorhodospiraceae, within the order Chromatiales, class Gammaproteobacteria in the phylum Proteobacteria.…”

“…T were cultured on SM15 agar medium at 37 C for 10 days, the same conditions used by León et al (2014León et al ( , 2015 for the previously described species of the genus Spiribacter. The extraction and analysis of fatty acids were performed according to the recommendations of the MIDI system.…”

“…Samples were collected in sterile containers, transported within 4-5 h of their collection to the laboratory and plated on a modified SM medium, previously described by León et al (2014), designated as SMM medium. The composition of the SMM medium was (g l À1 ): NaCl, 46.8; MgCl 2 Á 6H 2 O, 19.5; MgSO 4 Á 7H 2 O, 30.5; CaCl 2 , 0.5; KCl, 3.0; NaHCO 3 , 0.1; NaBr, 0.35; casein digest, 5.0; and sodium pyruvate, 1.1.…”