María José León

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 M19-40T, 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 M19-40T 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 name Spiribacter salinus gen. nov., sp. nov., is proposed, with strain M19-40T (= CECT 8282T = IBRC-M 10768T = LMG 27464T) being the type strain.

Genomes of “Spiribacter”, a streamlined, successful halophilic bacterium

BackgroundThalassosaline waters produced by the concentration of seawater are widespread and common extreme aquatic habitats. Their salinity varies from that of sea water (ca. 3.5%) to saturation for NaCl (ca. 37%). Obviously the microbiota varies dramatically throughout this range. Recent metagenomic analysis of intermediate salinity waters (19%) indicated the presence of an abundant and yet undescribed gamma-proteobacterium. Two strains belonging to this group have been isolated from saltern ponds of intermediate salinity in two Spanish salterns and were named “Spiribacter”.ResultsThe genomes of two isolates of “Spiribacter” have been fully sequenced and assembled. The analysis of metagenomic datasets indicates that microbes of this genus are widespread worldwide in medium salinity habitats representing the first ecologically defined moderate halophile. The genomes indicate that the two isolates belong to different species within the same genus. Both genomes are streamlined with high coding densities, have few regulatory mechanisms and no motility or chemotactic behavior. Metabolically they are heterotrophs with a subgroup II xanthorhodopsin as an additional energy source when light is available.ConclusionsThis is the first bacterium that has been proven by culture independent approaches to be prevalent in hypersaline habitats of intermediate salinity (half a way between the sea and NaCl saturation). Predictions from the proteome and analysis of transporter genes, together with a complete ectoine biosynthesis gene cluster are consistent with these microbes having the salt-out-organic-compatible solutes type of osmoregulation. All these features are also consistent with a well-adapted fully planktonic microbe while other halophiles with more complex genomes such as Salinibacter ruber might have particle associated microniches.

Draft Genome of Spiribacter salinus M19-40, an Abundant Gammaproteobacterium in Aquatic Hypersaline Environments

ABSTRACT We have previously used a de novo metagenomic assembly approach to describe the presence of an abundant gammaproteobacterium comprising nearly 15% of the microbial community in an intermediate salinity solar saltern pond. We have obtained this microbe in pure culture and describe the genome sequencing of the halophilic photoheterotrophic microbe, Spiribacter salinus M19-40.

Larsenia salina gen. nov., sp. nov., a new member of the family Halomonadaceae based on multilocus sequence analysis.

Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18(T) and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3-25% (w/v) NaCl (optimal growth at 7.5-10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0-7.0) and at temperatures between 15 and 40°C (optimal at 37°C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985(T) (95.2-94.8%) and Chromohalobacter salexigens DSM 3043(T) (95.0-94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA-DNA hybridization between both strains was 82%, whereas the values between strain M1-18(T) and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C18:1ω7c/C18:1ω6c, C16:0, and C16:1ω7c/C16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18(T) (=CCM 8464=CECT 8192(T)=IBRC-M 10767(T)=LMG 27461(T)).

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

Four pink-pigmented, non-motile, Gram-staining-negative and moderately halophilic curved rods, designated strains SSL50T, SSL25, SSL97 and SSL4, were isolated from a saltern located in Isla Cristina, Huelva, south-west Spain. Phylogenetic analyses based on 16S rRNA gene sequences showed that they were members of the genus Spiribacter, most closely related to Spiribacter curvatus UAH-SP71T (99.3-99.5 % sequence similarity) and Spiribacter salinus M19-40T (96.5-96.7 %). Other related strains were Alkalilimnicola ehrlichii MLHE-1T (95.1-95.3 %), Arhodomonas recens RS91T (95.1-95.2 %) and Arhodomonas aquaeolei ATCC 49307T (95.0-95.1 %), all members of the family Ectothiorhodospiraceae. The major fatty acids were C18 : 1ω6c and/or C18 : 1ω7c, C16 : 0 and C12 : 0. The DNA G+C range was 64.0-66.3 mol%. The DNA-DNA hybridization values between strains SSL50T, SSL25, SSL97, SSL4 and S. piribacter. curvatus UAH-SP71T were 37-49 %. The average nucleotide identity (ANIb) values between the genome of strain SSL50T and those of the two other representatives of the genus Spiribacter, S. curvatus UAH-SP71T and S. salinus M19-40T, were 82.4 % and 79.1 %, respectively, supporting the proposal of a novel species of the genus Spiribacter. On the basis of the polyphasic analysis, the four new isolates are considered to represent a novel species of the genus Spiribacter, for which the name Spiribacter roseus sp. nov. is proposed. The type strain is SSL50T (=CECT 9117T=IBRC-M 11076T).

Metagenomic sequence of prokaryotic microbiota from an intermediate-salinity pond of a saltern in isla cristina, Spain.

ABSTRACT Marine salterns are artificial multipond systems designed for the commercial production of salt by evaporation of seawater. We report here the metagenomic sequence of the prokaryotic microbiota of a pond with intermediate salinity (21% total salts) of a saltern located in Isla Cristina, Huelva, southwest Spain.

Compatible Solute Synthesis and Import by the Moderate Halophile Spiribacter salinus: Physiology and Genomics

Members of the genus Spiribacter are found worldwide and are abundant in ecosystems possessing intermediate salinities between seawater and saturated salt concentrations. Spiribacter salinus M19-40 is the type species of this genus and its first cultivated representative. In the habitats of S. salinus M19-40, high salinity is a key determinant for growth and we therefore focused on the cellular adjustment strategy to this persistent environmental challenge. We coupled these experimental studies to the in silico mining of the genome sequence of this moderate halophile with respect to systems allowing this bacterium to control its potassium and sodium pools, and its ability to import and synthesize compatible solutes. S. salinus M19-40 produces enhanced levels of the compatible solute ectoine, both under optimal and growth-challenging salt concentrations, but the genes encoding the corresponding biosynthetic enzymes are not organized in a canonical ectABC operon. Instead, they are scrambled (ectAC; ectB) and are physically separated from each other on the S. salinus M19-40 genome. Genomes of many phylogenetically related bacteria also exhibit a non-canonical organization of the ect genes. S. salinus M19-40 also synthesizes trehalose, but this compatible solute seems to make only a minor contribution to the cytoplasmic solute pool under osmotic stress conditions. However, its cellular levels increase substantially in stationary phase cells grown under optimal salt concentrations. In silico genome mining revealed that S. salinus M19-40 possesses different types of uptake systems for compatible solutes. Among the set of compatible solutes tested in an osmostress protection growth assay, glycine betaine and arsenobetaine were the most effective. Transport studies with radiolabeled glycine betaine showed that S. salinus M19-40 increases the pool size of this osmolyte in a fashion that is sensitively tied to the prevalent salinity of the growth medium. It was amassed in salt-stressed cells in unmodified form and suppressed the synthesis of ectoine. In conclusion, the data presented here allow us to derive a genome-scale picture of the cellular adjustment strategy of a species that represents an environmentally abundant group of ecophysiologically important halophilic microorganisms.

Spiribacter aquaticus sp. nov., a novel member of the genus Spiribacter isolated from a saltern

A moderately halophilic bacterium, designated strain SP30T, was isolated from a solar saltern located in Santa Pola, Alicante, on the East coast of Spain. It was a Gram-stain-negative, strictly aerobic bacterium, able to grow in 7.5-25 % (w/v) NaCl and optimally in 12.5 % (w/v) NaCl. Phylogenetic analyses, based on 16S rRNA gene sequences, showed that the novel isolate is a member of the genus Spiribacter, with the most closely related species being Spiribacter roseus SSL50T (99.9 % sequence similarity) and Spiribacter curvatus UAH-SP71T (99.4 % sequence similarity). The 16S rRNA gene sequence similarity with the type species Spiribacter salinus M19-40T was 96.6 %. The DNA-DNA relatedness value between strain SP30T and S. roseus SSL50T and S. curvatus UAH-SP71T was 40 and 55 %, respectively; these values are lower than the 70 % threshold accepted for species delineation. The major fatty acids were C16:0, C18 : 1ω7c, C19 : 0 cyclo ω8c and C12 : 0. Similarly to other species of the genus Spiribacter, strain SP30Twas observed as curved rods and spiral cells. Metabolic versatility was reduced to the utilization of a few organic compounds as the sole carbon and energy sources, as with other members of Spiribacter. However, it differed in terms of colony pigmentation (brownish-yellow instead of pink) and in having a higher growth rate. Based on these data and on the phenotypic, genotypic and chemotaxonomic characterization, we propose the classification of strain SP30T as a novel species within the genus Spiribacter, with the name Spiribacter aquaticus sp. nov. The type strain is SP30T (=CECT 9238T=LMG 30005T).

Draft Genome Sequence of the Moderately Halophilic Bacterium Pseudoalteromonas ruthenica Strain CP76

ABSTRACT Pseudoalteromonas ruthenica strain CP76, isolated from a saltern in Spain, is a moderately halophilic bacterium belonging to the Gammaproteobacteria. Here we report the draft genome sequence, which consists of a 4.0-Mb chromosome, of this strain, which is able to produce the extracellular enzyme haloprotease CPI.

Marinobacter aquaticus sp. nov., a moderately halophilic bacterium from a solar saltern

A moderately halophilic bacterium designated strain M6-53T was isolated from water of a pond from a marine saltern located in Huelva, south-west Spain. Cells of the strain were Gram-stain-negative, strictly aerobic, motile, slightly curved rods, able to grow in media containing 5-25 % (w/v) NaCl (optimal growth at 10 %, w/v), at temperatures from 20 to 40 °C (optimally at 37 °C) and at pH 6.5-9 (optimally at pH 7.0). Phylogenetic analysis based on 16S rRNA gene sequences placed the new isolate within the genus Marinobacter, with the type strains of the most closely related species being Marinobacter persicus IBRC-M 10445T (98.5 % similarity), Marinobacter oulmenensis Set74T (97.2 %) and Marinobacter hydrocarbonoclasticus ATCC 49840T (97.1 %). The major fatty acids present in strain M6-53T were C18 : 1ω9c (29.5 %), C16 : 0 (26.7 %), C12 : 0 3-OH (15.1 %), C18 : 0 (10.2 %) and C16 : ω9c (9.6 %). The G+C content of the genomic DNA for this strain was determined to be 56.4 mol%. The DNA-DNA hybridization values between strain M6-53T and M. persicus CECT 7991T, M. oulmenensis CECT 7499T and M. hydrocarbonoclasticus DSM 50418 were 8, 41 and 38 %, respectively. These values are lower than the accepted 70 % threshold and showed that the new isolate represented a different species within the genus Marinobacter. Phylogenetic analysis based on the 16S rRNA gene sequence and the phenotypic, genotypic and chemotaxonomic features of this new isolate support the placement of strain M6-53T as a representative of a novel species of the genus Marinobacter, for which we propose the name Marinobacter aquaticus sp. nov., with strain M6-53T (=CECT 9228T=LMG 30006T) as the type strain.