Josefa Antón

Extremely Halophilic Bacteria in Crystallizer Ponds from Solar Salterns

ABSTRACT It is generally assumed that hypersaline environments with sodium chloride concentrations close to saturation are dominated by halophilic members of the domain Archaea, while Bacteriaare not considered to be relevant in this kind of environment. Here, we report the high abundance and growth of a new group of hitherto-uncultured Bacteria in crystallizer ponds (salinity, from 30 to 37%) from multipond solar salterns. In the present study, these Bacteria constituted from 5 to 25% of the total prokaryotic community and were affiliated with theCytophaga-Flavobacterium-Bacteroides phylum. Growth was demonstrated in saturated NaCl. A provisional classification of this new bacterial group as “Candidatus Salinibacter gen. nov.” is proposed. The perception that Archaea are the only ecologically relevant prokaryotes in hypersaline aquatic environments should be revised.

Archaeal Biodiversity in Crystallizer Ponds from a Solar Saltern: Culture versus PCR

The culturable haloarchaeal diversity in a crystallizer pond from a solar saltern has been analyzed and compared with the biodiversity directly retrieved by analysis of rRNA genes amplified from the environment. Two different sets of culture conditions have been assayed: solid medium with yeast extract as carbon source and liquid media with either yeast extract or a mixture of fishmeal, Spirulina sp., and Artemia salina. Seventeen colonies grown on plates with yeast extract incubated at 30°C were analyzed by 16S rDNA partial sequencing. Sixteen were closely related to haloarchaea of the genus Halorubrum; 13 of them to Halorubrum coriense, a haloarchaeon isolated from a solar saltern pond in Australia, which had not been previously isolated from the pond analyzed in this study; and one to Haloarcula marismortui. Liquid cultures were analyzed by ribosomal internal spacer analysis (RISA) and partial sequencing of the 16SrRNA genes. A total of 18 sequences were analyzed, 15 corresponding to RISA bands obtained from cultures, and 3 from the environmental sample used as inoculum. Thirteen sequences obtained from cultures were related to several Halorubrum species, and 2 to Haloarcula. One of the clones obtained directly from the environmental sample was distantly related to a Natronobacterium, whereas two were related to SPhT, the phylotype most frequently retrieved from this environment by culture independent techniques. Our results show an extremely low diversity for the haloarchaea retrieved by cultivation even when modifications to the standard technique are introduced.

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.

Prokaryotic Diversity in Zostera noltii-Colonized Marine Sediments

ABSTRACT The diversity of microorganisms present in a sediment colonized by the phanerogam Zostera noltii has been analyzed. Microbial DNA was extracted and used for constructing two 16S rDNA clone libraries for Bacteria and Archaea. Bacterial diversity was very high in these samples, since 57 different sequences were found among the 60 clones analyzed. Eight major lineages of the Domain Bacteria were represented in the library. The most frequently retrieved bacterial group (36% of the clones) was δ-Proteobacteria related to sulfate-reducing bacteria. The second most abundant group (27%) was γ-Proteobacteria, including five clones closely related to S-oxidizing endosymbionts. The archaeal clone library included members of Crenarchaeota and Euryarchaeota, with nine different sequences among the 15 analyzed clones, indicating less diversity when compared to the Bacteria organisms. None of these sequences was closely related to culturedArchaea organisms.

Is there a common water-activity limit for the three domains of life?

Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (aw) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650–0.605 aw. Here, we present evidence that halophilic prokayotes can grow down to water activities of <0.755 for Halanaerobium lacusrosei (0.748), Halobacterium strain 004.1 (0.728), Halobacterium sp. NRC-1 and Halococcus morrhuae (0.717), Haloquadratum walsbyi (0.709), Halococcus salifodinae (0.693), Halobacterium noricense (0.687), Natrinema pallidum (0.681) and haloarchaeal strains GN-2 and GN-5 (0.635 aw). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 aw for extreme, obligately halophilic Archaea and Bacteria. These were compared with minima for the most solute-tolerant Bacteria in high-sugar (or other non-saline) media (Mycobacterium spp., Tetragenococcus halophilus, Saccharibacter floricola, Staphylococcus aureus and so on) and eukaryotic microbes in saline (Wallemia spp., Basipetospora halophila, Dunaliella spp. and so on) and high-sugar substrates (for example, Xeromyces bisporus, Zygosaccharomyces rouxii, Aspergillus and Eurotium spp.). We also manipulated the balance of chaotropic and kosmotropic stressors for the extreme, xerophilic fungi Aspergillus penicilloides and X. bisporus and, via this approach, their established water-activity limits for mycelial growth (∼0.65) were reduced to 0.640. Furthermore, extrapolations indicated theoretical limits of 0.632 and 0.636 aw for A. penicilloides and X. bisporus, respectively. Collectively, these findings suggest that there is a common water-activity limit that is determined by physicochemical constraints for the three domains of life.

Reconstructing Viral Genomes from the Environment Using Fosmid Clones: The Case of Haloviruses

Background Metaviriomes, the viral genomes present in an environment, have been studied by direct sequencing of the viral DNA or by cloning in small insert libraries. The short reads generated by both approaches make it very difficult to assemble and annotate such flexible genomic entities. Many environmental viruses belong to unknown groups or prey on uncultured and little known cellular lineages, and hence might not be present in databases. Methodology and Principal Findings Here we have used a different approach, the cloning of viral DNA into fosmids before sequencing, to obtain natural contigs that are close to the size of a viral genome. We have studied a relatively low diversity extreme environment: saturated NaCl brines, which simplifies the analysis and interpretation of the data. Forty-two different viral genomes were retrieved, and some of these were almost complete, and could be tentatively identified as head-tail phages (Caudovirales). Conclusions and Significance We found a cluster of phage genomes that most likely infect Haloquadratum walsbyi, the square archaeon and major component of the community in these hypersaline habitats. The identity of the prey could be confirmed by the presence of CRISPR spacer sequences shared by the virus and one of the available strain genomes. Other viral clusters detected appeared to prey on the Nanohaloarchaea and on the bacterium Salinibacter ruber, covering most of the diversity of microbes found in this type of environment. This approach appears then as a viable alternative to describe metaviriomes in a much more detailed and reliable way than by the more common approaches based on direct sequencing. An example of transfer of a CRISPR cluster including repeats and spacers was accidentally found supporting the dynamic nature and frequent transfer of this peculiar prokaryotic mechanism of cell protection.

The metavirome of a hypersaline environment

Hypersaline environments harbour the highest number of virus-like particles reported for planktonic systems. However, very little is known about the genomic diversity of these virus assemblages since most of the knowledge on halophages is based on the analysis of a few isolates infecting strains of hyperhalophilic Archaea that may not be representatives of the natural microbiota. Here, we report the characterization, through a metagenomic approach, of the viral assemblage inhabiting a crystallizer pond (CR30) from a multi-pond solar saltern in Santa Pola (SE Spain). A total of 1.35 Mbp were cloned that yielded a total of 620 kb sequenced viral DNA. The metavirome was highly diverse and different from virus communities of marine and other aquatic environments although it showed some similarities with metaviromes from high-salt ponds in solar salterns in San Diego (SW USA), indicating some common traits between high-salt viromes. A high degree of diversity was found in the halophages as revealed by the presence of 2479 polymorphic nucleotides. Dinucleotide frequency analysis of the CR30 metavirome showed a good correlation with GC content and enabled the establishment of different groups, and even the assignment of their putative hosts: the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber.

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.

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.

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.