From metagenomics to pure culture: isolation and characterization of the moderately halophilic bacterium Spiribacter salinus gen. nov., sp. nov

Abstract

Recent 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.

FIG 1

Phylogenetic tree of 16S rRNA gene sequences based on the maximum parsimony algorithm showing the position of Spiribacter salinus M19-40^T 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 169^T 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.

FIG 2

Recruitment of Spiribacter salinus M19-40^T genome (1739487 bp) against metagenomes of hypersaline aquatic habitats of different salinities. (A) Metagenome from a Santa Pola saltern (13% salinity pond); (B) metagenome from a Santa Pola saltern (19% salinity pond); (C) metagenome from an Isla Cristina saltern (21% salinity pond). A restrictive cutoff of 95% identity in at least 50 bp of the length of the metagenomic reads was used.

FIG 3

Transmission electron microphotographs (A to D) of thin sections of cells of Spiribacter salinus M19-40^T grown on SM15 liquid medium at early exponential phase. Bars, 2 μm (A), 300 nm (B), 1 μm (C), and 500 nm (D).

FIG 4

Transmission electron microphotographs (A to D) of thin sections of cells of Spiribacter salinus M19-40^T grown on SM15 liquid medium at late stationary phase. Bars, 2 μm (A, B), 200 nm (C), and 500 nm (D).