Characterization of ecologically diverse viruses infecting co-occurring strains of cosmopolitan hyperhalophilic Bacteroidetes.

Hypersaline environments close to saturation harbor the highest density of virus-like particles reported for aquatic systems as well as low microbial diversity. Thus, they offer unique settings for studying virus-host interactions in nature. However, no viruses have been isolated so far infecting the two most abundant inhabitants of these systems (that is, the euryarchaeon Haloquadratum walsbyi and the bacteroidetes Salinibacter ruber). Here, using three different co-occurring strains, we have isolated eight viruses infecting the ubiquitous S. ruber that constitute three new different genera (named as 'Holosalinivirus', 'Kryptosalinivirus' and 'Kairosalinivirus') according to their genomic traits, different host range, virus-host interaction capabilities and abundances in natural systems worldwide. Furthermore, to get a more complete and comprehensive view of S. ruber virus assemblages in nature, a microcosm experiment was set with a mixture of S. ruber strains challenged with a brine virus concentrate, and changes of viral populations were monitored by viral metagenomics. Only viruses closely related to kairosalinivirus (strictly lytic and wide host range) were enriched, despite their low initial abundance in the natural sample. Metagenomic analyses of the mesocosms allowed the complete recovery of kairosalinivirus genomes using an ad hoc assembly strategy as common viral metagenomic assembly tools failed despite their abundance, which underlines the limitations of current approaches. The increase of this type of viruses was accompanied by an increase in the diversity of the group, as shown by contig recruitment. These results are consistent with a scenario in which host range, not only virus and host abundances, is a key factor in determining virus fate in nature.

Retinal-binding proteins mirror prokaryotic dynamics in multipond solar salterns.

Microbial opsin (i.e. retinal-binding protein) dynamics has been studied along a salinity gradient in Santa Pola solar salterns (Alicante, Spain) by using culture-independent approaches and statistical analyses. Five ponds of salinities ranging from 18% to above 40% were sampled nine times along a year. Forty-three opsin-like sequences were retrieved by denaturing gradient gel electrophoresis and clustered into 18 different phylogroups, indicating that their diversity was higher than expected according to previous data. Moreover, the statistical correlation between environmental factors controlling microbial community structure and dynamics of environmental rhodopsin proteins indicated almost identical temporal fluctuations between the opsin-related sequences and their corresponding putative 'producers' in nature. Although most sequences were related to others previously detected in hypersaline environments, some pond-specific opsins putatively belonged to previously uncharacterized hosts. Furthermore, we propose that subtle changes in the bacteriorhodopsin 'retinal proton binding pocket', which is key in the photocycle function, could be the molecular basis behind a fine 'photocycle-tuning' mechanism to avoid inter/intraspecies light-competition in hypersaline environments.

From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists.

The microbiota of multi-pond solar salterns around the world has been analyzed using a variety of culture-dependent and molecular techniques. However, studies addressing the dynamic nature of these systems are very scarce. Here we have characterized the temporal variation during 1 year of the microbiota of five ponds with increasing salinity (from 18% to >40%), by means of CARD-FISH and DGGE. Microbial community structure was statistically correlated with several environmental parameters, including ionic composition and meteorological factors, indicating that the microbial community was dynamic as specific phylotypes appeared only at certain times of the year. In addition to total salinity, microbial composition was strongly influenced by temperature and specific ionic composition. Remarkably, DGGE analyses unveiled the presence of most phylotypes previously detected in hypersaline systems using metagenomics and other molecular techniques, such as the very abundant Haloquadratum and Salinibacter representatives or the recently described low GC Actinobacteria and Nanohaloarchaeota. In addition, an uncultured group of Bacteroidetes was present along the whole range of salinity. Database searches indicated a previously unrecognized widespread distribution of this phylotype. Single-cell genome analysis of five members of this group suggested a set of metabolic characteristics that could provide competitive advantages in hypersaline environments, such as polymer degradation capabilities, the presence of retinal-binding light-activated proton pumps and arsenate reduction potential. In addition, the fairly high metagenomic fragment recruitment obtained for these single cells in both the intermediate and hypersaline ponds further confirm the DGGE data and point to the generalist lifestyle of this new Bacteroidetes group.

Culture-independent approaches for studying viruses from hypersaline environments.

Hypersaline close-to-saturation environments harbor an extremely high concentration of virus-like particles, but the number of haloviruses isolated so far is still very low. Haloviruses can be directly studied from natural samples by using different culture-independent techniques that include transmission electron microscopy, pulsed-field gel electrophoresis, and different metagenomic approaches. Here, we review the findings of these studies, with a main focus on the metagenomic approaches. The analysis of bulk viral nucleic acids directly retrieved from the environment allows estimations of viral diversity, activity, and dynamics and tentative host assignment. Results point to a diverse and active viral community in constant interplay with its hosts and to a "hypersalineness" quality common to viral assemblages present in hypersaline environments that are thousands of kilometers away from each other.

Metatranscriptomic analysis of extremely halophilic viral communities.

Hypersaline environments harbour the highest number of viruses reported for aquatic environments. In crystallizer ponds from solar salterns, haloviruses coexist with extremely halophilic Archaea and Bacteria and present a high diversity although little is known about their activity. In this work, we analyzed the viral expression in one crystallizer using a metatranscriptomic approach in which clones from a metaviromic library were immobilized in a microarray and used as probes against total mRNA extracted from the hypersaline community. This approach has two advantages: (i) it overcomes the fact that there is no straightforward, unambiguous way to extract viral mRNA from bulk mRNAs and (ii) it makes the sequencing of all mRNAs unnecessary. Transcriptomic data indicated that the halovirus assemblage was highly active at the time of sampling and the viral groups with the highest expression levels were those related to high GC content haloarchaea and Salinibacter representatives, which are minor components in the environment. Moreover, the changes in the viral expression pattern and in the numbers of free viral particles were analyzed after submitting the samples to two stress conditions: ultraviolet-radiation and dilution. Results showed that Archaea were more sensitive than Bacteria to these stress conditions. The overexpression in the predicted archaeal virus fraction raised and the total numbers of free viruses increased. Furthermore, we identified some very closely related viral clones, displaying single-nucleotide polymorphisms, which were expressed only under certain conditions. These clones could be part of very closely related virus genomes for which we propose the term 'ecoviriotypes'.

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.

Influence of proline on the thermostability of the active site and membrane arrangement of transmembrane proteins.

Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro50, Pro91, and Pro186 in the native membrane environment. This information has been complemented with the analysis of the respective crystallographic structures by the FoldX force field. Differential scanning calorimetry allowed us to determine distorted membrane arrangement for P50A and P186A. The protein stability was severely affected for P186A and P91A. In the case of Pro91, a single point mutation is capable of strongly slowing down the conformational diffusion along the denaturation coordinate, becoming a barrier-free downhill process above 371 K. Temperature derivative spectroscopy, applied for first time to study thermal stability of proteins, has been used to monitor the stability of the active site of bacteriorhodopsin. The mutation of Pro91 and Pro186 showed the most striking effects on the retinal binding pocket. These residues are the Pro in closer contact to the active site (activation energies for retinal release of 60.1 and 76.8 kcal/mol, respectively, compared to 115.8 kcal/mol for WT). FoldX analysis of the protein crystal structures indicates that the Pro-to-Ala mutations have both local and long-range effects on the structural stability of residues involved in the architecture of the protein and the active site and in the proton pumping function. Thus, this study provides a complete overview of the substitution effect of helix-embedded prolines in the thermodynamic and dynamic stability of a membrane protein, also related to its structure and function.

Purification and biological characterization of halocin H1 from Haloferax mediterranei M2a.

The production of halocins, bacteriocin-like proteins of ecological significance, is a frequent characteristic of species from the family Halobacteriaceae. Halocin H1, produced by Haloferax mediterranei strain M2a, is a single 31-kDa polypeptide. Its purification was achieved by combining two chromatographic systems: Sepharose 4B linked to bacitracin followed by hydroxylapatite Bio-gel HTP. Halocin H1 required concentrations of NaCl higher than 1.5 M to maintain its activity. Haoarchaeal strains showed a differential degree of sensitivity to the action of this halocin.

Purification and biological characterization of halocin H1 from Haloferax mediterranei M2a

The production of halocins, bacteriocin-like proteins of ecological significance, is a frequent characteristic of species from the family Halobacteriaceae. Halocin H1, produced by Haloferax mediterranei strain M2a, is a single 31-kDa polypeptide. Its purification was achieved by combining two chromatographic systems: Sepharose 4B linked to bacitracin followed by hydroxylapatite Bio-gel HTP. Halocin H1 required concentrations of NaCl higher than 1.5 M to maintain its activity. Haoarchaeal strains showed a differential degree of sensitivity to the action of this halocin (AU)

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