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In this study, fluorescence in situ hybridization (FISH) and PCR-amplified fragments of the 16SrDNA gene were used to determine prokaryotes diversity in Urmia Salt Lake. Prokaryote cell population in Urmia lake range from 3.1 ± 0.3 × 106, 2 ± 0.2 × 108, 4 ± 0.3 × 108, and 1.8 ± 0.2 × 108 cells ml−1 for water, soil, sediment, and salt samples by DAPI (4, 6-diamidino-2-phenylindole) direct count, respectively. The proportion of bacteria and archaea in the samples determinable by FISH ranged between 36.1 and 55% and 48.5 and 55.5%, respectively. According to the DGGE method, some bands were selected and separated from the gel, then amplified and sequenced. The results of sequences were related to two phyla Proteobacteria (16.6%) and Bacteroidetes (83.3%), which belonged to four genera Salinibacter, Mangroviflexus, Pseudomonas, and Cesiribacter, and the archaeal sequences were related to Euryarchaeota phyla and three genera Halonotius, Haloquadratum, and Halorubrum. According to our results, it seems that prokaryotic populations in this hypersaline environment are more diverse than expected, and bacteria are so abundant and diverse and form the metabolically active part of the microbial population inhabiting this extreme environment. Molecular dependent and independent approaches revealed a different aspect of this environment microbiota.

WOS:000703487400011 Tuz Lake is the second biggest lake with an extreme hypersaline environment (over 32% (w/v) salt) in Turkey. It is reported that the lake was of marine origin, and its formation began with tensional movements during the Late Cretaceous and the Late Eocene. We conducted this research to determine the prokaryotic diversity of Tuz Lake by metagenomics approach, which is 16S rRNA amplicon sequencing of microbial communities in their natural environments. Hence, next-generation sequencing and then bioinformatic analysis was used to investigate the microbial structure of halophilic Bacteria and Archaea in Tuz Lake. Metagenomic analysis of reads revealed that all the archaeal populations in Tuz Lake belonged to the Euryarchaeota and Nanoarchaeaeota phyla. Haloquadratum (34%), Haloparvum (31%), Halonotius (7%), Halorubrum (3%), Halapricum (2%), Halobellus (3%), Natronomonas (1%), Halococcus (1%), and Halobacterium (1%) were found as the dominant archaeal genera. The most abundant and diverse bacterial phyla were Bacteroidetes and Proteobacteria. The remaining bacterial taxa were assigned to Acetothermia, Cyanobacteria, Firmicutes, and Halanaerobiaeota. Salinibacter (3%), uncultured microorganisms (1%), Pseudomonas (

The microbiota of hypersaline environments close to saturation is generally dominated by extremely halophilic Archaea and therefore these systems harbor a high proportion of archaeal viruses that can be characterized using metagenomics. This approach has provided information regarding haloarchaeal viruses that were not previously isolated, such as the ones infecting the square euryarchaeon Haloquadratum sp. or the recently described Nanohalaorchaea.

Prokaryotic communities and physico-chemical characteristics of 30 brine samples from the thalassohaline Tuz Lake (Salt Lake), Deep Zone, Kayacik, Kaldirim, and Yavsan salterns (Turkey) were analyzed using 16S rRNA amplicon sequencing and standard methods, respectively. Archaea (98.41% of reads) was found to dominate in these habitats in contrast to the domain Bacteria (1.38% of reads). Representatives of the phylum Euryarchaeota were detected as the most predominant, while 59.48% and 1.32% of reads, respectively, were assigned to 18 archaeal genera, 19 bacterial genera, 10 archaeal genera, and one bacterial genus that were determined to be present, with more than 1% sequences in the samples. They were the archaeal genera Haloquadratum, Haloarcula, Halorhabdus, Natronomonas, Halosimplex, Halomicrobium, Halorubrum, Halonotius, Halolamina, Halobacterium, and Salinibacter within the domain Bacteria. The genera Haloquadratum and Halorhabdus were found in all sampling sites. While Haloquadratum, Haloarcula, and Halorhabdus were the most abundant genera, two uncultured Tuz Lake Halobacteria (TLHs) 1 and 2 were detected in high abundance, and an additional uncultured haloarchaeal TLH-3 was found as a minor abundant uncultured taxon. Their future isolation in pure culture would permit us to expand our knowledge on hypersaline thalassohaline habitats, as well as their ecological role and biomedical and biotechnological potential applications.

Extracellular DNA (eDNA) comprises all the DNA molecules outside cells. This component of microbial ecosystems may serve as a source of nutrients and genetic information. Hypersaline environments harbour one of the highest concentrations of eDNA reported for natural systems, which has been attributed to the physicochemical preservative effect of salts and to high viral abundance. Here, we compared centrifugation and filtration protocols for the extraction of dissolved DNA (dDNA, as opposed to eDNA that also includes DNA from free viral particles) from a solar saltern crystallizer pond (CR30) water sample. The crystallizer dDNA fraction has been characterized, for the first time, and compared with cellular and viral metagenomes from the same location. High‐speed centrifugation affected CR30 dDNA concentration and composition due to cell lysis, highlighting that protocol optimization should be the first step in dDNA studies. Crystallizer dDNA, which accounted for lower concentrations than those previously reported for hypersaline anoxic sediments, had a mixed viral and cellular origin, was enriched in archaeal DNA and had a distinctive taxonomic composition compared to that from the cellular assemblage of the same sample. Bioinformatic analyses indicated that nanohaloarchaeal viruses could be a cause for these differences. This research was supported by the Spanish Ministry of Science, Innovation and Universities grant MICROMATES (PGC2018-096956-B-C44), which was also supported with European Regional Development Fund (FEDER) funds, and by the Generalitat Valenciana grant PROMETEO/2017/129. B.A.-R. is a ACIF (Generalitat Valenciana) fellow.

The Cabo Rojo solar salterns are a hypersaline environment located in a tropical climate, where conditions remain stable throughout the year. These conditions can favor the establishment of steady microbial communities. Little is known about the microbial composition that thrives in hypersaline environments in the tropics. The main goal of this study was to assess the microbial diversity present in the crystallizer ponds of Cabo Rojo, in terms of structure and metabolic processes across time using metagenomic techniques. Three samplings (December 2014, March and July 2016) were carried out, where water samples (50 L each) were filtered through a Millipore pressurized filtering system. DNA was subsequently extracted using physical&ndash, chemical methods and sequenced using paired end Illumina technologies. The sequencing effort produced three paired end libraries with a total of 111,816,040 reads, that were subsequently assembled into three metagenomes. Out of the phyla detected, the microbial diversity was dominated in all three samples by Euryarchaeota, followed by Bacteroidetes and Proteobacteria. However, sample MFF1 (for Muestreo Final Fraternidad) exhibited a higher diversity, with 12 prokaryotic phyla detected at 34% NaCl (w/v), when compared to samples MFF2 and MFF3, which only exhibited three phyla. Precipitation events might be one of the contributing factors to the change in the microbial community composition through time. Diversity at genus level revealed a more stable community structure, with an overwhelming dominance of the square archaeon Haloquadratum in the three metagenomes. Furthermore, functional annotation was carried out in order to detect genes related to metabolic processes, such as carbon, nitrogen, and sulfur cycles. The presence of gene sequences related to nitrogen fixation, ammonia oxidation, sulfate reduction, sulfur oxidation, and phosphate solubilization were detected. Through binning methods, four putative novel genomes were obtained, including a possible novel genus belonging to the Bacteroidetes and possible new species for the genera Natronomonas, Halomicrobium, and Haloquadratum. Using a metagenomic approach, a 3-year study has been performed in a Caribbean hypersaline environment. When compared to other salterns around the world, the Cabo Rojo salterns harbor a similar community composition, which is stable through time. Moreover, an analysis of gene composition highlights the importance of the microbial community in the biogeochemical cycles at hypersaline environments.

Microorganisms inhabiting saline environments are an interesting ecological model for the study of the adaptation of organisms to extreme living conditions and constitute a precious resource of enzymes and bioproducts for biotechnological applications. We analyzed the microbial communities in nine ponds with increasing salt concentrations (salinity range 4.9&ndash, 36.0%) of the Saltern of Margherita di Savoia (Italy), the largest thalassohaline saltern in Europe. A deep-metabarcoding NGS procedure addressing separately the V5-V6 and V3-V4 hypervariable regions of the 16S rRNA gene of Bacteria and Archaea, respectively, and a CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) analysis allowed us to profile the dynamics of microbial populations at the different salt concentrations. Both the domains were detected throughout the saltern, even if the low relative abundance of Archaea in the three ponds with the lowest salinities prevented the construction of the relative amplicon libraries. The highest cell counts were recorded at 14.5% salinity for Bacteria and at 24.1% salinity for Archaea. While Bacteria showed the greatest number of genera in the first ponds (salinity range 4.9&ndash, 14.5%), archaeal genera were more numerous in the last ponds of the saltern (salinity 24.1&ndash, 36.0%). Among prokaryotes, Salinibacter was the genus with the maximum abundance (~49% at 34.6% salinity). Other genera detected at high abundance were the archaeal Haloquadratum (~43% at 36.0% salinity) and Natronomonas (~18% at 13.1% salinity) and the bacterial &ldquo, Candidatus Aquiluna&rdquo, (~19% at 14.5% salinity). Interestingly, &ldquo, had not been identified before in thalassohaline waters.

While the dynamics of microbial community assembly driven by environmental perturbations have been extensively studied, our understanding is far from complete, particularly for light-induced perturbations. Extremely halophilic communities thriving in coastal solar salterns are mainly influenced by two environmental factors—salt concentrations and high sunlight irradiation. By experimentally manipulating light intensity through the application of shading, we showed that light acts as a deterministic factor that ultimately drives the establishment of recurrent microbial communities under near-saturation salt concentrations. In particular, the stable and highly change-resistant communities that established under high-light intensities were dominated (>90% of metagenomic reads) by Haloquadratum spp. and Salinibacter spp. On the other hand, under 37-fold lower light intensity, different, less stable and change-resistant communities were established, mainly dominated by yet unclassified haloarchaea and relatively diverse photosynthetic microorganisms. These communities harboured, in general, much lower carotenoid pigment content than their high-irradiation counterparts. Both assemblage types appeared to be highly resilient, re-establishing when favourable conditions returned after perturbation (i.e. high-irradiation for the former communities and low-irradiation for the latter ones). Overall, our results revealed that stochastic processes were of limited significance to explain these patterns., This study was funded by the Spanish Ministry of Economy projects CGL2012‐39627‐C03‐03 CLG2015_66686‐C3‐1‐P and PGC2018‐096956‐B‐C41 (to R.R.M.), CGL2015_66686‐C3‐3‐P (to J.A.) and CGL2015_66686‐C3‐2‐P (to J.E.G.P.), which were also supported with European Regional Development Fund (FEDER) funds. R.A. was funded by the Max Planck Society. KTK's research was supported, in part, by the U.S. National Science Foundation (Award No. 1831582). T.V.P. received a pre‐doctoral fellowship (No. BES‐2013‐064420) from the Spanish Government Ministry for Finance and Competition. R.R.M. acknowledges the financial support of the sabbatical stay at Georgia Tech supported by the Grant PRX18/00048 of the Ministry of Sciences, Innovation and Universities.

Plasmids PL6A and PL6B are both carried by the C23(T) strain of the square archaeon Haloquadratum walsbyi, and are closely related (76% nucleotide identity), circular, about 6 kb in size, and display the same gene synteny. They are unrelated to other known plasmids and all of the predicted proteins are cryptic in function. Here we describe two additional PL6-related plasmids, pBAJ9-6 and pLT53-7, each carried by distinct isolates of Haloquadratum walsbyi that were recovered from hypersaline waters in Australia. A third PL6-like plasmid, pLTMV-6, was assembled from metavirome data from Lake Tyrell, a salt-lake in Victoria, Australia. Comparison of all five plasmids revealed a distinct plasmid family with strong conservation of gene content and synteny, an average size of 6.2 kb (range 5.8-7.0 kb) and pairwise similarities between 61-79%. One protein (F3) was closely similar to a protein carried by betapleolipoviruses while another (R6) was similar to a predicted AAA-ATPase of His 1 halovirus (His1V_gp16). Plasmid pLT53-7 carried a gene for a FkbM family methyltransferase that was not present in any of the other plasmids. Comparative analysis of all PL6-like plasmids provided better resolution of conserved sequences and coding regions, confirmed the strong link to haloviruses, and showed that their sequences are highly conserved among examples from Haloquadratum isolates and metagenomic data that collectively cover geographically distant locations, indicating that these genetic elements are widespread.

We examined the presence of bacteriorhodopsin and other retinal protein pigments in the microbial community of the saltern crystallizer ponds in Eilat, Israel, and assessed the effect of the retinal-based proton pumps on the metabolic activity. The biota of the hypersaline (~309 g salts l(-1)) brine consisted of ~2200 β-carotene-rich Dunaliella cells and ~3.5 × 10(7) prokaryotes ml(-1), most of which were flat, square or rectangular Haloquadratum-like archaea. No indications were obtained for massive presence of Salinibacter. We estimated a concentration of bacteriorhodopsin and bacteriorhodopsin-like pigments of 3.6 nmol l(-1). When illuminated, the community respiration activity of the brine samples in which oxygenic photosynthesis was inhibited by 3-(3-4-dichlorophenyl)-1,1-dimethylurea, decreased by 40-43 %. This effect was interpreted to be the result of competition between two energy yielding systems: the bacteriorhodopsin proton pump and the respiratory chain. The results presented have important implications for the interpretation of many published data on photosynthetic and respiratory activities in hypersaline environments.

Some of the most commonly occurring but difficult to isolate halophilic prokaryotes, Archaea as well as Bacteria, require or prefer pyruvate as carbon and energy source. The most efficient media for the enumeration and isolation of heterotrophic prokaryotes from natural environments, from freshwater to hypersaline, including the widely used R2A agar medium, contain pyruvate as a key ingredient. Examples of pyruvate-loving halophiles are the square, extremely halophilic archaeon Haloquadratum walsbyi and the halophilic gammaproteobacterium Spiribacter salinus. However, surprisingly little is known about the availability of pyruvate in natural environments and about the way it enters the cell. Some halophilic Archaea (Halorubrum saccharovorum, Haloarcula spp.) partially convert sugars and glycerol to pyruvate and other acids (acetate, lactate) which are excreted to the medium. Pyruvate formation from glycerol was also shown during a bloom of halophilic Archaea in the Dead Sea. However, no pyruvate transporters were yet identified in the genomes of halophilic Archaea, and altogether, our understanding of pyruvate transport in the prokaryote world is very limited. Therefore, the preference for pyruvate by fastidious and often elusive halophiles and the empirically proven enhanced colony recovery on agar media containing pyruvate are still poorly understood.

Recent studies based on metagenomics and other molecular techniques have permitted a detailed knowledge of the microbial diversity and metabolic activities of microorganisms in hypersaline environments. The current accepted model of community structure in hypersaline environments is that the square archaeon Haloquadratum waslbyi, the bacteroidete Salinibacter ruber and nanohaloarchaea are predominant members at higher salt concentrations, while more diverse archaeal and bacterial taxa are observed in habitats with intermediate salinities. Additionally, metagenomic studies may provide insight into the isolation and characterization of the principal microbes in these habitats, such as the recently described gammaproteobacterium Spiribacter salinus.

Hypersaline environments encompass aquatic and terrestrial habitats. While only a limited number of studies on the microbial diversity of saline soils have been carried out, hypersaline lakes and marine salterns have been thoroughly investigated, resulting in an aquatic-biased knowledge about life in hypersaline environments. To improve our understanding of the assemblage of microbes thriving in saline soils, we assessed the phylogenetic diversity and metabolic potential of the prokaryotic community of two hypersaline soils (with electrical conductivities of ~24 and 55 dS/m) from the Odiel saltmarshes (Spain) by metagenomics. Comparative analysis of these soil databases with available datasets from salterns ponds allowed further identification of unique and shared traits of microbial communities dwelling in these habitats. Saline soils harbored a more diverse prokaryotic community and, in contrast to their aquatic counterparts, contained sequences related to both known halophiles and groups without known halophilic or halotolerant representatives, which reflects the physical heterogeneity of the soil matrix. Our results suggest that Haloquadratum and certain Balneolaeota members may preferentially thrive in aquatic or terrestrial habitats, respectively, while haloarchaea, nanohaloarchaea and Salinibacter may be similarly adapted to both environments. We reconstructed 4 draft genomes related to Bacteroidetes, Balneolaeota and Halobacteria and appraised their metabolism, osmoadaptation strategies and ecology. This study greatly improves the current understanding of saline soils microbiota. España, GL2013-46941-P and CGL2017-83385-P

Hypersaline environments represent some of the most challenging settings for life on Earth. Extremely halophilic microorganisms have been selected to colonize and thrive in these extreme environments by virtue of a broad spectrum of adaptations to counter high salinity and osmotic stress. Although there is substantial data on microbial taxonomic diversity in these challenging ecosystems and their primary osmoadaptation mechanisms, less is known about how hypersaline environments shape the genomes of microbial inhabitants at the functional level. In this study, we analyzed the microbial communities in five ponds along the discontinuous salinity gradient from brackish to salt-saturated environments and sequenced the metagenome of the salt (halite) precipitation pond in the artisanal Cáhuil Solar Saltern system. We combined field measurements with spectrophotometric pigment analysis and flow cytometry to characterize the microbial ecology of the pond ecosystems, including primary producers and applied metagenomic sequencing for analysis of archaeal and bacterial taxonomic diversity of the salt crystallizer harvest pond. Comparative metagenomic analysis of the Cáhuil salt crystallizer pond against microbial communities from other salt-saturated aquatic environments revealed a dominance of the archaeal genus Halorubrum and showed an unexpectedly low abundance of Haloquadratum in the Cáhuil system. Functional comparison of 26 hypersaline microbial metagenomes revealed a high proportion of sequences associated with nucleotide excision repair, helicases, replication and restriction-methylation systems in all of them. Moreover, we found distinctive functional signatures between the microbial communities from salt-saturated (>30% [w/v] total salinity) compared to sub-saturated hypersaline environments mainly due to a higher representation of sequences related to replication, recombination and DNA repair in the former. The current study expands our understanding of the diversity and distribution of halophilic microbial populations inhabiting salt-saturated habitats and the functional attributes that sustain them., Frontiers in Microbiology, 9, ISSN:1664-302X

We report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC 3.2.1.20) from the archaeon Haloquadratum walsbyi. A 2349 bp region (Hqrw_2071) from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+), expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6.0. Enzyme activity values were the highest in the presence of 3 M NaCl or 3–4 M KCl. However, specific activity values were two-fold higher in the presence of 3–4 M KCl when compared to NaCl suggesting a cytoplasmic localization. Phylogenetic analyses, with respect to other alpha-glucosidases from members of the class Halobacteria, showed that the Hqr. walsbyi MalH was most similar (up to 41%) to alpha-glucosidases and alpha-xylosidases of Halorubrum. Moreover, computational analyses for the detection of functional domains, active and catalytic sites, as well as 3D structural predictions revealed a close relationship with an E. coli YicI-like alpha-xylosidase of the GH31 family. However, the purified enzyme did not show alpha-xylosidase activity. This narrower substrate range indicates a discrepancy with annotations from different databases and the possibility of specific substrate adaptations of halophilic glucosidases due to high salinity. To our knowledge, this is the first report on the characterization of an alpha-glucosidase from the halophilic Archaea, which could serve as a new model to gain insights into carbon metabolism in this understudied microbial group.

Microbial community succession was examined over a two-year period using spatially and temporally coordinated water chemistry measurements, metagenomic sequencing, phylogenetic binning and de novo metagenomic assembly in the extreme hypersaline habitat of Lake Tyrrell, Victoria, Australia. Relative abundances of Haloquadratum-related sequences were positively correlated with co-varying concentrations of potassium, magnesium and sulfate, but not sodium, chloride or calcium ions, while relative abundances of Halorubrum, Haloarcula, Halonotius, Halobaculum and Salinibacter-related sequences correlated negatively with Haloquadratum and these same ionic factors. Nanohaloarchaea and Halorhabdus-related sequence abundances were inversely correlated with each other, but not other taxonomic groups. These data, along with predicted gene functions from nearly-complete assembled population metagenomes, suggest different ecological phenotypes for Nanohaloarchaea and Halorhabdus-related strains versus other community members. Nucleotide percent G+C compositions were consistently lower in community metagenomic reads from summer versus winter samples. The same seasonal G+C trends were observed within taxonomically binned read subsets from each of seven different genus-level archaeal groups. Relative seasonal abundances were also linked to percent G+C for assembled population genomes. Together, these data suggest that extreme ionic conditions may exert selective pressure on archaeal populations at the level of genomic nucleotide composition, thus contributing to seasonal successional processes. Despite the unavailability of cultured representatives for most of the organisms identified in this study, effective coordination of physical and biological measurements has enabled discovery and quantification of unexpected taxon-specific, environmentally mediated factors influencing microbial community structure. © 2014 International Society for Microbial Ecology All rights reserved.

Deep Lake in Antarctica is a globally isolated, hypersaline system that remains liquid at temperatures down to -20 °C. By analyzing metagenome data and genomes of four isolates we assessed genome variation and patterns of gene exchange to learn how the lake community evolved. The lake is completely and uniformly dominated by haloarchaea, comprising a hierarchically structured, low-complexity community that differs greatly to temperate and tropical hypersaline environments. The four Deep Lake isolates represent distinct genera (~85% 16S rRNA gene similarity and ~73% genome average nucleotide identity) with genomic characteristics indicative of niche adaptation, and collectively account for ~72% of the cellular community. Network analysis revealed a remarkable level of intergenera gene exchange, including the sharing of long contiguous regions (up to 35 kb) of high identity (~100%). Although the genomes of closely related Halobacterium, Haloquadratum, and Haloarcula (>90% average nucleotide identity) shared regions of high identity between species or strains, the four Deep Lake isolates were the only distantly related haloarchaea to share long high-identity regions. Moreover, the Deep Lake high-identity regions did not match to any other hypersaline environment metagenome data. The most abundant species, tADL, appears to play a central role in the exchange of insertion sequences, but not the exchange of high-identity regions. The genomic characteristics of the four haloarchaea are consistent with a lake ecosystem that sustains a high level of intergenera gene exchange while selecting for ecotypes that maintain sympatric speciation. The peculiarities of this polar system restrict which species can grow and provide a tempo and mode for accentuating gene exchange.

Background: Haloquadratum walsbyi dominates saturated thalassic lakes worldwide where they can constitute up to 80-90% of the total prokaryotic community. Despite the abundance of the enigmatic square-flattened cells, only 7 isolates are currently known with 2 genomes fully sequenced and annotated due to difficulties to grow them under laboratory conditions. We have performed a transcriptomic analysis of one of these isolates, the Spanish strain HBSQ001 in order to investigate gene transcription under light and dark conditions. Results: Despite a potential advantage for light as additional source of energy, no significant differences were found between light and dark expressed genes. Constitutive high gene expression was observed in genes encoding surface glycoproteins, light mediated proton pumping by bacteriorhodopsin, several nutrient uptake systems, buoyancy and storage of excess carbon. Two low expressed regions of the genome were characterized by a lower codon adaptation index, low GC content and high incidence of hypothetical genes. Conclusions: Under the extant cultivation conditions, the square hyperhalophile devoted most of its transcriptome towards processes maintaining cell integrity and exploiting solar energy. Surface glycoproteins are essential for maintaining the large surface to volume ratio that facilitates light and organic nutrient harvesting whereas constitutive expression of bacteriorhodopsin warrants an immediate source of energy when light becomes available The research leading to these results has received funding from the European Union Seventh Framework Program (FP7/2007-2013- MaCuMBA project) under grant agreement n° 311,975 (HB and FRV; salary (in part), consumables and sequencing costs) Salaries of RR and ABMC were partly payed by the projects “VIREVO” CGL2016-76273-P [AEI/FEDER, UE], from Agencia Estatal de Investigación (AEI) y por la Unión Europea a través del Fondo Europeo de Desarrollo Regional - FEDER – “Una manera de hacer Europa” Acciones de dinamización “REDES DE EXCELENCIA” CONSOLIDER- CGL2015-71523-REDC from the Spanish Ministerio de Economía, Industria y Competitividad PROMETEO II/2014/012 “AQUAMET” from Conselleria d’Educació, Cultura i Esport. Direcció General d’Universitat, Estudis Superiors i Ciència. Generalitat Valenciana

Solar salterns are excellent model ecosystems for studying virus-microbial interactions because of their low microbial diversity, environmental stability, and high viral density. By using the power of CRISPR spacers to link viruses to their prokaryotic hosts, we explored virus-host interactions in geographically diverse salterns and related them to carbon cycling. Using taxonomic profiling, we identified hosts such as archaeal Haloquadratum, Halorubrum, and Haloarcula and bacterial Salinibacter, and we found that community composition related to not only salinity but also local environmental dynamics. Characterizing glycerol metabolism genes in these metagenomes suggested Halorubrum and Haloquadratum possess most dihydroxyacetone kinase genes while Salinibacter possesses most glycerol-3-phosphate dehydrogenase genes. We identified CRISPR spacers in the metagenomes with two different methods and found more spacers in the IC21 and C34 salterns compared with the SS19, SS33, and SS37 salterns, suggesting fewer types of CRISPR spacers in the Haloquadratum-majority salterns. After CRISPR detection, spacers were aligned against haloviral genomes to map virus to host. While most alignments linked viruses to Haloquadratum walsbyi, there were groups of interactions with the low abundance taxa Haloarcula and Haloferax. Further examination of the dinucleotide and trinucleotide usage differences between paired viruses and their hosts confirmed viruses and hosts had similar nucleotide usage signatures. Detection of cas genes in the salterns supported the possibility of CRISPR activity. Taken together, our studies suggest similar virus-host interactions exist in different solar salterns and that the glycerol metabolism gene dihydroxyacetone kinase is associated with Haloquadratum and Halorubrum.

A few extremely halophilic Archaea (Halobacterium salinarum, Haloquadratum walsbyi, Haloferax mediterranei, Halorubrum vacuolatum, Halogeometricum borinquense, Haloplanus spp.) possess gas vesicles that bestow buoyancy on the cells. Gas vesicles are also produced by the anaerobic endospore-forming halophilic Bacteria Sporohalobacter lortetii and Orenia sivashensis. We have extensive information on the properties of gas vesicles in Hbt. salinarum and Hfx. mediterranei and the regulation of their formation. Different functions were suggested for gas vesicle synthesis: buoying cells towards oxygen-rich surface layers in hypersaline water bodies to prevent oxygen limitation, reaching higher light intensities for the light-driven proton pump bacteriorhodopsin, positioning the cells optimally for light absorption, light shielding, reducing the cytoplasmic volume leading to a higher surface-area-to-volume ratio (for the Archaea) and dispersal of endospores (for the anaerobic spore-forming Bacteria). Except for Hqr. walsbyi which abounds in saltern crystallizer brines, gas-vacuolate halophiles are not among the dominant life forms in hypersaline environments. There only has been little research on gas vesicles in natural communities of halophilic microorganisms, and the few existing studies failed to provide clear evidence for their possible function. This paper summarizes the current status of the different theories why gas vesicles may provide a selective advantage to some halophilic microorganisms.

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.

Prokaryotic diversity in Aran-Bidgol salt lake, a thalasohaline lake in Iran, was studied by fluorescence in situ hybridization (FISH), cultivation techniques, denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of 16S rRNA genes and 16S rRNA gene clone library analysis. Viable counts obtained (2.5-4 × 10(6) cells mL(-1)) were similar to total cell abundance in the lake determined by DAPI direct count (3-4×10(7) cells mL(-1)). The proportion of Bacteria to Archaea in the community detectable by FISH was unexpectedly high and ranged between 1:3 and 1:2. We analyzed 101 archaeal isolates and found that most belonged to the genera Halorubrum (55%) and Haloarcula (18%). Eleven bacterial isolates obtained in pure culture were affiliated with the genera Salinibacter (18.7%), Salicola (18.7%) and Rhodovibrio (35.3%). Analysis of inserts of 100 clones from the eight 16S rRNA clone libraries constructed revealed 37 OTUs. The majority (63%) of these sequences were not related to any previously identified taxa. Within this sampling effort we most frequently retrieved phylotypes related to Halorhabdus (16% of archaeal sequences obtained) and Salinibacter (36% of bacterial sequences obtained). Other prokaryotic groups that were abundant included representatives of Haloquadratum, the anaerobic genera Halanaerobium and Halocella, purple sulfur bacteria of the genus Halorhodospira and Cyanobacteria.

The solar salterns located in the Odiel marshlands, in southwest Spain, are an excellent example of a hypersaline environment inhabited by microbial populations specialized in thriving under conditions of high salinity, which remains poorly explored. Traditional culture-dependent taxonomic studies have usually under-estimated the biodiversity in saline environments due to the difficulties that many of these species have to grow at laboratory conditions. Here we compare two molecular methods to profile the microbial population present in the Odiel saltern hypersaline water ponds (33% salinity). On the one hand, the construction and characterization of two clone PCR amplified-16S rRNA libraries, and on the other, a high throughput 16S rRNA sequencing approach based on the Illumina MiSeq platform. The results reveal that both methods are comparable for the estimation of major genera, although massive sequencing provides more information about the less abundant ones. The obtained data indicate that Salinibacter ruber is the most abundant genus, followed by the archaea genera, Halorubrum and Haloquadratum. However, more than 100 additional species can be detected by Next Generation Sequencing (NGS). In addition, a preliminary study to test the biotechnological applications of this microbial population, based on its ability to produce and excrete haloenzymes, is shown.

Summary Remarkable morphological diversity of virus-like par- ticles was observed by transmission electron micros- copy in a hypersaline water sample from Lake Retba, Senegal. The majority of particles morphologically resembled hyperthermophilic archaeal DNA viruses isolated from extreme geothermal environments. Some hypersaline viral morphotypes have not been previously observed in nature, and less than 1% of observed particles had a head-and-tail morphology, which is typical for bacterial DNA viruses. Culture- independent analysis of the microbial diversity in the sample suggested the dominance of extremely halophilic archaea. Few of the 16S sequences corre- sponded to known archeal genera (Haloquadratum, Halorubrum and Natronomonas), whereas the major- ity represented novel archaeal clades. Three sequences corresponded to a new basal lineage of the haloarchaea. Bacteria belonged to four major phyla, consistent with the known diversity in saline environments. Metagenomic sequencing of DNA from the purified virus-like particles revealed very few similarities to the NCBI non-redundant database at either the nucleotide or amino acid level. Some of the identifiable virus sequences were most similar to previously described haloarchaeal viruses, but no sequence similarities were found to archaeal viruses from extreme geothermal environments. A large pro- portion of the sequences had similarity to previously sequenced viral metagenomes from solar salterns.

To measure community respiration by the heterotrophic Archaea (dominated by Haloquadratum) and Bacteria (Salinibacter) in the NaCl-saturated crystallizer brines of the solar salterns in Eilat, Israel, and to obtain information on the substrates preferred by the community as energy sources, we used 2 complementary approaches: monitoring of changes in oxygen concentration using planar optode sensors in short (up to 30 min) experiments, and long-term (up to 40-50 h) incubations using Winkler titration to assess changes in oxygen levels. Respiration rates measured were -3 fmol cell -1 h -1 . Respiration was markedly stimulated by glycerol, dihydroxyacetone and pyruvate, but not by yeast extract, succinate, and fumarate. These findings are discussed in view of genomic information on the dominant heterotrophic organisms in the community as well as the outcome of earlier studies on the behavior of halophilic prokaryotes in situ and in laboratory cultures. Despite the low in situ respiration rate, the oxygen uptake studies added information on the activities of the heterotrophic communities in salt-saturated ecosystems and on the substrates metabolized by the microorganisms present.

Antibiotics and bile salts have been used to differentiate between heterotrophic activity of halophilic Archaea and Bacteria in saltern ponds. In NaCl-saturated brines of crystallizer ponds, most activity was attributed to Archaea. Following the recent isolation of Haloquadratum, the dominant archaeon in the salterns (reported to be sensitive to chloramphenicol and erythromycin), and the discovery of Salinibacter, a representative of the Bacteria, in the same ecosystem, reevaluation of the earlier data is required. The authors measured amino acid incorporation by Haloquadratum and Salinibacter suspended in crystallizer brine to investigate the suitability of antibiotics and bile salts to distinguish between archaeal and bacterial activities. The amino acid uptake rate per cell in Salinibacter was two orders of magnitude lower than that of Haloquadratum under the same conditions. Salinibacter was inhibited by chloramphenicol, erythromycin, and deoxycholate, but not by taurocholate. Erythromycin did not inhibit incorporation by Haloquadratum, but moderate inhibition was found by chloramphenicol at 10–50 μg mL−1. Deoxycholate was highly inhibitory, but only partial inhibition was obtained in the presence of 25 μg mL−1 taurocholate. Inhibition by chloramphenicol and taurocholate increased with increasing salt concentration. Erythromycin and taurocholate proved most valuable to differentiate between archaeal and bacterial activities in saltern brines.

© 2015 International Society for Microbial Ecology. Marine microbial communities experience daily fluctuations in light and temperature that can have important ramifications for carbon and nutrient cycling. Elucidation of such short time scale community-wide dynamics is hindered by system complexity. Hypersaline aquatic environments have lower species richness than marine environments and can be well-defined spatially, hence they provide a model system for diel cycle analysis. We conducted a 3-day time series experiment in a well-defined pool in hypersaline Lake Tyrrell, Australia. Microbial communities were tracked by combining cultivation-independent lipidomic, metagenomic and microscopy methods. The ratio of total bacterial to archaeal core lipids in the planktonic community increased by up to 58% during daylight hours and decreased by up to 32% overnight. However, total organism abundances remained relatively consistent over 3 days. Metagenomic analysis of the planktonic community composition, resolved at the genome level, showed dominance by Haloquadratum species and six uncultured members of the Halobacteriaceae. The post 0.8 μm filtrate contained six different nanohaloarchaeal types, three of which have not been identified previously, and cryo-transmission electron microscopy imaging confirmed the presence of small cells. Notably, these nano-sized archaea showed a strong diel cycle, with a pronounced increase in relative abundance over the night periods. We detected no eukaryotic algae or other photosynthetic primary producers, suggesting that carbon resources may derive from patchily distributed microbial mats at the sediment-water interface or from surrounding land. Results show the operation of a strong community-level diel cycle, probably driven by interconnected temperature, light abundance, dissolved oxygen concentration and nutrient flux effects.

The square haloarchaea of Walsby (SHOW group) dominate hypersaline microbial communities but have not been cultured since their discovery 25 years ago. We show that natural water dilution cultures can be used to isolate members of this group and, once in pure culture, they can be grown in standard halobacterial media. Cells display a square morphology and contain gas vesicles and poly-beta-hydroxybutyrate (PHB) granules. The 16S rRNA gene sequence was99% identical to other SHOW group sequences. They prefer high salinities (23-30%), and can grow with a doubling time of 1-2 days in rich media. The ability to culture SHOW group organisms makes it possible to study, in a comprehensive way, the microbial ecology of salt lakes.

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10 6 CFU/ml) were obtained on solid media. Long incubation times (≥8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae , including members related to species of the genera Haloferax , Halorubrum , and Natronomonas . In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.

The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR approach) was applied to brine samples of a solar saltern crystallizer pond from Mallorca (Spain) where the simultaneous occurrence of Salinibacter spp. and the conspicuous square Archaea had been detected. Radioactively labeled bicarbonate, acetate, glycerol, and an amino acid mixture were tested as substrates for the microbial populations inhabiting such brines. The results indicated that hitherto uncultured 'square Archaea' do actively incorporate amino acids and acetate. However, Salinibacter spp. only showed amino acid incorporation in pure culture, but no evidence of such activity in their natural environment could be demonstrated. No glycerol incorporation was observed for any component of the microbial community., This work was supported by the following institutions' research projects: the Spanish Ministry of Science and Technology with grants BOS2000-1123C02-01 and BOS2000-1123C02-02 (to R.R.M. and J.A.). R.R.M. acknowledges the grant of a Marina Bueno Fellowship (CSIC-DFG), which covered travel expenses to perform the current project

Halophiles are among the best model microbes to study cellular adaptation. Haloferax, Haloarcula, Haloquadratum, Halobacteriales and many other halophiles living in the Great Salt Lake can survive extremely high salt concentrations. Many of them cannot survive if moved from hypersaline to non-saline habitats, and die from immediate cell lysis. The unique biological survivability of these bacteria in the saline condition is dictated by the phylogenetic dexterity at the given environmental constraints. Microbial adaptation has played a major role when bacteria in all forms branched out into different environmental niches, arising from the hypersaline conditions of the primordial sea. Their pervasive nature, created by ever-changing environmental conditions, was acquired by microbes from millions of years of making. The adaptation of enzymes during the course of their evolutionary development and some metabolic differences helped them expand and achieve environmental diversity. Metabolic processes, like osmoregulation in halotolerant cells, dictate the regulation of the bacterial cell membrane. For example, glycerol metabolism has been linked to osmoregulation in some halophilic microbes. Some mechanisms behind osmoregulation in halophiles are extremely energetic since they live in hostile environments. Pathways of such regulations, including de novo synthesis of solutes anabolically and/or extraction of solutes environmentally, help them to produce secondary metabolites like poly-β-hydroxyalkanoates. How halophiles maintain high metabolic similarity to other non-saline bacteria while showing different survivability under heavy salt stress remains an important question. This review attempts to outline progress in our understanding of their environmental diversity and biological survivability.

© 2015 International Society for Microbial Ecology All rights reserved. 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., This work was supported by the projects CGL2012-39627-C03-01 and 02 of the Spanish Ministry of Economy and Competitiveness, which were also co-financed with FEDER support from the European Union. TG group research is funded in part by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2012-37161), a grant from the Qatar National Research Fund grant (NPRP 5-298-3-086) and a grant from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC (grant agreement no. ERC-2012-StG-310325)

Hypersaline systems near salt saturation levels represent an extreme environment, in which organisms grow and survive near the limits of life. One of the abundant members of the microbial communities in hypersaline systems is the square archaeon,Haloquadratum walsbyi. Utilizing a short-read metagenome from Lake Tyrrell, a hypersaline ecosystem in Victoria, Australia, we performed a comparative genomic analysis ofH. walsbyito better understand the extent of variation between strains/subspecies. Results revealed that previously isolated strains/subspecies do not fully describe the complete repertoire of the genomic landscape present inH. walsbyi. Rearrangements, insertions, and deletions were observed for the Lake Tyrrell derivedHaloquadratumgenomes and were supported by environmentalde novosequences, including shifts in the dominant genomic landscape of the two most abundant strains. Analysis pertaining to halomucins indicated that homologs for this large protein are not a feature common for all species ofHaloquadratum. Further, we analyzed ATP-binding cassette transporters (ABC-type transporters) for evidence of niche partitioning between different strains/subspecies. We were able to identify unique and variable transporter subunits from all five genomes analyzed and thede novoenvironmental sequences, suggesting that differences in nutrient and carbon source acquisition may play a role in maintaining distinct strains/subspecies.

Bacterial and archaeal assemblages have been studied in a multipond solar saltern using a range of microbial ecology techniques by four laboratories simultaneously. These include 16S rDNA sequencing from both denaturing gradient gel electrophoresis (DGGE) and clone libraries, and culturing methods. Water samples from eight ponds were analysed, covering a salinity range from near sea water (4% salt) to saturated sodium chloride (37% salt; ponds called crystallizers). Clone libraries focused on ponds with salinity of 8%, 22% and 32%. Although different cloning strategies were able to retrieve the same type of dominant sequences, there were differing degrees of success with less abundant sequences. Thus, the use of two sets of primers recovered a higher number of phylotypes. Bacterial and archaeal isolates were, however, different from any of the retrieved environmental sequences. For Bacteria, most sequences in the 8% salt pond were related to organisms of marine origin. Thus, representatives of the alpha‐, beta‐, gamma‐ and epsilon‐subdivisions of Proteobacteria, the Cytophaga–Flavobacterium–Bacteroides group (CFB), high‐G+C Gram‐positive bacteria and cyanobacteria were found. In the 22% salt pond, alpha‐ and gamma‐Proteobacteria, cyanobacteria and CFB were the only groups found, and most of them were related to specialized halophilic bacteria. From the 32% salt pond, only CFB were found, and most of the sequences retrieved clustered with Salinibacter ruber, an extremely halophilic bacterium. A decrease in the richness of bacterial genera was therefore apparent along the gradient. Archaea behaved quite similarly. In the lowest salinity ponds, sequences were related to environmental clones of Marine Archaea Group III (Thermoplasmales relatives) and to unclassified branches of Euryarchaeaota. In the 8%, 22% and 32% ponds, most of the clones were related to different cultured strains of Halobacteriaceae. Finally, most sequences from the crystallizers clustered with the uncultured square archaeon SPhT. Crenarchaeaota were not detected. Despite the fact that higher prokaryotic richness was apparent in the lower salinity ponds than in the crystallizers, the diversity index from clone libraries calculated according to Shannon and Weaver did not show this trend. This was because diversity in the crystallizers can be considered as ‘microdiversity’, the co‐existence of several closely related clones of Bacteria (the S. ruber cluster) and Archaea (the SPhT cluster). Regardless of the changes in abundance, both Bacteria and Archaea showed the same pattern; as salinity increased, the number of different clusters decreased, and only one cluster became dominant. Both clusters, however, showed a considerable degree of microdiversity. The meaning of such microdiversity remains to be determined

Water bodies with NaCl concentrations approaching saturation are often populated by dense microbial communities. Red halophilic Archaea of the family Halobacteriaceae dominate in such environments. The application of molecular biological techniques, in particular the use of approaches based on the characterization of ribosomal RNA sequences, has greatly contributed to our understanding of the community structure of halophilic Archaea in hypersaline ecosystems. Analyses of lipids extracted from the environment have also provided useful information. This article reviews our present understanding of the community structure of halophilic Archaea in saltern crystallizer ponds, in the Dead Sea, in African hypersaline soda lakes, and in other hypersaline water bodies. It was recently shown that red heterotrophic Bacteria of the genus Salinibacter, which are no less salt-dependent and salt-tolerant than the most halophilic among the Archaea, may coexist with the halophilic archaeal community. Our latest insights into their distribution in hypersaline ecosystems are presented as well.

Heterotrophic prokaryotic communities that inhabit saltern crystallizer ponds are typically dominated by two species, the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber , regardless of location. These organisms behave as ‘microbial weeds’ as defined by Cray et al . ( Microb Biotechnol 6 : 453–492, 2013) that possess the biological traits required to dominate the microbiology of these open habitats. Here, we discuss the enigma of the less abundant Haloferax mediterranei , an archaeon that grows faster than any other, comparable extreme halophile. It has a wide window for salt tolerance, can grow on simple as well as on complex substrates and degrade polymeric substances, has different modes of anaerobic growth, can accumulate storage polymers, produces gas vesicles, and excretes halocins capable of killing other Archaea. Therefore, Hfx. mediterranei is apparently more qualified as a ‘microbial weed’ than Haloquadratum and Salinibacter . However, the former differs because it produces carotenoid pigments only in the lower salinity range and lacks energy-generating retinal-based, light-driven ion pumps such as bacteriorhodopsin and halorhodopsin. We discuss these observations in relation to microbial weed biology in, and the open-habitat ecology of, hypersaline systems.

We analyzed the prokaryotic community structure of a saltern pond with 21 % total salts located in Isla Cristina, Huelva, Southwest Spain, close to the Atlantic ocean coast. For this purpose, we constructed a metagenome (designated as IC21) obtained by pyrosequencing consisting of 486 Mb with an average read length of 397 bp and compared it with other metagenomic datasets obtained from ponds with 19, 33 and 37 % total salts acquired from Santa Pola marine saltern, located in Alicante, East Spain, on the Mediterranean coast. Although the salinity in IC21 is closer to the pond with 19 % total salts from Santa Pola saltern (designated as SS19), IC21 is more similar at higher taxonomic levels to the pond with 33 % total salts from Santa Pola saltern (designated as SS33), since both are predominated by the phylum Euryarchaeota. However, there are significant differences at lower taxonomic levels where most sequences were related to the genus Halorubrum in IC21 and to Haloquadratum in SS33. Within the Bacteroidetes, the genus Psychroflexus is the most abundant in IC21 while Salinibacter dominates in SS33. Sequences related to bacteriorhodopsins and halorhodopsins correlate with the abundance of Haloquadratum in Santa Pola SS19 to SS33 and of Halorubrum in Isla Cristina IC21 dataset, respectively. Differences in composition might be attributed to local ecological conditions since IC21 showed a decrease in the number of sequences related to the synthesis of compatible solutes and in the utilization of phosphonate.

Analysis of the pigments extracted from solar saltern crystallizer ponds in Santa Pola near Alicante and on the Balearic island of Mallorca, Spain, showed that 5-7.5% of the total prokaryotic pigment absorbance could be attributed to a novel carotenoid or carotenoid-like compound. This unidentified pigment was identical to the sole pigment present in Salinibacter ruber, the only described member of a newly discovered genus of red halophilic Bacteria related to the Cytophaga-Flavobacterium-Bacteroides group. On the basis of fluorescence in situ hybridization experiments it has been shown that Salinibacter is an important component of the microbial community of Spanish saltern ponds. The red color of saltern crystallizer ponds may thus not only be due to red halophilic Archaea and to beta-carotene-rich Dunaliella cells as previously assumed, but may contain a bacterial contribution as well. The Salinibacter pigment was not detected in samples collected from crystallizer ponds of the salterns of Eilat, Israel, and only traces of it may have been present in the Newark, CA, USA, salterns. The community structure of the prokaryote community inhabiting saltern crystallizers thus shows significant geographic variations. Polar lipid analyses of the biomass collected from the Santa Pola salterns showed that the total contribution of Salinibacter and other Bacteria to the total biomass was minor, the most important component of the community being halophilic Archaea.

It is generally assumed that hypersaline environments with sodium chloride concentrations close to saturation are dominated by halophilic members of the domain Archaea , while Bacteria are 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 the Cytophaga-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.

A fluorescence in situ hybridization (FISH) protocol suitable for the identification of prokaryotes inhabiting hypersaline environments was developed and applied to several crystallizer ponds with salinities above 36% from a multipond solar saltern in Alicante, Spain. Two morphotypes were abundant in these environments: rods and square or square-like prokaryotes that could be affiliated to Bacteria and Archaea, respectively, by FISH with domain-specific probes. FISH with a newly designed probe proved that the archaeal 16S rDNA sequence most frequently recovered from the crystallizers, SPhT, originated from the dominant square-like prokaryotes. These uncultured prokaryotes have the morphology of Walsby's square bacteria. Additionally, FISH with a probe targeted to the genus Haloarcula, members of which are frequently isolated from this environment, indicated that this genus accounts for less than 0.1% of the total prokaryotic community.

A multi-pond saltern that creates a gradient of salt concentrations has been studied with respect to some characteristics of the resulting environments and their microbial populations. The increase in salt concentration was correlated with increase in diurnal temperature and biomass present and with decrease in oxygen concentrations. Many types of organisms below 15% (w/v) total salts, were found, many of them normal inhabitants of seawater and even freshwater. Most organisms over 15% salts were halophilic. The salt concentrations comprised two ranges, each characterized by different microbial populations. First, between 15 and 30% salts, the populations ofDunaliella increased, reaching large numbers; moderately halophilic eubacteria and some fast-growing halobacteria predominated as heterotrophic microorganisms and, among the first, thePseudomonas-Alteromonas-Alcaligenes group andVibrio were the more abundant taxonomic groups; and gram-positive cocci appeared mainly over 25% salts. Phototrophic bacteria, both oxygenic and anoxygenic, were also found in this range, and among the anoxygenic type,Chromatium species andRodospirillum salexigens were probably predominant. Second, over 30% salts the diversity decreased greatly, all organisms found at the lower salt concentrations disappeared, and instead large populations of halobacteria developed. Over 50% salts, only three species of halobacteria were found.

The goal of this study was to use environmental sequencing of 16S rRNA and bop genes to compare the diversity of planktonic bacteria and archaea across ponds with increasing salinity in the Exportadora de Sal (ESSA) evaporative saltern in Guerrero Negro, Baja CA S., Mexico. We hypothesized that diverse communities of heterotrophic bacteria and archaea would be found in the ESSA ponds, but that bacterial diversity would decrease relative to archaea at the highest salinities. Archaeal 16S rRNA diversity was higher in Ponds 11 and 12 (370 & 380 g l-1 total salts respectively) compared to Pond 9 (180 g l-1 total salts). Both Pond 11 and 12 communities had high representation (47 and 45% of clones respectively) by Haloquadratum walsbyi-like (99% similarity) lineages. The archaeal community in Pond 9 was dominated (79%) by a single uncultured phylotype with 99% similarity to sequences recovered from the Sfax saltern in Tunisia. This pattern was mirrored in bop gene diversity with greater numbers of highly supported phylotypes including many Haloquadratum-like sequences from the two highest salinity ponds. In Pond 9, most bop sequences, were not closely related to sequences in databases. Bacterial 16S rRNA diversity was higher than archaeal in both Pond 9 and Pond 12 samples, but not Pond 11, where a non-Salinibacter lineage within the Bacteroidetes >98% similar to environmental clones recovered from Lake Tuz in Turkey and a saltern in Chula Vista, CA was most abundant (69% of community). This OTU was also the most abundant in Pond 12, but only represented 14% of clones in the more diverse pond. The most abundant OTU in Pond 9 (33% of community) was 99% similar to an uncultured gammaproteobacterial clone from the Salton Sea. Results suggest that the communities of saltern bacteria and archaea vary even in ponds with similar salinity and further investigation into the ecology of diverse, uncultured halophile communities is warranted.

Square, gas vacuole-containing Archaea of the type first described by Walsby were found to dominate in a saltern crystallizer pond in Eilat, Israel. To obtain information on the taxonomic position of these yet uncultured bacteria, we analyzed the polar lipids present in the microbial community in the saltern brine. In addition to phosphatidylglycerol, phosphatidylglycerophosphate and phosphatidylglycerosulfate we found one glycolipid, chromatographically identical with the sulfated diglycosyl diether lipid found as the major glycolipid in Haloferax species. As the square bacteria contributed at least 85% of the total membrane surface in the biota of the sample examined, we concluded, based on polar lipid composition, that these organisms are unrelated to the genera Halobacterium and Haloarcula, and probably belong to a new genus.

We have explored the use of optical oxygen electrodes to study oxygenic photosynthesis and heterotrophic activities in crystallizer brines of the salterns in Eilat, Israel. Monitoring oxygen uptake rates in the dark enables the identification of organic substrates that are preferentially used by the community. Addition of glycerol (the osmotic solute synthesized by Dunaliella) or dihydroxyacetone (produced from glycerol by Salinibacter) enhanced respiration rates. Pyruvate, produced from glycerol or from some sugars by certain halophilic Archaea also stimulated community respiration. Fumarate had a sparing effect on respiration, possibly as many halophilic Archaea can use fumarate as a terminal electron acceptor in respiration. Calculating the photosynthetic activity of Dunaliella by monitoring oxygen concentration changes during light/dark incubations is not straightforward as light also affects respiration of some halophilic Archaea and Bacteria due to action of light-driven proton pumps. When illuminated, community respiration of brine samples in which oxygenic photosynthesis was inhibited by DCMU decreased by ~40%. This effect was interpreted as the result of competition between two energy yielding systems: the bacteriorhodopsin proton pump and the respiratory chain of the prokaryotes. These findings have important implications for the interpretation of other published data on photosynthetic and respiratory activities in hypersaline environments.

Summary The new methods based on PCR amplification and sequencing of 16S rRNA genes from DNA samples extracted directly from the environment allow the description of microbial diversity in natural ecosystems without the need for cultivation. We have applied this technique to an extreme environment presumed to have very low diversity: the crystallizer ponds of a marine saltern (salinity over NaCl saturation). The molecular methodology has shown that indeed very low diversity can be found here. Bacteria (formerly eubacteria) are largely minoritary, and only a cluster of closely related sequences was found, all distant relatives of the α-Proteohacteria (82% to Rhodopseudoinonas marina). Halophilic Archaea were shown by hybridization to be, as expected, the largest component of biomass in this environment. All the archaeal clones sequenced again were highly similar to each other suggesting that they are probably members of the same genus. However, all the sequences diverged considerably from those of the described genera of halophilic Archaea. In fact the data are consistent with the idea that the 16S rDNA genes directly amplified from the saltern correspond to members of an undescribed genus which seems to be abundant in the sample. This is remarkable since many collection strains sequenced come specifically from this same saltern. Furthermore, 16S rDNA obtained from archaeal cultures isolated from the same sample had no homology to the sequences obtained by PCR amplification, instead they appear to be members of the well known genus Haloarcula. However, this is in agreement with the findings of other authors who by culture have obtained organisms different from those indicated by the sequences retrieved directly by PCR. Possible explanations are discussed.

Hypersaline lakes with salt concentrations exceeding 250 g/l are often characterized by very dense communities of halophilic microorganisms imparting a red coloration to the brines. Such red waters can be found in the North Arm of Great Salt Lake , Utah, in crystallizer ponds of solar salterns for the production of salt from seawater, and in many extremely hypersaline alkaline lakes. At times even the magnesium chloride-rich waters of the Dead Sea have become red as a result of massive development of pigmented salt-loving microorganisms.

We describe the microbiota of two hypersaline saltern ponds, one of intermediate salinity (19%) and a NaCl saturated crystallizer pond (37%) using pyrosequencing. The analyses of these metagenomes (nearly 784 Mb) reaffirmed the vast dominance of Haloquadratum walsbyi but also revealed novel, abundant and previously unsuspected microbial groups. We describe for the first time, a group of low GC Actinobacteria, related to freshwater Actinobacteria, abundant in low and intermediate salinities. Metagenomic assembly revealed three new abundant microbes: a low-GC euryarchaeon with the lowest GC content described for any euryarchaeon, a high-GC euryarchaeon and a gammaproteobacterium related to Alkalilimnicola and Nitrococcus. Multiple displacement amplification and sequencing of the genome from a single archaeal cell of the new low GC euryarchaeon suggest a photoheterotrophic and polysaccharide-degrading lifestyle and its relatedness to the recently described lineage of Nanohaloarchaea. These discoveries reveal the combined power of an unbiased metagenomic and single cell genomic approach.

Here, we combined flow cytometry (FCM) and phylogenetic analyses after cell sorting to characterize the dominant groups of the prokaryotic assemblages inhabiting two ponds of increasing salinity: a crystallizer pond (TS) with a salinity of 390 g/L, and the non-crystallizer pond (M1) with a salinity of 200 g/L retrieved from the solar saltern of Sfax in Tunisia. As expected, FCM analysis enabled the resolution of high nucleic acid content (HNA) and low nucleic acid content (LNA) prokaryotes. Next, we performed a taxonomic analysis of the bacterial and archaeal communities comprising the two most populated clusters by phylogenetic analyses of 16S rRNA gene clone library. We show for the first time that the presence of HNA and LNA content cells could also be extended to the archaeal populations. Archaea were detected in all M1 and TS samples, whereas representatives of Bacteria were detected only in LNA for M1 and HNA for TS. Although most of the archaeal sequences remained undetermined, other clones were most frequently affiliated to Haloquadratum and Halorubrum. In contrast, most bacterial clones belonged to the Alphaproteobacteria class (Phyllobacterium genus) in M1 samples and to the Bacteroidetes phylum (Sphingobacteria and Salinibacter genus) in TS samples. Electronic supplementary material The online version of this article (doi:10.1007/s00792-011-0364-5) contains supplementary material, which is available to authorized users.

Bile salts (deoxycholate, taurocholate) were used to estimate the contribution of bacteria of the Halobacterium group to bacterial community size and activity at different salinities as found in a multi-pond saltern. Low concentrations of bile salts cause lysis of halophilic archaebacteria of the Halobacterium group, while halophilic eubacteria and halococci remain microscopically intact. Upon addition of bile salts, total bacterial numbers (as estimated microscopically) in saltern ponds at salinities below 250 g/l did not decrease, while above this salinity bacterial numbers decreased by 30–50%. To estimate the contribution of halobacteria to overall heterotrophic activity, the effect of bile salt addition was tested on the incorporation of labelled amino acids. In saltern ponds of a salinity below 250 g/l activity was not greatly inhibited by taurocholate, while at salinity above 300 g/l taurocholate completely abolished incorporation of amino acids.