Characterization of heterotrophic prokaryote subgroups in the Sfax coastal solar salterns by combining flow cytometry cell sorting and phylogenetic analysis.
ABSTRACT: 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.
Project description: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.
Project description:Recent studies on archaeal diversity in few salterns have revealed heterogeneity between sites and unique structures of separate places that hinder drawing of generalized conclusions. Investigations on the archaeal community composition in P18, the biggest crystallizer pond in Pomorie salterns (PS) (34% salinity), demonstrated unusually high number of presented taxa in hypersaline environment. Archaeal clones were grouped in 26 different operational taxonomic units (OTUs) assigned to 15 different genera from two orders, Halobacteriales and Haloferacales. All retrieved sequences were related to culturable halophiles or unculturable clones from saline (mostly hypersaline) niches. New sequences represented 53.9% of archaeal OTUs. Some of them formed separate branches with 90% similarity to the closest neighbor. Present results significantly differed from the previous investigations in regard to the number of presented genera, the domination of some genera not reported before in such extreme niche, and the identification of previously undiscovered 16S rRNA sequences.
Project description: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.
Project description:Cáhuil Lagoon in central Chile harbors distinct microbial communities in various solar salterns that are arranged as interconnected ponds with increasing salt concentrations. Here, we report the metagenome of the 3.0- to 0.2-µm fraction of the microbial community present in a crystallizer pond with 34% salinity.
Project description:Here we used flow cytometry (FCM) and filtration paired with amplicon sequencing to determine the abundance and composition of small low nucleic acid (LNA)-content bacteria in a variety of freshwater ecosystems. We found that FCM clusters associated with LNA-content bacteria were ubiquitous across several ecosystems, varying from 50 to 90% of aquatic bacteria. Using filter-size separation, we separated small LNA-content bacteria (passing 0.4?µm filter) from large bacteria (captured on 0.4?µm filter) and characterized communities with 16S amplicon sequencing. Small and large bacteria each represented different sub-communities within the ecosystems' community. Moreover, we were able to identify individual operational taxonomical units (OTUs) that appeared exclusively with small bacteria (434 OTUs) or exclusively with large bacteria (441 OTUs). Surprisingly, these exclusive OTUs clustered at the phylum level, with many OTUs appearing exclusively with small bacteria identified as candidate phyla (i.e. lacking cultured representatives) and symbionts. We propose that LNA-content bacteria observed with FCM encompass several previously characterized categories of bacteria (ultramicrobacteria, ultra-small bacteria, candidate phyla radiation) that share many traits including small size and metabolic dependencies on other microorganisms.
Project description: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 and 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.
Project description:Increasing efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve activity (ARNA), which in turn decreases ERSNA via activation of the renorenal reflexes in the overall goal of maintaining low ERSNA. We now examined whether the ERSNA-induced increases in ARNA are modulated by dietary sodium and the role of endothelin (ET). The ARNA response to reflex increases in ERSNA was enhanced in high (HNa)- vs. low-sodium (LNa) diet rats, 7,560 +/- 1,470 vs. 900 +/- 390%.s. The norepinephrine (NE) concentration required to increase PGE(2) and substance P release from isolated renal pelvises was 10 pM in HNa and 6,250 pM in LNa diet rats. In HNa diet pelvises 10 pM NE increased PGE(2) release from 67 +/- 6 to 150 +/- 13 pg/min and substance P release from 6.7 +/- 0.8 to 12.3 +/- 1.8 pg/min. In LNa diet pelvises 6,250 pM NE increased PGE(2) release from 64 +/- 5 to 129 +/- 22 pg/min and substance P release from 4.5 +/- 0.4 to 6.6 +/- 0.7 pg/min. In the renal pelvic wall, ETB-R are present on unmyelinated Schwann cells close to the afferent nerves and ETA-R on smooth muscle cells. ETA-receptor (R) protein expression in the renal pelvic wall is increased in LNa diet. In HNa diet, renal pelvic administration of the ETB-R antagonist BQ788 reduced ERSNA-induced increases in ARNA and NE-induced release of PGE(2) and substance P. In LNa diet, the ETA-R antagonist BQ123 enhanced ERSNA-induced increases in ARNA and NE-induced release of substance P without altering PGE(2) release. In conclusion, activation of ETB-R and ETA-R contributes to the enhanced and suppressed interaction between ERSNA and ARNA in conditions of HNa and LNa diet, respectively, suggesting a role for ET in the renal control of ERSNA that is dependent on dietary sodium.
Project description:The microbial community inhabiting Sfax solar salterns on the east coast of Tunisia has been studied by means of different molecular and culture-dependent tools that have unveiled the presence of novel microbial groups as well as a community structure different from that of other coastal hypersaline environments. We have focused on the study of the viral assemblages of these salterns and their changes along the salinity gradient and over time. Viruses from three ponds (C4, M1, and TS) encompassing salinities from moderately hypersaline to saturated (around 14, 19, and 35%, respectively) were sampled in May and October 2009 and analyzed by transmission electron microscopy (TEM) and pulsed-field gel electrophoresis (PFGE). Additionally, for all three October samples and the May TS sample, viral metagenomic DNA was cloned in fosmids, end sequenced, and analyzed. Viral concentration, as well as virus-to-cell ratios, increased along the salinity gradient, with around 10(10) virus-like particles (VLPs)/ml in close-to-saturation ponds, which represents the highest viral concentration reported so far for aquatic systems. Four distinct morphologies could be observed with TEM (spherical, tailed, spindled, and filamentous) but with various proportions in the different samples. Metagenomic analyses indicated that every pond harbored a distinct viral assemblage whose G+C content could be roughly correlated with that of the active part of the microbial community that may have constituted the putative hosts. As previously reported for hypersaline metaviromes, most sequences did not have matches in the databases, although some were conserved among the Sfax metaviromes. BLASTx, BLASTp, and dinucleotide frequency analyses indicated that (i) factors additional to salinity could be structuring viral communities and (ii) every metavirome had unique gene contents and dinucleotide frequencies. Comparison with hypersaline metaviromes available in the databases indicated that the viral assemblages present in close-to-saturation environments located thousands of kilometers apart presented some common traits among them in spite of their differences regarding the putative hosts. A small core metavirome for close-to-saturation systems was found that contained 7 sequences of around 100 nucleotides (nt) whose function was not hinted at by in silico search results, although it most likely represents properties essential for hyperhalophilic viruses.
Project description:Heterotrophic bacteria play a major role in organic matter cycling in the ocean. Although the high abundances and relatively fast growth rates of coastal surface bacterioplankton make them suitable sentinels of global change, past analyses have largely overlooked this functional group. Here, time series analysis of a decade of monthly observations in temperate Atlantic coastal waters revealed strong seasonal patterns in the abundance, size and biomass of the ubiquitous flow-cytometric groups of low (LNA) and high nucleic acid (HNA) content bacteria. Over this relatively short period, we also found that bacterioplankton cells were significantly smaller, a trend that is consistent with the hypothesized temperature-driven decrease in body size. Although decadal cell shrinking was observed for both groups, it was only LNA cells that were strongly coherent, with ecological theories linking temperature, abundance and individual size on both the seasonal and interannual scale. We explain this finding because, relative to their HNA counterparts, marine LNA bacteria are less diverse, dominated by members of the SAR11 clade. Temperature manipulation experiments in 2012 confirmed a direct effect of warming on bacterial size. Concurrent with rising temperatures in spring, significant decadal trends of increasing standing stocks (3% per year) accompanied by decreasing mean cell size (-1% per year) suggest a major shift in community structure, with a larger contribution of LNA bacteria to total biomass. The increasing prevalence of these typically oligotrophic taxa may severely impact marine food webs and carbon fluxes by an overall decrease in the efficiency of the biological pump.
Project description:Organism size reduction with increasing temperature has been suggested as a universal response to global warming. Since genome size is usually correlated to cell size, reduction of genome size in unicells could be a parallel outcome of warming at ecological and evolutionary time scales. In this study, the short-term response of cell size and nucleic acid content of coastal marine prokaryotic communities to temperature was studied over a full annual cycle at a NE Atlantic temperate site. We used flow cytometry and experimental warming incubations, spanning a 6°C range, to analyze the hypothesized reduction with temperature in the size of the widespread flow cytometric bacterial groups of high and low nucleic acid content (HNA and LNA bacteria, respectively). Our results showed decreases in size in response to experimental warming, which were more marked in 0.8 ?m pre-filtered treatment rather than in the whole community treatment, thus excluding the role of protistan grazers in our findings. Interestingly, a significant effect of temperature on reducing the average nucleic acid content (NAC) of prokaryotic cells in the communities was also observed. Cell size and nucleic acid decrease with temperature were correlated, showing a common mean decrease of 0.4% per °C. The usually larger HNA bacteria consistently showed a greater reduction in cell and NAC compared with their LNA counterparts, especially during the spring phytoplankton bloom period associated to maximum bacterial growth rates in response to nutrient availability. Our results show that the already smallest planktonic microbes, yet with key roles in global biogeochemical cycling, are likely undergoing important structural shrinkage in response to rising temperatures.