Cristina Sánchez-Porro

New Abundant Microbial Groups in Aquatic Hypersaline Environments

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.

Biogeography of the ubiquitous marine bacterium Alteromonas macleodii determined by multilocus sequence analysis

Twenty‐three isolates of the widely distributed marine bacteria Alteromonas macleodii have been analysed by multilocus sequence analysis combined with phylogenetic and multivariate statistical analyses. The strains originated from the Pacific Ocean, Mediterranean Sea, English Channel, Black Sea and Thailand. Using the nucleotide sequences of nine loci for each of the 23 isolates, a robust identification was achieved of different clades within the single species. Strains generally clustered with the depth in the water column from which the isolate originated. Strains also showed more recombination with isolates from the same vicinity, suggesting that genetic exchange plays a role in diversification of planktonic marine prokaryotes. This study thus shows for the first time for a large set of isolates of a species of planktonic marine prokaryotes that multilocus sequence analysis overcomes the problems associated with the analysis of individual marker genes or presence of extensive recombination events. It can thus achieve intraspecific identification to the level of genotypes and, by comparison with relevant environmental data, ecotypes.

Halophilic and Halotolerant Micro-Organisms from Soils

Hypersaline environments are extreme habitats in which several other factors, in addition to high salt content, may limit the growth of organisms. These additional factors include temperature, pH, pressure, oxygen, nutrient availability, and solar radiations (Rodriguez-Valera 1988). Hypersaline environments comprise hypersaline waters and soils . Hypersaline waters are defined as those environments that have higher concentrations of salts than seawater (Rodriguez-Valera 1988). However, depending on their origin, the salt composition may differ from that of seawater and on that basis hypersaline water habitats are categorized as thalassohaline , when the relative amounts of the different inorganic salts are approximately equal to those present in seawater, or as athalassohaline , if the proportions of the different salts are markedly different from those of seawater. The later environments are more heterogeneous and may have very different origins. Examples of thalassohaline water habitats, which are typically chroride types, are the Great Salt Lake or the solar salterns used for the industrial production of marine salt by evaporation of seawater; among the athalassohaline waters are the Dead Sea, the Wadi Natrun, Lake Magadi, and several other soda lakes. In contrast to the hypersaline waters, the hypersaline soils are not well defined and in fact there is no clear definition of a saline or hypersaline soil. They are widely represented in our planet. Because most soils contain small amounts of soluble salts, a soil would be considered as hypersaline when its salt concentration is above a certain threshold (Rodriguez-Valera 1988). According to Kaurichev (1980), soils containing more than 0.2% (w/v) soluble salt should be considered as saline soils. Micro-organisms show quite different responses to salt . According to the particular salt concentration required for their optimal growth, several physiological groups of micro-organisms are considered : (i) nonhalophiles require less than 1%

Microbial diversity of hypersaline environments: a metagenomic approach.

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.

Halomonas titanicae sp. nov., a halophilic bacterium isolated from the RMS Titanic

A Gram-negative, heterotrophic, aerobic, non-endospore-forming, peritrichously flagellated and motile bacterial strain, designated BH1(T), was isolated from samples of rusticles, which are formed in part by a consortium of micro-organisms, collected from the RMS Titanic wreck site. The strain grew optimally at 30-37°C, pH 7.0-7.5 and in the presence of 2-8 % (w/v) NaCl. We carried out a polyphasic taxonomic study in order to characterize the strain in detail. Phylogenetic analyses based on 16S rRNA gene sequence comparison indicated that strain BH1(T) clustered within the branch consisting of species of Halomonas. The most closely related type strains were Halomonas neptunia (98.6 % 16S rRNA sequence similarity), Halomonas variabilis (98.4 %), Halomonas boliviensis (98.3 %) and Halomonas sulfidaeris (97.5 %). Other closely related species were Halomonas alkaliphila (96.5 % sequence similarity), Halomonas hydrothermalis (96.3 %), Halomonas gomseomensis (96.3 %), Halomonas venusta (96.3 %) and Halomonas meridiana (96.2 %). The major fatty acids of strain BH1(T) were C(18 : 1)ω7c (36.3 %), C(16 : 0) (18.4 %) and C(19 : 0) cyclo ω8c (17.9 %). The DNA G+C content was 60.0 mol% (T(m)). Ubiquinone 9 (Q-9) was the major lipoquinone. The phenotypic features, fatty acid profile and DNA G+C content further supported the placement of strain BH1(T) in the genus Halomonas. DNA-DNA hybridization values between strain BH1(T) and H. neptunia CECT 5815(T), H. variabilis DSM 3051(T), H. boliviensis DSM 15516(T) and H. sulfidaeris CECT 5817(T) were 19, 17, 30 and 29 %, respectively, supporting the differential taxonomic status of BH1(T). On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain BH1(T) is considered to represent a novel species, for which the name Halomonas titanicae sp. nov. is proposed. The type strain is BH1(T) (=ATCC BAA-1257(T) =CECT 7585(T) =JCM 16411(T) =LMG 25388(T)).

Halorubrum chaoviator sp. nov., a haloarchaeon isolated from sea salt in Baja California, Mexico, Western Australia and Naxos, Greece

Three halophilic isolates, strains Halo-G*T, AUS-1 and Naxos II, were compared. Halo-G* was isolated from an evaporitic salt crystal from Baja California, Mexico, whereas AUS-1 and Naxos II were isolated from salt pools in Western Australia and the Greek island of Naxos, respectively. Halo-G*T had been exposed previously to conditions of outer space and survived 2 weeks on the Biopan facility. Chemotaxonomic and molecular comparisons suggested high similarity between the three strains. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the strains clustered with Halorubrum species, showing sequence similarities of 99.2-97.1%. The DNA-DNA hybridization values of strain Halo-G*T and strains AUS-1 and Naxos II are 73 and 75%, respectively, indicating that they constitute a single species. The DNA relatedness between strain Halo-G*T and the type strains of 13 closely related species of the genus Halorubrum ranged from 39 to 2%, suggesting that the three isolates constitute a different genospecies. The G+C content of the DNA of the three strains was 65.5-66.5 mol%. All three strains contained C20C20 derivatives of diethers of phosphatidylglycerol, phosphatidylglyceromethylphosphate and phosphatidylglycerolsulfate, together with a sulfated glycolipid. On the basis of these results, a novel species that includes the three strains is proposed, with the name Halorubrum chaoviator sp. nov. The type strain is strain Halo-G*T (=DSM 19316T=NCIMB 14426T=ATCC BAA-1602T).

Comparison of prokaryotic community structure from Mediterranean and Atlantic saltern concentrator ponds by a metagenomic approach

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.

From Metagenomics to Pure Culture: Isolation and Characterization of the Moderately Halophilic Bacterium Spiribacter salinus gen. nov., sp. nov.

ABSTRACT Recent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dominated by both Euryarchaeota and halophilic bacteria, with a gammaproteobacterium closely related to the genera Alkalilimnicola and Arhodomonas being one of the most predominant microorganisms, making up to 15% of the total prokaryotic population. Here we used several strategies and culture media in order to isolate this organism in pure culture. We report the isolation and taxonomic characterization of this new, never before cultured microorganism, designated M19-40T, isolated from a saltern located in Isla Cristina, Spain, using a medium with a mixture of 15% salts, yeast extract, and pyruvic acid as the carbon source. Morphologically small curved cells (young cultures) with a tendency to form long spiral cells in older cultures were observed in pure cultures. The organism is a Gram-negative, nonmotile bacterium that is strictly aerobic, non-endospore forming, heterotrophic, and moderately halophilic, and it is able to grow at 10 to 25% (wt/vol) NaCl, with optimal growth occurring at 15% (wt/vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that strain M19-40T has a low similarity with other previously described bacteria and shows the closest phylogenetic similarity with species of the genera Alkalilimnicola (94.9 to 94.5%), Alkalispirillum (94.3%), and Arhodomonas (93.9%) within the family Ectothiorhodospiraceae. The phenotypic, genotypic, and chemotaxonomic features of this new bacterium showed that it constitutes a new genus and species, for which the name Spiribacter salinus gen. nov., sp. nov., is proposed, with strain M19-40T (= CECT 8282T = IBRC-M 10768T = LMG 27464T) being the type strain.

Transfer of Teichococcus ludipueritiae and Muricoccus roseus to the genus Roseomonas, as Roseomonas ludipueritiae comb. nov. and Roseomonas rosea comb. nov., respectively, and emended description of the genus Roseomonas

Phylogenetic analyses based on 16S rRNA gene sequences revealed that Teichococcus ludipueritiae and Muricoccus roseus are closely related to the species of the genus Roseomonas. The type strain of Teichococcus ludipueritiae, 170/96(T), exhibited 16S rRNA gene sequence similarity levels of 96.4 % to Roseomonas cervicalis ATCC 49957(T), 95.0 % to Roseomonas aquatica TR53(T), 94.5 % to Muricoccus roseus 173/96(T), 93.4 % to Roseomonas mucosa ATCC BAA-692(T) and 93.5 % to Roseomonas gilardii subsp. gilardii ATCC 49956(T), while Muricoccus roseus 173/96(T) showed 16S rRNA gene sequence similarity values of 95.7 % to R. mucosa ATCC BAA-692(T), 95.7 % to R. aquatica TR53(T) and 95.3 % to R. gilardii subsp. gilardii ATCC 49956(T) and R. gilardii subsp. rosea ATCC BAA-691(T). Different phylogenetic analysis methods (neighbour-joining, maximum-likelihood and maximum-parsimony) confirmed that both species are within the Roseomonas branch. Neither polyamine patterns (spermidine predominant) nor major characteristics in the polar lipid profiles distinguished the two species from representatives of the genus Roseomonas. The fatty acid composition of the two species exhibited alphaproteobacterial characteristics but, like Roseomonas species, they also showed considerable amounts of the rarely encountered C(18 : 1) 2-OH. On the other hand, they showed some phenotypic differences, but their features are compatible with the transfer of these two species to the genus Roseomonas. We propose the reclassification of Teichococcus ludipueritiae and Muricoccus roseus as Roseomonas ludipueritiae comb. nov. (type strain 170/96(T) =CIP 107418(T) =DSM 14915(T)) and Roseomonas rosea comb. nov. (type strain 173/96(T) =CIP 107419(T) =DSM 14916(T)), respectively. Emended descriptions of the genus Roseomonas and the species Roseomonas gilardii (and its subspecies Roseomonas gilardii subsp. gilardii and Roseomonas gilardii subsp. rosea), Roseomonas aquatica, Roseomonas cervicalis, Roseomonas mucosa and Roseomonas lacus are provided.

Bacillus iranensis sp. nov., a moderate halophile from a hypersaline lake.

A Gram-positive, moderately halophilic rod, designated X5BT, was isolated from saline mud of the hypersaline lake Aran-Bidgol in Iran. Strain X5BT was a strictly aerobic, motile bacterium that produced ellipsoidal endospores at a central-subterminal position in non-swollen sporangia. The isolate grew at pH 7.0-10.0 (optimum pH 7.5), at 25-45 °C (optimum 35 °C) and with 2.5-15 % (w/v) NaCl (optimum 5-7.5 %). On the basis of 16S rRNA gene sequences, strain X5BT belonged to the genus Bacillus and showed highest similarity with Bacillus persepolensis HS136T (95.6 % 16S rRNA gene sequence similarity) and Bacillus salarius BH169T (95.5 %). The DNA G+C content was 42.4 mol%. The major cellular fatty acids were anteiso-C15:0 and iso-C15:0 and the polar lipid profile consisted of phosphatidylglycerol, diphosphatidylglycerol, three phospholipids and two glycolipids. The diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid and the isoprenoid quinones were MK-7 (92 %), MK-6 (6 %) and MK-5 (2 %). On the basis of phylogenetic, chemotaxonomic and phenotypic data, a novel species of the genus Bacillus is proposed, with the name Bacillus iranensis sp. nov. The type strain is X5BT (=IBRC 10446T=DSM 23995T).