High-quality draft genome sequences of Pseudomonas monteilii DSM 14164T, Pseudomonas mosselii DSM 17497T, Pseudomonas plecoglossicida DSM 15088T, Pseudomonas taiwanensis DSM 21245T and Pseudomonas vranovensis DSM 16006T: taxonomic considerations.

Pseudomonas is the bacterial genus of Gram-negative bacteria with the highest number of recognized species. It is divided phylogenetically into three lineages and at least 11 groups of species. The Pseudomonas putida group of species is one of the most versatile and best studied. It comprises 15 species with validly published names. As a part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA) project, we present the genome sequences of the type strains of five species included in this group: Pseudomonas monteilii (DSM 14164T), Pseudomonas mosselii (DSM 17497T), Pseudomonas plecoglossicida (DSM 15088T), Pseudomonas taiwanensis (DSM 21245T) and Pseudomonas vranovensis (DSM 16006T). These strains represent species of environmental and also of clinical interest due to their pathogenic properties against humans and animals. Some strains of these species promote plant growth or act as plant pathogens. Their genome sizes are among the largest in the group, ranging from 5.3 to 6.3 Mbp. In addition, the genome sequences of the type strains in the Pseudomonas taxonomy were analysed via genome-wide taxonomic comparisons of ANIb, gANI and GGDC values among 130 Pseudomonas strains classified within the group. The results demonstrate that at least 36 genomic species can be delineated within the P. putida phylogenetic group of species.

High quality draft genome sequences of Pseudomonas fulva DSM 17717(T), Pseudomonas parafulva DSM 17004(T) and Pseudomonas cremoricolorata DSM 17059(T) type strains.

Pseudomonas has the highest number of species out of any genus of Gram-negative bacteria and is phylogenetically divided into several groups. The Pseudomonas putida phylogenetic branch includes at least 13 species of environmental and industrial interest, plant-associated bacteria, insect pathogens, and even some members that have been found in clinical specimens. In the context of the Genomic Encyclopedia of Bacteria and Archaea project, we present the permanent, high-quality draft genomes of the type strains of 3 taxonomically and ecologically closely related species in the Pseudomonas putida phylogenetic branch: Pseudomonas fulva DSM 17717(T), Pseudomonas parafulva DSM 17004(T) and Pseudomonas cremoricolorata DSM 17059(T). All three genomes are comparable in size (4.6-4.9 Mb), with 4,119-4,459 protein-coding genes. Average nucleotide identity based on BLAST comparisons and digital genome-to-genome distance calculations are in good agreement with experimental DNA-DNA hybridization results. The genome sequences presented here will be very helpful in elucidating the taxonomy, phylogeny and evolution of the Pseudomonas putida species complex.

Phylogenomics and systematics in Pseudomonas.

The genus Pseudomonas currently contains 144 species, making it the genus of Gram-negative bacteria that contains the largest number of species. Currently, multilocus sequence analysis (MLSA) is the preferred method for establishing the phylogeny between species and genera. Four partial gene sequences of housekeeping genes (16S rRNA, gyrB, rpoB, and rpoD) were obtained from 112 complete or draft genomes of strains related to the genus Pseudomonas that were available in databases. These genes were analyzed together with the corresponding sequences of 133 Pseudomonas type strains of validly published species to assess their correct phylogenetic assignations. We confirmed that 30% of the sequenced genomes of non-type strains were not correctly assigned at the species level in the accepted taxonomy of the genus and that 20% of the strains were not identified at the species level. Most of these strains had been isolated and classified several years ago, and their taxonomic status has not been updated by modern techniques. MLSA was also compared with indices based on the analysis of whole-genome sequences that have been proposed for species delineation, such as tetranucleotide usage patterns (TETRA), average nucleotide identity (ANIm, based on MUMmer and ANIb, based on BLAST) and genome-to-genome distance (GGDC). TETRA was useful for discriminating Pseudomonas from other genera, whereas ANIb and GGDC clearly separated strains of different species. ANIb showed the strongest correlation with MLSA. The correct species classification is a prerequisite for most diversity and evolutionary studies. This work highlights the necessity for complete genomic sequences of type strains to build a phylogenomic taxonomy and that all new genome sequences submitted to databases should be correctly assigned to species to avoid taxonomic inconsistencies.

Draft Genome Sequence of Pseudomonas azotifigens Strain DSM 17556T (6H33bT), a Nitrogen Fixer Strain Isolated from a Compost Pile.

Pseudomonas azotifigens strain 6H33b(T) is a nitrogen fixer isolated from a hyperthermal compost pile in 2005 by Hatayama and collaborators. Here we report the draft genome, which has an estimated size of 5.0 Mb, exhibits an average G+C content of 66.73%, and is predicted to encode 4,536 protein-coding genes and 100 RNA genes.

Draft Genome Sequence of Pseudomonas stutzeri Strain B1SMN1, a Nitrogen-Fixing and Naphthalene-Degrading Strain Isolated from Wastewater.

Pseudomonas stutzeri strain B1SMN1 is a naphthalene-degrading and simultaneously nitrogen-fixing strain isolated from a wastewater sample taken at a lagooning treatment plant in Menorca (Balearic Islands, Spain). Here we report the draft genome sequence of P. stutzeri B1SMN1. It is composed of a chromosome of an estimated size of 5.2 Mb and two plasmids of 44,324 bp and 56,118 bp.

High metabolomic microdiversity within co-occurring isolates of the extremely halophilic bacterium Salinibacter ruber.

Salinibacter ruber is an extremely halophilic member of the Bacteroidetes that thrives in crystallizer ponds worldwide. Here, we have analyzed two sets of 22 and 35 co-occurring S. ruber strains, newly isolated respectively, from 100 microliters water samples from crystalizer ponds in Santa Pola and Mallorca, located in coastal and inland Mediterranean Spain and 350 km apart from each other. A set of old strains isolated from the same setting were included in the analysis. Genomic and taxonomy relatedness of the strains were analyzed by means of PFGE and MALDI-TOF, respectively, while their metabolomic potential was explored with high resolution ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT/MS). Overall our results show a phylogenetically very homogeneous species expressing a very diverse metabolomic pool. The combination of MALDI-TOF and PFGE provides, for the newly isolated strains, the same scenario presented by the previous studies of intra-specific diversity of S. ruber using a more restricted number of strains: the species seems to be very homogeneous at the ribosomal level while the genomic diversity encountered was rather high since no identical genome patterns could be retrieved from each of the samples. The high analytical mass resolution of ICR-FT/MS enabled the description of thousands of putative metabolites from which to date only few can be annotated in databases. Some metabolomic differences, mainly related to lipid metabolism and antibiotic-related compounds, provided enough specificity to delineate different clusters within the co-occurring strains. In addition, metabolomic differences were found between old and new strains isolated from the same ponds that could be related to extended exposure to laboratory conditions.

Draft Genome of Pseudomonas stutzeri Strain NF13, a Nitrogen Fixer Isolated from the Galapagos Rift Hydrothermal Vent.

Pseudomonas stutzeri strain NF13 was isolated from a water sample taken at a hydrothermal vent in the Galapagos rift. It was selected for its ability to metabolize sulfur compounds and to grow diazotrophically. Here, we report the first draft genome of a member of genomovar 19 of the species.

Complete genome sequence of the naphthalene-degrading bacterium Pseudomonas stutzeri AN10 (CCUG 29243).

Pseudomonas stutzeri AN10 (CCUG 29243) can be considered a model strain for aerobic naphthalene degradation. We report the complete genome sequence of this bacterium. Its 4.71-Mb chromosome provides insights into other biodegradative capabilities of strain AN10 (i.e., benzoate catabolism) and suggests a high number of horizontal gene transfer events.

Genome sequence of Pseudomonas stutzeri strain JM300 (DSM 10701), a soil isolate and model organism for natural transformation.

Pseudomonas stutzeri strain JM300 (DSM 10701) is a denitrifying soil isolate and a model organism for natural transformation in bacteria. Here we report the first complete genome sequence of JM300, the reference strain of genomovar 8 for the species.

Draft genome of Pseudomonas stutzeri strain ZoBell (CCUG 16156), a marine isolate and model organism for denitrification studies.

Pseudomonas stutzeri strain ZoBell, formerly a strain of Pseudomonas perfectomarina (CCUG 16156 = ATCC 14405), is a model organism for denitrification. It was isolated by ZoBell in 1944 from a marine sample, and here we report the first genome draft of a strain assigned to genomovar 2 of the species P. stutzeri.

Spatial and seasonal prokaryotic community dynamics in ponds of increasing salinity of Sfax solar saltern in Tunisia.

The spatial and seasonal dynamics of the halophilic prokaryotic community was investigated in five ponds from Sfax solar saltern (Tunisia), covering a salinity gradient ranging from 20 to 36%. Fluorescence in situ hybridization indicated that, above 24% salinity, the prokaryotic community shifted from bacterial to archaeal dominance with a remarkable increase in the proportion of detected cells. Denaturing gradient gel electrophoresis (DGGE) profiles were rather similar in all the samples analyzed, except in the lowest salinity pond (around 20% salt) where several specific archaeal and bacterial phylotypes were detected. In spite of previous studies on these salterns, DGGE analysis unveiled the presence of microorganisms not previously described in these ponds, such as Archaea related to Natronomonas or bacteria related to Alkalimnicola, as well as many new sequences of Bacteroidetes. Some phylotypes, such as those related to Haloquadratum or to some Bacteroidetes, displayed a strong dependence of salinity and/or magnesium concentrations, which in the case of Haloquadratum could be related to the presence of ecotypes. Seasonal variability in the prokaryotic community composition was focused on two ponds with the lowest (20%) and the highest salinity (36%). In contrast to the crystallized pond, where comparable profiles between autumn 2007 and summer 2008 were obtained, the non-crystallized pond showed pronounced seasonal changes and a sharp succession of "species" during the year. Canonical correspondence analysis of biological and physicochemical parameters indicated that temperature was a strong factor structuring the prokaryotic community in the non-crystallizer pond, that had salinities ranging from 20 to 23.8% during the year.

Bacterial diversity in dry modern freshwater stromatolites from Ruidera Pools Natural Park, Spain.

Ruidera Pools Natural Park, Spain, constitutes one of the most representative systems of carbonate precipitation in Europe. The prokaryotic community of a dry modern stromatolite recovered from the park has been analyzed by molecular techniques that included denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis, together with microscopic observations from the sample and cultures. Ribosomal RNA was directly extracted to study the putatively active part of the microbial community present in the sample. A total of 295 16S rRNA gene sequences were analyzed. Libraries were dominated by sequences related to Cyanobacteria, most frequently to the genus Leptolyngbya. A diverse and abundant assemblage of non-cyanobacterial sequences was also found, including members of Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Acidobacteria,Planctomycetes and Chloroflexi groups. No amplification was obtained when using archaeal primers. The results showed that at the time of sampling, when the pool was dry, the bacterial community of the stromatolites was dominated by groups of highly related Cyanobacteria, including new groups that had not been previously reported, although a high diversity outside this phylogenetic group was also found. The results indicated that part of the Cyanobacteria assemblage was metabolically active and could thus play a role in the mineralization processes inside the stromatolites.

Fine-scale evolution: genomic, phenotypic and ecological differentiation in two coexisting Salinibacter ruber strains.

Genomic and metagenomic data indicate a high degree of genomic variation within microbial populations, although the ecological and evolutive meaning of this microdiversity remains unknown. Microevolution analyses, including genomic and experimental approaches, are so far very scarce for non-pathogenic bacteria. In this study, we compare the genomes, metabolomes and selected ecological traits of the strains M8 and M31 of the hyperhalophilic bacterium Salinibacter ruber that contain ribosomal RNA (rRNA) gene and intergenic regions that are identical in sequence and were simultaneously isolated from a Mediterranean solar saltern. Comparative analyses indicate that S. ruber genomes present a mosaic structure with conserved and hypervariable regions (HVRs). The HVRs or genomic islands, are enriched in transposases, genes related to surface properties, strain-specific genes and highly divergent orthologous. However, the many indels outside the HVRs indicate that genome plasticity extends beyond them. Overall, 10% of the genes encoded in the M8 genome are absent from M31 and could stem from recent acquisitions. S. ruber genomes also harbor 34 genes located outside HVRs that are transcribed during standard growth and probably derive from lateral gene transfers with Archaea preceding the M8/M31 divergence. Metabolomic analyses, phage susceptibility and competition experiments indicate that these genomic differences cannot be considered neutral from an ecological perspective. The results point to the avoidance of competition by micro-niche adaptation and response to viral predation as putative major forces that drive microevolution within these Salinibacter strains. In addition, this work highlights the extent of bacterial functional diversity and environmental adaptation, beyond the resolution of the 16S rRNA and internal transcribed spacers regions.

Distribution, abundance and diversity of the extremely halophilic bacterium Salinibacter ruber.

Since its discovery in 1998, representatives of the extremely halophilic bacterium Salinibacter ruber have been found in many hypersaline environments across the world, including coastal and solar salterns and solar lakes. Here, we review the available information about the distribution, abundance and diversity of this member of the Bacteroidetes.

Prokaryotic diversity in Tuz Lake, a hypersaline environment in Inland Turkey.

Tuz Lake is an inland thalassohaline water body located in central Anatolia that contributes to 60% of the total salt production in Turkey per year. The microbiota inhabiting this lake has been studied by FISH, denaturing gradient gel electrophoresis of PCR-amplified fragments of 16S rRNA genes, and 16S rRNA gene clone library analysis. Total cell counts per milliliter (1.38 x 10(7)) were in the range of the values normally found for hypersaline environments. The proportion of Bacteria to Archaea in the community detectable by FISH was one to three. 16S rRNA gene clone libraries indicated that the archaeal assemblage was dominated by members of the Square Haloarchaea of the Walsby group, although some other groups were also found. Bacteria were dominated by members of the Bacteroidetes, including Salinibacter ruber-related phylotypes. Because members of Bacteroidetes are widely present in different hypersaline environments, a phylogenetic analysis of 16S rRNA gene sequences from Bacteroidetes retrieved from these environments was carried out in order to ascertain whether they formed a unique cluster. Sequences retrieved from Tuz Lake and a group of sequences from other hypersaline environments clustered together in a branch that could be considered as the 'halophilic branch' within the Bacteroidetes phylum.

Metabolic evidence for biogeographic isolation of the extremophilic bacterium Salinibacter ruber.

The biogeography of prokaryotes and the effect of geographical barriers as evolutionary constraints are currently subjected to great debate. Some clear-cut evidence for geographic isolation has been obtained by genetic methods but, in many cases, the markers used are too coarse to reveal subtle biogeographical trends. Contrary to eukaryotic microorganisms, phenotypic evidence for allopatric segregation in prokaryotes has never been found. Here we present, for the first time, a metabolomic approach based on ultrahigh resolution mass spectrometry to reveal phenotypic biogeographical discrimination. We demonstrate that strains of the cosmopolitan extremophilic bacterium Salinibacter ruber, isolated from different sites in the world, can be distinguished by means of characteristic metabolites, and that these differences can be correlated to their geographical isolation site distances. The approach allows distinct degrees of discrimination for isolates at different geographical scales. In all cases, the discriminative metabolite patterns were quantitative rather than qualitative, which may be an indication of geographically distinct transcriptional or posttranscriptional regulations.

Metagenomic approach to the study of halophages: the environmental halophage 1.

Hypersaline environments, such as crystallizer ponds of solar salterns, show one of the highest concentration of viruses reported for aquatic systems. All the halophages characterized so far are isolates obtained by cultivation from described haloarchaeal species that have only low abundance in the environment. We employed a culture-independent metagenomic approach to analyse for the first time complete genomes in the halophage community and explored the in situ diversity by transmission electron microscopy and pulsed-field gel electrophoresis. We report the genomic sequence of a not yet isolated halophage (named as environmental halophage 1 'EHP-1') whose DNA was obtained from crystallizer samples with a salinity of 31%. The sequenced genome has a size of 35 kb and a G + C content around 51%. The G + C content is lower than that of previously characterized halophages. However, G + C content and codon usage in EHP-1 are similar to the recently cultivated and sequenced Haloquadratum walsbyi, the major prokaryotic component in solar salterns around the world. Forty open reading frames have been predicted, including genes that putatively code for proteins involved in DNA replication (ribonucleotide reductases, thymidylate kinase) normally found in lytic viruses.

Phylogenetic position of Salinibacter ruber based on concatenated protein alignments.

A total of 22 genes from the genome of Salinibacter ruber strain M31 were selected in order to study the phylogenetic position of this species based on protein alignments. The selection of the genes was based on their essential function for the organism, dispersion within the genome, and sufficient informative length of the final alignment. For each gene, an individual phylogenetic analysis was performed and compared with the resulting tree based on the concatenation of the 22 genes, which rendered a single alignment of 10,757 homologous positions. In addition to the manually chosen genes, an automatically selected data set of 74 orthologous genes was used to reconstruct a tree based on 17,149 homologous positions. Although single genes supported different topologies, the tree topology of both concatenated data sets was shown to be identical to that previously observed based on small subunit (SSU) rRNA gene analysis, in which S. ruber was placed together with Bacteroidetes. In both concatenated data sets the bootstrap was very high, but an analysis with a gradually lower number of genes indicated that the bootstrap was greatly reduced with less than 12 genes. The results indicate that tree reconstructions based on concatenating large numbers of protein coding genes seem to produce tree topologies with similar resolution to that of the single 16S rRNA gene trees. For classification purposes, 16S rRNA gene analysis may remain as the most pragmatic approach to infer genealogic relationships.

Intraspecific comparative analysis of the species Salinibacter ruber.

Salinibacter ruber is the first extremely halophilic member of the Bacteria domain of proven environmental relevance in hypersaline brines at or approaching NaCl saturation, that has been brought to pure culture. A collection of 17 strains isolated from five different geographical locations (Mallorca, Alicante, Ebro Delta, Canary Islands, and Peruvian Andes) were studied following the currently accepted taxonomic approach. Additionally, random amplification of genomic DNA led to the phenetic analysis of the intraspecific diversity. Altogether the taxonomic study indicated that S. ruber remained highly homogeneous beyond any geographical barrier. However, genomic fingerprints indicated that populations from different isolation sites could still be discriminated.