Nested PCR and New Primers for Analysis of Sulfate-Reducing Bacteria in Low-Cell-Biomass Environments▿ †

New primers were designed for the amplification of dsrAB genes by nested PCR to investigate the diversity of sulfate-reducing prokaryotes (SRP) in environments with low bacterial cell density. The success of the nested PCR for the determination of SRP diversity was estimated by terminal-restriction fragment length polymorphism analysis in the Reigous, a small creek at an inactive mine (Carnoulès, France), which constitutes an extreme acidic arsenic-rich environment. Nested PCR limits were evaluated in dsrAB-rich sediments, and this technique was compared to direct PCR using either known primers (DSR1F/DSR4R) or new primers (dsr619AF/dsr1905BR). The comparison of clone libraries revealed that, even if the levels of diversity observed were not identical, nested PCR did not reduce the diversity compared to that of direct DSR1F/DSR4R PCR. Clone sequences were affiliated mainly with the Desulfobacteraceae and Desulfohalobiaceae families. Many sequences (∼30%) were related to a deeply branching lineage unaffiliated with any cultured SRP. Although this dsrAB cluster was found in all libraries, the new primers better amplified this lineage, providing more information on this unknown bacterial group. Thanks to these new primers in nested PCR, the SRP community from Carnoulès could be characterized. Specific SRP populations were obtained according to environmental characteristics. Desulfomicrobiaceae-related sequences were recovered in samples with low pH, low levels of dissolved oxygen, and high As content, while sequences belonging to the deeply branching group were found in a less extreme sample. Furthermore, for the first time, dsrAB sequences related to the latter group were recovered from freshwater

Combination of high throughput cultivation and dsrA sequencing for assessment of sulfate-reducing bacteria diversity in sediments

International audienceImproving the knowledge on sulfate-reducing bacteria (SRB) diversity and ecophysiology will permit a better understanding on their key roles in aquatic ecosystems. Therefore, their diversity was evaluated in estuarine sediments by a polyphasic approach including dsrA gene cloning and sequencing (156 clones) and high-throughput isolations in 384-well microplates (177 strains). Using the related thresholds of 95% (DsrA amino acid sequences) and 97% (16S rRNA gene sequences) for sequence similarity, SRB were grouped into 60 and 22 operational taxonomic units, respectively. Both approaches poorly overlapped and rather complemented each other. The clone library was dominated by sequences related to the Desulfobacteraceae, while only one isolate belonged to this family. Most of the strains were affiliated to the genera Desulfopila and Desulfotalea within the Desulfobulbaceae. Desulfopila-related strains exhibited a high phylogenetic microdiversity and represented numerically significant populations. In contrast, Desulfovibrio isolates were less abundant but displayed a high phylogenetic diversity. Three hundred and eighty-four-well microplate isolations enhanced significantly the number of isolates handled. As a consequence, 15 new taxa sharing less than 98% sequence similarity (16S rRNA gene) with their closest relatives were obtained. This polyphasic approach allowed to obtain a high phylogenetic diversity and thus a better view of sulfate-reducing communities in intertidal sediments. © 2012 Federation of European Microbiological Societies

Coupling fluorescent probes to characterize S-containing compounds in a sulfate reducing bacteria involved in Hg methylation

The microbial methylation of inorganic mercury Hg(II) is governed by S-containing compounds such as thiols (RSH) and sulfides (S2−). Various S-containing molecules in an environmental or culture medium can be difficult to assess because of the complexity of the medium, poor stability, and low concentration ranges of sulfide and thiol compounds. Here, we applied two fluorescence spectroscopy-based methods using α, β-unsaturated ethanoylcoumarin fluorophore (DHC) for the quantification of sulfides, and monobromo (trimethylammonio) bimane (qBBr) to quantify total thiol concentrations (in extracellular and bacterial cell fractions). The potential interferences of both organic and inorganic compounds from the matrix were evaluated. In the presence of Hg species, both methods allowed the quantification of free sulfides or thiols (not forming complexes with Hg). The two methods were highly sensitive, with detection limits of 100 nM and 20 nM for thiols and sulfides, respectively. They also exhibited high selectivity for the detection of thiols or sulfides against other tested matrix compounds. Finally, both methods were applied to characterize S-containing compounds in a culture of Pseudodesulfovibrio hydrargyri strain BerOc1, a methylating sulfate-reducing bacterium (SRB) exposed to 0.1 mM of cysteine. During bacterial growth, we used (i) DHC probe to quantify sulfide concentration in the bulk fraction, (ii) qBBr for total extracellular thiols and total thiols adsorbed on the cells, and (iii) liquid chromatography-tandem mass spectrometry to track cysteine degradation and characterize other thiols. The time series until the end of BerOc1 growth showed biodegradation of cysteine, and biosynthesis of sulfides and other thiol compounds

Pseudodesulfovibrio hydrargyri sp. nov., a mercury-methylating bacterium isolated from a brackish sediment

International audienceThe strain BerOc1T was isolated from brackish sediments contaminated with hydrocarbons and heavy metals. This strain has been used as a model strain of sulfate-reducer to study the biomethylation of mercury. The cells are vibrio-shaped, motile and not sporulated. Phylogeny and physiological traits placed this strain within the genus Pseudodesulfovibrio. Optimal growth was obtained at 30 °C, 1.5 % NaCl and pH 6.0-7.4. The estimated G+C content of the genomic DNA was 62.6 mol%. BerOc1T used lactate, pyruvate, fumarate, ethanol and hydrogen. Terminal electron acceptors used were sulfate, sulfite, thiosulfate and DMSO. Only pyruvate could be used without a terminal electron acceptor. The major fatty acids were C18 : 0, anteiso-C15 : 0, C16 : 0 and C18 : 1ω7. The name Pseudodesulfovibrio hydrargyri sp. nov. is proposed for the type strain BerOc1T (DSM 10384T=JCM 31820T

Bacterial diversity of an acid mine drainage beside the Xichú River (Mexico) accessed by culture-dependent and culture-independent approaches

International audienceXichú River is a Mexican river located in an environmental preservation area called Sierra Gorda Biosphere Reserve. Around it, there are tons of abandoned mine residues that represent a serious environmental issue. Sediment samples of Xichú River, visibly contaminated by flows of an acid mine drainage, were collected to study their prokaryotic diversity. The study was based on both cultural and non-cultural approaches. The analysis of total 16S rRNA gene by MiSEQ sequencing allowed to identify 182 Operational Taxonomic Units. The community was dominated by Pseudomonadota, Bacteroidota, “Desulfobacterota” and Acidobacteriota (27, 21, 19 and 16%, respectively). Different culture conditions were used focusing on the isolation of anaerobic bacteria, including sulfate-reducing bacteria (SRB) and arsenate-reducing bacteria (ARB). Finally, 16 strains were isolated. Among them, 12 were phylogenetically identified, with two strains being SRB, belonging to the genus Solidesulfovibrio (“Desulfobacterota”), while ten are ARB belonging to the genera Azospira (Pseudomonadota), Peribacillus (Bacillota), Raineyella and Propionicimonas (Actinomycetota). The isolate representative of Raineyella genus probably corresponds to a new species, which, besides arsenate, also reduces nitrate, nitrite, and fumarate

The bacterial diversity on steam vents from Paricutín and Sapichu volcanoes

International audienceVapor steam vents are prevailing structures on geothermal sites in which local geochemical conditions allow the development of extremophilic microorganisms. We describe the structure of the prokaryotic community able to grow on the walls and rocks of such microecosystems in two terrestrial Mexican volcanoes: Paricutín (PI and PII samples) and its satellite Sapichu (S sample). The investigated samples showed similar diversity indices, with few dominant OTUs (abundance > 1%): 21, 16 and 23, respectively for PI, PII and S. However, each steam vent showed a particular community profile: PI was dominated by photosynthetic bacteria (Cyanobacteria and Chloroflexia class), PII by Actinobacteria and Proteobacteria, and S by Ktedonobacteria class, Acidobacteria and Cyanobacteria phyla. Concerning the predicted metabolic potential, we found a dominance of cellular pathways, especially the ones for energy generation with metabolisms for sulfur respiration, nitrogen fixation, methanogenesis, carbon fixation, photosynthesis, and metals, among others. We suggest a different maturity stage for the three studied fumaroles, from the youngest (PI) to the oldest (S and PII), also influenced by the temperature and other geochemical parameters. Furthermore, four anaerobic strains were isolated, belonging to Clostridia class (Clostridium sphenoides, C. swellfunanium and Anaerocolumna cellulosilytica) and to Bacilli class (Paenibacillus azoreducens)

Degradation of the

Since March 2001, samples of the remaining oil from the wreck of the “Erika” have been collected along the Atlantic coastline in order to assess the natural degradation rate. Four years after the sinking of the tanker, chemical analyses of the oil revealed the influence of environmental parameters on the degradation kinetics. Among the diverse parameters controlling the fate of oil in the environment, biodegradation by microorganisms is known to play an important role. To investigate the role of microorganisms on “Erika” oil degradation, microbial mats from the Guérande salt marches were maintained in slurries containing the pollutant. From these slurries experiments, a low biodegradation rate of the “Erika” oil was detected indicating the degradation capacities of microbial mats. Biodiversity studies were conducted to further understand the biodegradation processes. Microbial mats from the Guérande salterns were maintained in microcosms to evaluate the impact of “Erika” oil on bacterial communities. Molecular analysis based on 16S rRNA and pufM encoding genes allowed fingerprinting of the bacterial and purple anoxygenic bacterial (PAB) communities respectively. These studies revealed bacterial diversity and communities changes showing the adaptation of microorganisms to the “Erika”

Linking Microbial Activities and Low-Molecular-Weight Thiols to Hg Methylation in Biofilms and Periphyton from High-Altitude Tropical Lakes in the Bolivian Altiplano

International audienceThe sources and factors controlling concentrations of monomethylmercury (MMHg) in aquatic ecosystems need to be better understood. Here, we investigated Hg transformations in sediments, periphyton associated with green algae's or aquatic plants, and benthic biofilms from the Lake Titicaca hydrosystem and compared them to the occurrence of active methylating microorganisms and extracellular Hg ligands. Intense Hg methylation was found in benthic biofilms and green algae's periphyton, while it remained low in sediments and aquatic plants' periphyton. Demethylation varied between compartments but remained overall in the same range. Hg methylation was mainly carried out by sulfate reducers, although methanogens also played a role. Its variability between compartments was first explained by the presence or absence of the hgcAB genes. Next, both benthic biofilm and green algae's periphyton exhibited a great diversity of extracellular low-molecular-weight (LMW) thiols (13 or 14 compounds) present at a range of a few nmol L−1 or μmol L−1 but clearly dominated by cysteine and 3-mercaptopropionic acid. Hg methylation was overall positively correlated to the total thiol concentrations, albeit to different extents according to the compartment and conditions. This work is the first examining the interplay between active methylating bacterial communities and extracellular ligands in heterotrophic biofilms and supports the involvement of LMW thiols in Hg methylation in real aquatic systems