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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. [ABSTRACT FROM AUTHOR]

Tinto River (Huelva, Spain) is a natural acidic rock drainage (ARD) environment produced by the bio-oxidation of metallic sulfides from the Iberian Pyritic Belt. This study quantified the abundance of diverse microbial populations inhabiting ARD-related sediments from two physicochemically contrasting sampling sites (SN and JL dams). Depth profiles of total cell numbers differed greatly between the two sites yet were consistent in decreasing sharply at greater depths. Although catalyzed reporter deposition fluorescence in situ hybridization with domain-specific probes showed that Bacteria (>98%) dominated over Archaea (<2%) at both sites, important differences were detected at the class and genus levels, reflecting differences in pH, redox potential, and heavy metal concentrations. At SN, where the pH and redox potential are similar to that of the water column (pH 2.5 and +400 mV), the most abundant organisms were identified as iron-reducing bacteria: Acidithiobacillus spp. and Acidiphilium spp., probably related to the higher iron solubility at low pH. At the JL dam, characterized by a banded sediment with higher pH (4.2 to 6.2), more reducing redox potential (--210 mV to 50 mV), and a lower solubility of iron, members of sulfate-reducing genera Syntrophobacter, Desulfosporosinus, and Desulfurella were dominant. The latter was quantified with a newly designed CARD-FISH probe. In layers where sulfate-reducing bacteria were abundant, pH was higher and redox potential and levels of dissolved metals and iron were lower. These results suggest that the attenuation of ARD characteristics is biologically driven by sulfate reducers and the consequent precipitation of metals and iron as sulfides. [ABSTRACT FROM AUTHOR]

The phylogeny, abundance, and biogeography of the NOR5/OM60 clade was investigated. This clade includes "Congregibacter litoralis" strain KT71, the first cultured representative of marine aerobic anoxygenic phototrophic Gammaproteobacteria. More than 500 16S rRNA sequences affiliated with this clade were retrieved from public databases. By comparative sequence analysis, 13 subclades could be identified, some of which are currently restricted to discrete habitat types. Almost all sequences in the largest subclade NOR5-1 and related subclade NOR5-4 originated from marine surface water samples. Overall, most of the NOR5/OM60 sequences were retrieved from marine coastal settings, whereas there were fewer from open-ocean surface waters, deep-sea sediment, freshwater, saline lakes and soil. The abundance of members of the NOR5/OM60 clade in various marine sites was determined by fluorescence in situ hybridization using a newly designed and optimized probe set. Relative abundances in coastal marine waters off Namibia and the Yangtze estuary were up to 3% of the total 4',6-diamidino-2-phenylindole (DAPI) counts, and in the German Bight off Helgoland the abundance was even up to 7%. In an open-ocean North Atlantic transect, between Iceland and the Azores, the NOR5/OM60 group was much less abundant (0.1-0.5%). Interestingly, the surface layer of North Sea intertidal sediments was very rich in NOR5/OM60, with absolute numbers >10(8) cells cm(-3) (or 4% of the total DAPI). An analysis of the frequencies of NOR5/OM60 16S rRNA genes in the Global Ocean Survey datasets provided further support for a marine cosmopolitan occurrence of NOR5/OM60, and a clear preference for coastal marine waters.

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

Five brightly red-pigmented, motile, rod-shaped, extremely halophilic bacteria were isolated from saltern crystallizer ponds in Alicante (two strains) and Mallorca (three strains), Spain. They grew optimally at salt concentrations between 20 and 30% and did not grow below 15% salts. Thus, these isolates are among the most halophilic organisms known within the domain Bacteria. The temperature optimum was 37-47 degrees C. A single, yet to be identified pigment was present, with an absorption maximum at 482 nm and a shoulder at 506-510 nm. The G+C content of the DNA was 66.3-67.7 mol% and, together, they formed a homogeneous genomic group with DNA-DNA similarities above 70%. The 16S rRNA gene sequences were almost identical to sequences recovered earlier from the saltern biomass by amplification of bacterial small-subunit rRNA genes from DNA extracted from the environment. This phylotype, earlier described as 'Candidatus Salinibacter', was shown by fluorescence in situ hybridization to contribute between 5 and 25% of the prokaryote community of the saltern crystallizers. We have therefore succeeded in isolating a bacterium from the natural environment that, although being a major component of the community, was previously known by its phylotype only. Isolation of the organism now allows formal description of a novel genus and species, for which we propose the name Salinibacter ruber gen. nov., sp. nov. The type strain is strain M31T (= DSM 13855T = CECT 5946T).