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

Draft genome sequence of desulfovibrio BerOc1, a mercury-methylating Strain

Desulfovibrio BerOc1 is a sulfate-reducing bacterium isolated from the Berre lagoon (French Mediterranean coast). BerOc1 is able to methylate and demethylate mercury. The genome size is 4,081,579 bp assembled into five contigs. We identified the hgcA and hgcB genes involved in mercury methylation, but not those responsible for mercury demethylation

Distribution of sulfate-reducing communities from estuarine to marine bay waters

Estuaries are highly dynamic ecosystems in which freshwater and seawater mix together. Depending on tide and river inflows, particles originating from rivers or from the remobilization of sediments accumulate in the water column. Due to the salinity gradient and the high heterotrophic activity in the estuarine plume, hypoxic and anoxic microniches may form in oxygenated waters, sustaining favorable conditions for resuspended anaerobic microorganisms. In this context, we tested the hypothesis that anaerobic sulfate-reducing prokaryotes may occur in the water column of the Adour River. Using 16S ribosomal RNA (rRNA) and dsrAB-based terminal restriction fragment length polymorphism (T-RFLP) techniques, we characterized total prokaryotic and sulfate-reducing communities along a gradient from estuarine to marine bay waters.[br/] Sulfate-reducing prokaryotes were further characterized by the description of dsrB genes and the cultivation of sulfidogenic anaerobic microorganisms. As a result, physical-chemical parameters had a significant effect on water bacterial diversity and community structure along the studied gradient. The concentration of cultured sulfidogenic microorganisms ranged from 1 to 60 x 10(3) cells l(-1) in the water column. Sulfate-reducing prokaryotes occurring in estuarine waters were closely related to microorganisms previously detected in freshwater sediments, suggesting an estuarine origin, mainly by the remobilization of the sediments. In the marine bay station, sediment-derived sulfate-reducing prokaryotes were not cultured anymore, probably due to freshwater dilution, increasing salinity and extended oxic stress.[br/] Nevertheless, isolates related to the type strain Desulfovibrio oceani were cultured from the diluted plume and deep marine waters, indicating the occurrence of autochthonous sulfate-reducing bacteri

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

Impact of an urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas spp.

cited By 241International audienceIn order to evaluate the impact of an urban effluent on antibiotic resistance of freshwater bacterial populations, water samples were collected from the Arga river (Spain), upstream and downstream from the wastewater discharge of the city of Pamplona. Strains of Enterobacteriaceae (representative of the human and animal commensal flora) (110 isolates) and Aeromonas (typically waterborne bacteria) (118 isolates) were selected for antibiotic susceptibility testing. Most of the Aeromonas strains (72%) and many of the Enterobacteriaceae (20%) were resistant to nalidixic acid. Singly nalidixic acid-resistant strains were frequent regardless of the sampling site for Aeromonas, whereas they were more common upstream from the discharge for enterobacteria. The most common resistances to antibiotics other than quinolones were to tetracycline (24.3%) and beta-lactams (20.5%) for Enterobacteriaceae and to tetracycline (27.5%) and co-trimoxazole (26.6%) for Aeromonas. The rates of these antibiotic resistances increased downstream from the discharge at similar degrees for the two bacterial groups; it remained at high levels for enterobacteria but decreased along the 30-km study zone for Aeromonas. Genetic analysis of representative strains demonstrated that these resistances were mostly (enterobacteria) or exclusively (Aeromonas) chromosomally mediated. Moreover, a reference strain of Aeromonas caviae (CIP 7616) could not be transformed with conjugative R plasmids of enterobacteria. Thus, the urban effluent resulted in an increase of the rates of resistance to antibiotics other than quinolones in the riverine bacterial populations, despite limited genetic exchanges between enterobacteria and Aeromonas. Quinolone resistance probably was selected by heavy antibiotic discharges of unknown origin upstream from the urban effluent

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

Effect of Exogenous and Endogenous Sulfide on The Production and The Export of Methylmercury by Sulfate Reducing Bacteria _ Preprint version

International audienceMercury (Hg) is a global pollutant of environmental and health concern; its methylated form, methylmercury (MeHg) is a potent neurotoxin. Sulfur-containing molecules play a role in MeHg production by microorganisms. While sul des are considered to limit Hg methylation, sulfate and cysteine were shown to favor this process. However, these two forms can be endogenously converted by microorganisms into sul de. Here, we explore the effect of sul de (produced by the cell or supplied exogenously) on Hg methylation. For this purpose, Pseudodesulfovibrio hydrargyri BerOc1 was cultivated in non-sul dogenic conditions with addition of cysteine and sul de as well as in sul dogenic conditions. We report that Hg methylation depends on sul de concentration in the culture rather than on the initial form of sulfur (cysteine, sul de or sulfate) added, and was independent of hgcA expression. Interestingly, MeHg production was maximal at 0.1-0.5 mM of sul des. Besides, a strong positive correlation between MeHg in the extracellular medium and the increase of sul de concentrations was observed, suggesting a facilitated MeHg export with sul de and/or higher desorption from the cell. We demonstrate that sul des (exogenous or endogenous) play a key role in controlling mercury methylation, and should be considered when investigating the impact of Hg on natural environments

Genome insights of mercury methylation among Desulfovibrio and Pseudodesulfovibrio strains

International audienceMercury methylation converts inorganic mercury into the toxic methylmercury, and the consequences of this transformation are worrisome for human health and the environment. This process is performed by anaerobic microorganisms, such as several strains related to Pseudodesulfovibrio and Desulfovibrio genera. In order to provide new insights into the molecular mechanisms of mercury methylation, we performed a comparative genomic analysis on mercury methylators and non-methylators from (Pseudo)Desulfovibrio strains. Our results showed that (Pseudo)Desulfovibrio species are phylogenetically and metabolically distant and consequently, these genera should be divided into various genera. Strains able to perform methylation are affiliated with one branch of the phylogenetic tree, but, except for hgcA and hgcB genes, no other specific genetic markers were found among methylating strains. hgcA and hgcB genes can be found adjacent or separated, but proximity between those genes does not promote higher mercury methylation. In addition, close examination of the non-methylator Pseudodesulfovibrio piezophilus C1TLV30 strain, showed a syntenic structure that suggests a recombination event and may have led to hgcB depletion. The genomic analyses identify also arsR gene coding for a putative regulator upstream hgcA. Both genes are cotranscribed suggesting a role of ArsR in hgcA expression and probably a role in mercury methylation