Biogeography at the limits of life: Do extremophilic microbial communities show biogeographical regionalization?

Aim Biogeographical regions are the fundamental geographical units for grouping Earth's biodiversity. Biogeographical regionalization has been demonstrated for many higher taxa, such as terrestrial plants and vertebrates, but not in microbial communities. Therefore, we sought to test empirically whether microbial communities, or taxa, show patterns consistent with biogeographical regionalization. Location Within halite (NaCl) crystals from coastal solar salterns of western Europe, the Mediterranean and east Africa. Time period Modern (2006–2013). Major taxa studied Archaea. Methods Using high-throughput Illumina amplicon sequencing, we generated the most high-resolution characterization of halite-associated archaeal communities to date, using samples from 17 locations. We grouped communities into biogeographical clusters based on community turnover to test whether these communities show biogeographical regionalization. To examine whether individual taxa, rather than communities, show biogeographical patterns, we also tested whether the relative abundance of individual genera may be indicative of a community's biogeographical origins using machine learning methods, specifically random forest classification. Results We found that the rate of community turnover was greatest over subregional spatial scales (< 500 km), whereas at regional spatial scales the turnover was independent of geographical distance. Biogeographical clusters of communities were either not statistically robust or lacked spatial coherence, inconsistent with biogeographical regionalization. However, we identified several archaeal genera that were good indicators of biogeographical origin, providing classification error rates of < 10%. Main conclusions Overall, our results provide little support for the concept of biogeographical regions in these extremophilic microbial communities, despite the fact that some taxa do show biogeographical patterns. We suggest that variable dispersal ability among the halite-associated Archaea may disrupt biogeographical patterns at the community level, preventing the formation of biogeographical regions. This means that the processes that lead to the formation of biogeographical regions operate differentially on individual microbial taxa rather than on entire communities

Metabolism of halophilic archaea

In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature

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 (a w) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650-0.605 a w. 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 a w). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 a w 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 a w 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

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.This work was supported by the projects CLG2009-12651-C02-01 and 02; and CE-CSD2007-0005 of the Spanish Ministry of Science and Innovation, and all three projects were also co-financed with FEDER support from the European Union. JBE was financed by the Government of the Balearic Islands, Ministry of Economy and Finances

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'. © 2011 International Society for Microbial Ecology. All rights reserved.This work was supported by projects CGL2006-12714-CO2-01 and -02, and AYA2008-04013 of the Spanish Ministry of Science and Innovation (MICINN), which include funding from the European Union (‘FEDER’) and ACOM09/155 from the Generalitat Valenciana.Peer Reviewe