Ana Beatriz Fernández

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.

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.

Microbial Diversity in Sediment Ecosystems (Evaporites Domes, Microbial Mats, and Crusts) of Hypersaline Laguna Tebenquiche, Salar de Atacama, Chile.

We combined nucleic acid-based molecular methods, biogeochemical measurements, and physicochemical characteristics to investigate microbial sedimentary ecosystems of Laguna Tebenquiche, Atacama Desert, Chile. Molecular diversity, and biogeochemistry of hypersaline microbial mats, rhizome-associated concretions, and an endoevaporite were compared with: The V4 hypervariable region of the 16S rRNA gene was amplified by pyrosequencing to analyze the total microbial diversity (i.e., bacteria and archaea) in bulk samples, and in addition, in detail on a millimeter scale in one microbial mat and in one evaporite. Archaea were more abundant than bacteria. Euryarchaeota was one of the most abundant phyla in all samples, and particularly dominant (97% of total diversity) in the most lithified ecosystem, the evaporite. Most of the euryarchaeal OTUs could be assigned to the class Halobacteria or anaerobic and methanogenic archaea. Planctomycetes potentially also play a key role in mats and rhizome-associated concretions, notably the aerobic organoheterotroph members of the class Phycisphaerae. In addition to cyanobacteria, members of Chromatiales and possibly the candidate family Chlorotrichaceae contributed to photosynthetic carbon fixation. Other abundant uncultured taxa such as the candidate division MSBL1, the uncultured MBGB, and the phylum Acetothermia potentially play an important metabolic role in these ecosystems. Lithifying microbial mats contained calcium carbonate precipitates, whereas endoevoporites consisted of gypsum, and halite. Biogeochemical measurements revealed that based on depth profiles of O2 and sulfide, metabolic activities were much higher in the non-lithifying mat (peaking in the least lithified systems) than in lithifying mats with the lowest activity in endoevaporites. This trend in decreasing microbial activity reflects the increase in salinity, which may play an important role in the biodiversity.

Bacterial Diversity in Microbial Mats and Sediments from the Atacama Desert

The Atacama Desert has extreme environmental conditions that allow the development of unique microbial communities. The present paper reports the bacterial diversity of microbial mats and sediments and its mineralogical components. Some physicochemical conditions of the water surrounding these ecosystems have also been studied trying to determine their influence on the diversity of these communities. In that way, mats and sediments distributed among different hypersaline lakes located in salt flats of the Atacama Desert were subjected to massive parallel sequencing of the V4 region of the 16S rRNA genes of Bacteria. A higher diversity in sediment than in mat samples have been found. Lakes that harbor microbial mats have higher salinity than lakes where mats are absent. Proteobacteria and/or Bacteroidetes are the major phyla represented in all samples. An interesting item is the finding of a low proportion or absence of Cyanobacteria sequences in the ecosystems studied, suggesting the possibility that other groups may be playing an essential role as primary producers in these extreme environments. Additionally, the large proportion of 16S rRNA gene sequences that could not be classified at the level of phylum indicates potential new phyla present in these ecosystems.

Lentibacillus persicus sp. nov., a moderately halophilic species isolated from a saline lake

A Gram-staining-positive, moderately halophilic bacterium, designated strain Amb31(T), was isolated from water of the hypersaline lake Aran-Bidgol in Iran and characterized taxonomically using a polyphasic approach. Cells were rods, motile and able to produce ellipsoidal endospores at a central position in swollen sporangia. Strain Amb31(T) was facultatively anaerobic and catalase- and oxidase-positive. The strain grew in a complex medium supplemented with 3-25 % (w/v) NaCl (optimum 7.5-10 %). Optimal growth was at 30-35 degrees C and pH 7.5. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain Amb31(T) belonged to the genus Lentibacillus; it exhibited 16S rRNA gene sequence similarity values of 96.8 and 96.4 % to Lentibacillus salicampi SF-20(T) and Lentibacillus salinarum AHS-1(T), respectively, and values of 95.9-94.7 % to the type strains of other recognized species of Lentibacillus. The cell-wall peptidoglycan of strain Amb31(T) was based on meso-diaminopimelic acid and MK-7 was the respiratory isoprenoid quinone. The major fatty acids were anteiso-C(15 : 0) (44.7 %), iso-C(16 : 0) (21.4 %) and anteiso-C(17 : 0) (15.9 %) and the polar lipid pattern consisted of phosphatidylglycerol, diphosphatidylglycerol, five phospholipids and a glycolipid. The DNA G+C content was 44.1 mol%. All these features confirmed the placement of strain Amb31(T) within the genus Lentibacillus and the strain could be clearly differentiated from strains of the other species of Lentibacillus on the basis of several phenotypic, genotypic and chemotaxonomic features. DNA-DNA relatedness with the type strain of the most closely related strain, L. salicampi DSM 16425(T), was 28 %. Therefore, strain Amb31(T) represents a novel species of the genus Lentibacillus, for which the name Lentibacillus persicus sp. nov. is proposed. The type strain is Amb31(T) (=CCM 7683(T) =CECT 7524(T) =DSM 22530(T) =LMG 25304(T)).

Draft Genome of Spiribacter salinus M19-40, an Abundant Gammaproteobacterium in Aquatic Hypersaline Environments

ABSTRACT We have previously used a de novo metagenomic assembly approach to describe the presence of an abundant gammaproteobacterium comprising nearly 15% of the microbial community in an intermediate salinity solar saltern pond. We have obtained this microbe in pure culture and describe the genome sequencing of the halophilic photoheterotrophic microbe, Spiribacter salinus M19-40.

Metagenome Sequencing of Prokaryotic Microbiota from Two Hypersaline Ponds of a Marine Saltern in Santa Pola, Spain

ABSTRACT Marine salterns are composed of several shallow ponds with a salinity gradient, from seawater to salt saturation, with gradually changing microbial populations. Here, we report the metagenome sequencing of the prokaryotic microbiota of two ponds with 13% and 33% salinity from a saltern in Santa Pola, Spain.

Metagenomic sequence of prokaryotic microbiota from an intermediate-salinity pond of a saltern in isla cristina, Spain.

ABSTRACT Marine salterns are artificial multipond systems designed for the commercial production of salt by evaporation of seawater. We report here the metagenomic sequence of the prokaryotic microbiota of a pond with intermediate salinity (21% total salts) of a saltern located in Isla Cristina, Huelva, southwest Spain.

Prokaryotic diversity and biogeochemical characteristics of benthic microbial ecosystems at La Brava, a hypersaline lake at Salar de Atacama, Chile

Benthic microbial ecosystems of Laguna La Brava, Salar de Atacama, a high altitude hypersaline lake, were characterized in terms of bacterial and archaeal diversity, biogeochemistry, (including O2 and sulfide depth profiles and mineralogy), and physicochemical characteristics. La Brava is one of several lakes in the Salar de Atacama where microbial communities are growing in extreme conditions, including high salinity, high solar insolation, and high levels of metals such as lithium, arsenic, magnesium, and calcium. Evaporation creates hypersaline conditions in these lakes and mineral precipitation is a characteristic geomicrobiological feature of these benthic ecosystems. In this study, the La Brava non-lithifying microbial mats, microbialites, and rhizome-associated concretions were compared to each other and their diversity was related to their environmental conditions. All the ecosystems revealed an unusual community where Euryarchaeota, Crenarchaeota, Acetothermia, Firmicutes and Planctomycetes were the most abundant groups, and cyanobacteria, typically an important primary producer in microbial mats, were relatively insignificant or absent. This suggests that other microorganisms, and possibly novel pathways unique to this system, are responsible for carbon fixation. Depth profiles of O2 and sulfide showed active production and respiration. The mineralogy composition was calcium carbonate (as aragonite) and increased from mats to microbialites and rhizome-associated concretions. Halite was also present. Further analyses were performed on representative microbial mats and microbialites by layer. Different taxonomic compositions were observed in the upper layers, with Archaea dominating the non-lithifying mat, and Planctomycetes the microbialite. The bottom layers were similar, with Euryarchaeota, Crenarchaeota and Planctomycetes as dominant phyla. Sequences related to Cyanobacteria were very scarce. These systems may contain previously uncharacterized community metabolisms, some of which may be contributing to net mineral precipitation. Further work on these sites might reveal novel organisms and metabolisms of biotechnological interest.