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

Colin Y., Gury J., Monperrus M., Gentes S., Ayala Borda P., Goni-Urriza M., Guyoneaud R.

Mercury methylation and demethylation processes govern the fate of methylmercury in aquatic ecosystems. Under anoxic conditions, methylation activity is mainly of biological origin and is often the result of sulfate-reducing bacteria. In this study, the use of a luminescent biosensor for screening methylmercury production was validated by exposing the reporter strain to methylating or non-methylating Desulfovibrio strains. The sensitivity of the biosensor to methylmercury was shown to depend on sulfate-reducing bacterial growth conditions. Bioluminescence was measured using 1-10 mM of sulfides. As the sulfide level increased, luminescence decreased by 40-70%, respectively. Nevertheless, assuming an average of 5 mM of sulfide produced during sulfate-reducing growth, a mercury methylation potential of over 4% was detected when using 185 nM of inorganic mercury. Due to technical limitations, mercury speciation has, to date, only been investigated in a small number of bacterial strains, and no consistent phylogenetic distribution has been identified. Here, the biosensor was further used to assess the Hg methylation capacities of an additional 21 strains related to the Desulfobulbaceae. Seven of them were identified as methylmercury producers. Cultivation procedures combined with bacterial biosensors could provide innovative tools to identify new methylator clades amongst the prokaryotes.

Gentès S., Taupiac J., Colin Y., André J., Guyoneaud R.

Macrophyte floating roots are considered as hotspots for methylmercury (MeHg) production in aquatic ecosystems through microbial activity. Nevertheless, very little is known about periphyton bacterial communities and mercury (Hg) methylators in such ecological niches. The ability to methylate inorganic Hg is broadly distributed among prokaryotes; however, sulfate-reducers have been reported to be the most important MeHg producers in macrophyte floating roots. In the present work, the periphyton bacterial communities colonizing Ludwigia sp. floating roots were investigated through molecular methods. Among the 244 clones investigated, anaerobic microorganisms associated with the sulfur biogeochemical cycle were identified. Notably, members of the sulfur-oxidizing prokaryotes and the anoxygenic, purple non-sulfur bacteria (Rhodobacteraceae, Comamonadaceae, Rhodocyclaceae, Hyphomicrobiaceae) and the sulfate reducers (Desulfobacteraceae, Syntrophobacteraceae, and Desulfobulbaceae) were detected. In addition, 15 sulfate-reducing strains related to the Desulfovibrionaceae family were isolated and their Hg-methylation capacity was tested using a biosensor. The overall results confirmed that Hg methylation is a strain-specific process since the four strains identified as new Hg-methylators were closely related to non-methylating isolates. This study highlights the potential involvement of periphytic bacteria in Hg methylation when favorable environmental conditions are present in such ecological micro-niches.

Yoon S., Ha S., Lim J., Kwon S., Chun J.

Average nucleotide identity (ANI) is a category of computational analysis that can be used to define species boundaries of Archaea and Bacteria. Calculating ANI usually involves the fragmentation of genome sequences, followed by nucleotide sequence search, alignment, and identity calculation. The original algorithm to calculate ANI used the BLAST program as its search engine. An improved ANI algorithm, called OrthoANI, was developed to accommodate the concept of orthology. Here, we compared four algorithms to compute ANI, namely ANIb (ANI algorithm using BLAST), ANIm (ANI using MUMmer), OrthoANIb (OrthoANI using BLAST) and OrthoANIu (OrthoANI using USEARCH) using >100,000 pairs of genomes with various genome sizes. By comparing values to the ANIb that is considered a standard, OrthoANIb and OrthoANIu exhibited good correlation in the whole range of ANI values. ANIm showed poor correlation for ANI of <90%. ANIm and OrthoANIu runs faster than ANIb by an order of magnitude. When genomes that are larger than 7 Mbp were analysed, the run-times of ANIm and OrthoANIu were shorter than that of ANIb by 53- and 22-fold, respectively. In conclusion, ANI calculation can be greatly sped up by the OrthoANIu method without losing accuracy. A web-service that can be used to calculate OrthoANIu between a pair of genome sequences is available at http://www.ezbiocloud.net/tools/ani . For large-scale calculation and integration in bioinformatics pipelines, a standalone JAVA program is available for download at http://www.ezbiocloud.net/tools/orthoaniu .

Goñi Urriza M., Gassie C., Bouchez O., Klopp C., Guyoneaud R.

ABSTRACT 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.

Berlendis S., Ranchou-Peyruse M., Fardeau M., Lascourrèges J., Joseph M., Ollivier B., Aüllo T., Dequidt D., Magot M., Ranchou-Peyruse A.

Two novel strictly anaerobic bacteria, strains Bs105T and Bs107T, were isolated from a deep aquifer-derived hydrocarbonoclastic community. The cells were rod-shaped, not motile and had terminal spores. Phylogenetic affiliation and physiological properties revealed that these isolates belong to two novel species of the genus Desulfotomaculum. Optimal growth temperatures for strains Bs105T and Bs107T were 42 and 45 °C, respectively. The estimated G+C content of the genomic DNA was 42.9 and 48.7 mol%. For both strains, the major cellular fatty acid was palmitate (C16 : 0). Specific carbon fatty acid signatures of Gram-positive bacteria (iso-C17 : 0) and sulfate-reducing bacteria (C17 : 0cyc) were also detected. An insertion was revealed in one of the two 16S rRNA gene copies harboured by strain Bs107T. Similar insertions have previously been highlighted among moderately thermophilic species of the genus Desulfotomaculum. Both strains shared the ability to oxidize aromatic acids (Bs105T: hydroquinone, acetophenone, para-toluic acid, 2-phenylethanol, trans-cinnamic acid, 4-hydroxybenzaldehyde, benzyl alcohol, benzoic acid 4-hydroxybutyl ester; Bs107T: ortho-toluic acid, benzoic acid 4-hydroxybutyl ester). The names Desulfotomaculum aquiferis sp. nov. and Desulfotomaculum profundi sp. nov. are proposed for the type strains Bs105T (=DSM 24088T=JCM 31386T) and Bs107T (=DSM 24093T=JCM 31387T).

Cao J., Gayet N., Zeng X., Shao Z., Jebbar M., Alain K.

A novel sulfate-reducing bacterium, strain J2T, was isolated from a serpentinized peridotite sample from the Indian Ocean. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain J2T clustered with the genus Desulfovibrio within the family Desulfovibrionaceae , but it showed low similarity (87.95 %) to the type species Desulfovibrio desulfuricans DSM 642T. It was most closely related to Desulfovibrio portus MSL79T (96.96 %), followed by Desulfovibrio aespoeensis Aspo-2T (96.11 %), Desulfovibrio piezophilus C1TLV30T (96.04 %) and Desulfovibrio profundus DSM 11384T (95.17 %). Other available sequences shared less than 93.33 % 16S rRNA gene sequence similarity. Cells were Gram-staining-negative, anaerobic, motile vibrios (2–6×0.4–0.6 µm). Growth was observed at salinities ranging from 0.2 to 6 % (optimum 2.5 %), from pH 5 to 8 (optimum pH 6.5–7) and at temperatures between 9 and 40 °C (optimum 30–35 °C). J2T was piezophilic, growing optimally at 10 MPa (range 0–30 MPa). J2T used lactate, malate, pyruvate, formate and hydrogen as energy sources. Sulfate, thiosulfate, sulfite, fumarate and nitrate were used as terminal electron acceptors. Lactate and pyruvate were fermented. The main fatty acids were iso-C15 : 0, anteiso-C15 : 0, summed feature 9 (iso-C17 : 1ω9c and/or C16 : 0 10-methyl) and iso-C17 : 0. The DNA G+C content of strain J2T was 63.5 mol%. The combined genotypic and phenotypic data show that strain J2T represents a novel species of a novel genus in the family Desulfovibrionaceae , for which the name Pseudodesulfovibrio indicus gen. nov., sp. nov. is proposed, with the type strain J2T (=MCCC 1A01867T = DSM 101483T). We also propose the reclassification of D. piezophilus as Pseudodesulfovibrio piezophilus comb. nov., D. profundus as Pseudodesulfovibrio profundus comb. nov., D. portus as Pseudodesulfovibrio portus comb. nov. and D. aespoeensis as Pseudodesulfovibrio aespoeensis comb. nov.

Cao J., Maignien L., Shao Z., Alain K., Jebbar M.

ABSTRACT The complete genome sequence of Desulfovibrio indicus J2 T , a member of the family Desulfovibrionaceae , consists of 3,966,573-bp in one contig and encodes 3,461 predicted genes, 5 noncoding RNAs, 3 rRNAs operons, and 52 tRNA-encoding genes. The genome is consistent with a heterotrophic, anaerobic lifestyle including the sulfate reduction pathway.

Goñi-Urriza M., Corsellis Y., Lanceleur L., Tessier E., Gury J., Monperrus M., Guyoneaud R.

The proteins encoded by the hgcA and hgcB genes are currently the only ones known to be involved in the mercury methylation by anaerobic microorganisms. However, no studies have been published to determine the relationships between their expression level and the net/gross methylmercury production. This study aimed to decipher the effect of growth conditions on methylmercury production and the relationships between hgcA and hgcB expression levels and net methylation. Desulfovibrio dechloroacetivorans strain BerOc1 was grown under sulfidogenic conditions with different carbon sources and electron donors as well as under fumarate respiration. A good correlation was found between the biomass production and the methylmercury production when the strain was grown under sulfate-reducing conditions. Methylmercury production was much higher under fumarate respiration when no sulfide was produced. During exponential growth, hgcA and hgcB gene expression levels were only slightly higher in the presence of inorganic mercury, and it was difficult to conclude whether there was a significant induction of hgcA and hgcB genes by inorganic mercury. Besides, no relationships between hgcA and hgcB expression levels and net mercury methylation could be observed when the strain was grown either under sulfate reduction or fumarate respiration, indicating that environmental factors had more influence than expression levels.

Perrot V., Bridou R., Pedrero Z., Guyoneaud R., Monperrus M., Amouroux D.

Inorganic mercury (iHg) methylation in aquatic environments is the first step leading to monomethylmercury (MMHg) bioaccumulation in food webs and might play a role in the Hg isotopic composition measured in sediments and organisms. Methylation by sulfate reducing bacteria (SRB) under sulfate-reducing conditions is probably one of the most important sources of MMHg in natural aquatic environments, but its influence on natural Hg isotopic composition remains to be ascertained. In this context, the methylating SRB Desulfovibrio dechloracetivorans (strain BerOc1) was incubated under sulfate reducing and fumarate respiration conditions (SR and FR, respectively) to determine Hg species specific (MMHg and IHg) isotopic composition associated with methylation and demethylation kinetics. Our results clearly establish Hg isotope mass-dependent fractionation (MDF) during biotic methylation (-1.20 to +0.58‰ for δ(202)Hg), but insignificant mass-independent fractionation (MIF) (-0.12 to +0.15‰ for Δ(201)Hg). During the 24h of the time-course experiments Hg isotopic composition in the produced MMHg becomes significantly lighter than the residual IHg after 1.5h and shows similar δ(202)Hg values under both FR and SR conditions at the end of the experiments. This suggests a unique pathway responsible for the MDF of Hg isotopes during methylation by this strain regardless the metabolism of the cells. After 9 h of experiment, significant simultaneous demethylation is occurring in the culture and demethylates preferentially the lighter Hg isotopes of MMHg. Therefore, depending on their methylation/demethylation capacities, SRB communities in natural sulfate reducing conditions likely have a significant and specific influence on the Hg isotope composition of MMHg (MDF) in sediments and aquatic organisms.

Figueras M.J., Beaz-Hidalgo R., Hossain M.J., Liles M.R.

ABSTRACT The average nucleotide identity (ANI) determines if two genomes belong to the same species. Using ANI, we detected mislabeled genomes and recommend verifying with ANI and multilocus phylogenetic analysis the species affiliations of the announced genomes. The slightly different results obtained with different ANI calculation software can potentially mislead taxonomic inferences.

Rodriguez-R L.M., Konstantinidis K.T.

Tamura K., Stecher G., Peterson D., Filipski A., Kumar S.

We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.

Pedrero Z., Bridou R., Mounicou S., Guyoneaud R., Monperrus M., Amouroux D.

Microbial activity is recognized to play an important role on Hg methylation in aquatic ecosystems. However, the mechanism at the cellular level is still poorly understood. In this work subcellular partitioning and transformation of Hg species in two strains: Desulfovibrio sp. BerOc1 and Desulfovibrio desulfuricans G200 (which exhibit different Hg methylation potential) are studied as an approach to the elucidation of Hg methylation/demethylation processes. The incubation with isotopically labeled Hg species ((199)Hgi and Me(201)Hg) not only allows the determination of methylation and demethylation rates simultaneously, but also the comparison of the localization of the originally added and resulting species of such metabolic processes. A dissimilar Hg species distribution was observed. In general terms, monomethylmercury (MeHg) is preferentially localized in the extracellular fraction; meanwhile inorganic mercury (Hgi) is associated to the cells. The investigation of Hg binding biomolecules on the cytoplasmatic and extracellular fractions (size exclusion chromatography coupled to ICP-MS) revealed noticeable differences in the pattern corresponding to the Hg methylating and nonmethylating strains.

Gilmour C.C., Elias D.A., Kucken A.M., Brown S.D., Palumbo A.V., Schadt C.W., Wall J.D.

ABSTRACT We propose the use of Desulfovibrio desulfuricans ND132 as a model species for understanding the mechanism of microbial Hg methylation. Strain ND132 is an anaerobic dissimilatory sulfate-reducing bacterium (DSRB), isolated from estuarine mid-Chesapeake Bay sediments. It was chosen for study because of its exceptionally high rates of Hg methylation in culture and its metabolic similarity to the lost strain D. desulfuricans LS, the only organism for which methylation pathways have been partially defined. Strain ND132 is an incomplete oxidizer of short-chain fatty acids. It is capable of respiratory growth using fumarate as an electron acceptor, supporting growth without sulfide production. We used enriched stable Hg isotopes to show that ND132 simultaneously produces and degrades methylmercury (MeHg) during growth but does not produce elemental Hg. MeHg produced by cells is mainly excreted, and no MeHg is produced in spent medium. Mass balances for Hg and MeHg during the growth of cultures, including the distribution between filterable and particulate phases, illustrate how medium chemistry and growth phase dramatically affect Hg solubility and availability for methylation. The available information on Hg methylation among strains in the genus Desulfovibrio is summarized, and we present methylation rates for several previously untested species. About 50% of Desulfovibrio strains tested to date have the ability to produce MeHg. Importantly, the ability to produce MeHg is constitutive and does not confer Hg resistance. A 16S rRNA-based alignment of the genus Desulfovibrio allows the very preliminary assessment that there may be some evolutionary basis for the ability to produce MeHg within this genus.

Brown S.D., Gilmour C.C., Kucken A.M., Wall J.D., Elias D.A., Brandt C.C., Podar M., Chertkov O., Held B., Bruce D.C., Detter J.C., Tapia R., Han C.S., Goodwin L.A., Cheng J.-., et. al.

Le Bars M., Monperrus M., Barrouilhet S., Petrel M., Goñi-Urriza M., Isaure M.

Bidzhieva S.K., Tourova T.P., Grouzdev D.S., Samigullina S.R., Sokolova D.S., Poltaraus A.B., Avtukh A.N., Tereshina V.M., Mardanov A.V., Zhaparov N.S., Nazina T.N.

Sulfidogenic bacteria cause numerous issues in the oil industry since they produce sulfide, corroding steel equipment, reducing oil quality, and worsening the environmental conditions in oil fields. The purpose of this work was to isolate and taxonomically identify the sulfidogenic bacteria responsible for the corrosion of steel equipment at the Karazhanbas oil field (Kazakhstan). In this study, we characterized five sulfidogenic strains of the genera Pseudodesulfovibrio, Oleidesulfovibrio, and Acetobacterium isolated from the formation water of the Karazhanbas oil field (Kazakhstan). Sulfate-reducing strain 9FUST revealed 98.9% similarity of the 16S rRNA gene sequence with the closely related strain ‘Pseudodesulfovibrio methanolicus’ 5S69T and was studied in detail to enhance the taxonomic resolution. Strain 9FUST grew optimally at 23–28 °C, pH 6.5, and 0–2% (w/v) NaCl. The strain used lactate, pyruvate, methanol, ethanol, fructose, ribose, and H2/CO2 (in the presence of acetate) as carbon and energy sources for sulfate reduction. Iso-C17:1 ω11, C15:0, iso-C15:0, and C16:0 were the predominant fatty acids. The genome is 4.20 Mbp with a G + C content of 64.0%. The average nucleotide identity and digital DNA–DNA hybridization values with Pseudodesulfovibrio spp. genomes were 72.5–91.6% (<95%) and 18.5–45.0% (<70%), respectively, and supported our conclusion that 9FUST (=VKM B-3654T = KCTC 25498T) belonged to a novel Pseudodesulfovibrio species, for which the name Pseudodesulfovibrio karagichevae sp. nov. is proposed. Pangenome analysis of sixteen Pseudodesulfovibrio species and functional annotation analysis of identified genes revealed complete modules of enzymes of the main metabolic pathways, characteristic of bacteria of this genus, and unique genes highlighting the adaptations of strain 9FUST in carbohydrate metabolism, nutrient uptake, and environmental stress response. Isolation of these strains expands our understanding of the diversity of sulfidogens in oil reservoirs and can be used to test the effectiveness of biocides used in an oil field.

Bidzhieva S.K., Tourova T.P., Kadnikov V.V., Samigullina S.R., Sokolova D.S., Poltaraus A.B., Avtukh A.N., Tereshina V.M., Beletsky A.V., Mardanov A.V., Nazina T.N.

The search for the microorganisms responsible for sulfide formation and corrosion of steel equipment in the oil fields of Tatarstan (Russia) resulted in the isolation of a new halotolerant strictly anaerobic sulfate-reducing bacterium, strain 5S69T. The cells were motile curved Gram-negative rods. Optimal growth was observed in the presence of 2.0–4.0% (w/v) NaCl, at pH 6.5, and at 23–28 °C under sulfate-reducing conditions. The isolate was capable of chemoorganotrophic growth with sulfate and other sulfoxides as electron acceptors, resulting in sulfide formation; and of pyruvate fermentation resulting in formation of H2 and acetate. The strain utilized lactate, pyruvate, ethanol, methanol, fumarate, and fructose, as well as H2/CO2/acetate for sulfate reduction. The genome size of the type strain 5S69T was 4.16 Mb with a G + C content of 63.0 mol%. On the basis of unique physiological properties and results of the 16S rRNA gene-based phylogenetic analysis, phylogenomic analysis of the 120 conserved single copy proteins and genomic indexes (ANI, AAI, and dDDH), assigning the type strain 5S69T ((VKM B-3653T = KCTC 25499T) to a new species within the genus Pseudodesulfovibrio, is suggested, with the proposed name Pseudodesulfovibrio methanolicus sp. nov. Genome analysis of the new isolate showed several genes involved in sulfate reduction and its sulfide-producing potential in oil fields with high saline formation water.

Scuvée D., Goñi-Urriza M., Tessier E., Gassie C., Ranchou-Peyruse M., Amouroux D., Guyoneaud R., Khalfaoui-Hassani B.

Molybdate inhibits sulfate respiration in sulfate-reducing bacteria (SRB). It is used as an inhibitor to indirectly evaluate the role of SRB in mercury methylation in the environment. Here, the SRB Pseudodesulfovibrio hydrargyri BerOc1 was used to assess the effect of molybdate on cell growth and mercury methylation under various metabolic conditions. Geobacter sulfurreducens PCA was used as the non-SRB counterpart strain with the ability to methylate mercury. While PCA growth and methylation are not affected by molybdate, 1 mM of molybdate inhibits BerOc1 growth under sulfate respiration (50% inhibition) but also under fumarate respiration (complete inhibition). Even more surprising, mercury methylation of BerOc1 is totally inhibited at 0.1 mM of molybdate when grown under sulfate or fumarate respiration with pyruvate as the electron donor. As molybdate is expected to reduce cellular ATP level, the lower Hg methylation observed with pyruvate could be the consequence of lower energy production. Although molybdate alters the expression of hgcA (mercury methylation marker) and sat (involved in sulfate reduction and molybdate sensitivity) in a metabolism-dependent manner, no relationship with mercury methylation rates could be found. Our results show, for the first time, a specific mercury methylation inhibition by molybdate in SRB.

Slobodkina G., Merkel A., Novikov A., Slobodkin A.

Terrestrial mud volcanoes (TMVs), surface expressions of a deep-subterranean sedimentary volcanism, are widespread throughout the world. The methane and sulfur cycles are recognized as the most important biogeochemical cycles in these environments. Only few anaerobic bacterial strains were recovered from TMVs. We have isolated a novel sulfate-reducing bacterium (strain SB368T) from TMV located at Taman Peninsula, Russia. Optimum growth of strain SB368T was observed at 30 °C, pH 8.0 and 1% NaCl. Strain SB368T utilized lactate, pyruvate and fumarate in the presence of sulfate, sulfite or thiosulfate. Growth with molecular hydrogen was observed only in the presence of acetate. Fermentative growth occurred on pyruvate. Phylogenetic analysis revealed that strain SB368T belongs to the genus Pseudodesulfovibrio but is distinct from all described species. Based on its genomic and phenotypic properties, a new species, Pseudodesulfovibrio pelocollis sp. nov. is proposed with strain SB368T (= DSM 111087 T = VKM B-3585 T) as a type strain.

Gaikwad S.L., Pore S.D., Dhakephalkar P.K., Dagar S.S., Soni R., Kaur M.P., Rawat H.N.

Characterization and documentation of strain MCM B-1480T, a novel sulfate-reducing bacterium isolated from produced water of India's western offshore hydrocarbon reservoir.Strain MCM B-1480T was unequivocally identified using a polyphasic approach routinely followed in bacterial systematics. The morphological and biochemical characterization of strain MCM B-1480T was carried out using standard microbiological techniques.MCM B-1480T was a Gram-stain-negative, motile, non-spore-forming, curved-rod-shaped bacterium. MCM B-1480T could grow at temperatures between 20 and 60 °C (optimum 37 °C), pH 6-8 (optimum 7), and required 1-6% NaCl (optimum 3%) for growth. Strain MCM B-1480T was reducing sulfate to produce hydrogen sulfide during growth. This strain used lactate and pyruvate as prominent electron donors, whereas sulfate, sulfite, thiosulfate, and nitrate served as electron acceptors. MCM B-1480T shared maximum 16S rRNA gene sequence homology of 98.65% with the members of the genus Pseudodesulfovibrio. The G + C content of the 3.87 Mb MCM B-1480T genome was 60.39%. Digital DDH (27.7%) and average nucleotide identity (ANI 84%) with the closest phylogenetic affiliate (less than 70% and 95%, respectively) reaffirmed its distinctiveness. The major cellular fatty acids components, namely iso-C15:0, anteiso-C15:0, C16:0, and anteiso-C17:0, differentiated strain MCM B-1480T from other species of Pseudodesulfovibrio. Genome annotation revealed the presence of genes encoding dissimilatory sulfate reduction and nitrate reduction in strain MCM B-1480T.The polyphasic studies, including SSU rRNA gene sequencing, average nucleotide identity, Digital DNA-DNA hybridization, cell wall fatty acids analysis, etc., identified strain MCM B-1480T as a novel taxon and Pseudodesulfovibrio thermohalotolerans sp. nov. was proposed (= JCM 39269T = MCC 4711T).

Kondo R.

A novel sulphate-reducing bacterium, strain SYKT, was isolated from a xenic culture of an anaerobic protist obtained from a sulphidogenic sediment of the saline Lake Hiruga in Fukui, Japan. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that SYKT clustered with the members of the genus Pseudodesulfovibrio . The closest relative of strain SYKT was Pseudodesulfovibrio sediminis SF6T, with 16S rRNA gene sequence identity of 97.43 %. Digital DNA–DNA hybridisation and average nucleotide identity values between SYKT and species of the genus Pseudodesulfovibrio fell below the respective thresholds for species delineation, indicating that SYKT represents a novel species of the genus Pseudodesulfovibrio . Cells measured 1.7–3.7×0.2–0.5 µm in size and were Gram-stain-negative, obligately anaerobic, motile by means of a single polar flagellum and had a curved rod or sigmoid shape. Cell growth was observed under saline conditions from pH 6.0 to 9.5 (optimum pH 8.0–9.0) and at a temperature of 10–30 °C (optimum 25 °C). SYKT used lactate, pyruvate, fumarate, formate and H2 as electron donors. It used sulphate, sulphite, thiosulphate and sulphur as terminal electron acceptors. Pyruvate and fumarate were fermented. Major cellular fatty acids were anteiso-C15 : 0, C16 : 0, anteiso-C17 : 1ω9c, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The DNA G+C content of SYKT was 49.4 mol%. On the basis of the the genetic and phenotypic features, SYKT was determined to represent a novel species of the genus Pseudodesulfovibrio for which the name Pseudodesulfovibrio nedwellii sp. nov. is proposed with type strain SYKT (=DSM 114958T=JCM 35746T).

Gionfriddo C.M., Soren A.B., Wymore A.M., Hartnett D.S., Podar M., Parks J.M., Elias D.A., Gilmour C.C.

This work reveals a link between microbial mercury methylation and arsenic resistance and may hold clues to the evolution of mercury methylation genes ( hgcAB ). Microbes with hgcAB produce methylmercury, a strong neurotoxin that readily accumulates in the food web.

Gionfriddo C.M., Soren A.B., Wymore A., Hartnett D.S., Podar M., Parks J.M., Elias D.A., Gilmour C.C.

ABSTRACTThehgcABgene pair encodes mercury (Hg) methylation capability in a diverse group of microorganisms, but its evolution and transcriptional regulation remain unknown. Working from the possibility that the evolutionary function of HgcAB may not be Hg methylation, we test a possible link to arsenic resistance. Using model Hg-methylatorPseudodesulfovibrio mercuriiND132, we specifically evaluated transcriptional control ofhgcABby a putative ArsR encoded upstream and co-transcribed withhgcAB. This regulator shares homology with ArsR repressors of arsenic resistance and S-adenosyl-homocysteine (SAH) responsive regulators of methionine biosynthesis but is distinct from other ArsR/SahR inPseudodesulfovibrio mercuriiND132. Using qPCR and RNA-seq analyses we confirmed this ArsR regulateshgcABtranscription, and is responsive to arsenic and SAH. Additionally, RNA-seq indicated a possible link betweenhgcABactivity and arsenic transformations byPseudodesulfovibrio mercuriiND132, with significant up-regulation of other ArsR-regulated arsenic resistance operons alongsidehgcAB. Interestingly, wild-type ND132 was less sensitive to AsV (but not AsIII) than anhgcABknockout strain, supporting the idea thathgcABmay be linked to arsenic resistance. Arsenic significantly impacted Hg-methylation rates by ND132, however, responses varied with culture conditions. Differences in growth and overall metabolic activity did not account for arsenic impacts on methylation. One goal of this research is to better predict MeHg production in nature. However, we found thathgcABgene and transcript abundance was not a good predictor of Hg-methylation rates. Our finding thathgcABactivity is linked to arsenic may hold clues to the possible environmental drivers of horizontal transfer ofhgcAB.IMPORTANCEThis work reveals a link between microbial mercury methylation and arsenic resistance and may hold clues to the evolution of mercury methylation genes (hgcAB). Microbes withhgcABproduce methylmercury, a strong neurotoxin that readily accumulates in the food web. This study addresses a critical gap in our understanding about the environmental factors that controlhgcABexpression. We show thathgcABexpression is controlled by an ArsR-like regulator responsive to both arsenic and S-adenosyl-homocysteine in our model organism,Pseudodesulfovibrio mercuriiND132. Exposure to arsenic also significantly impactedPseudodesulfovibrio mercuriiND132 mercury methylation rates. However, expression ofhgcABwas not always a good predictor of Hg methylation rates, highlighting the roles of Hg bioavailability and other biochemical mechanisms in methylmercury production. This study improves our understanding of the controls onhgcABexpression which is needed to better predict environmental methylmercury production.

Barrouilhet S., Monperrus M., Tessier E., Khalfaoui-Hassani B., Guyoneaud R., Isaure M., Goñi-Urriza M.

Mercury (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 sulfides 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 sulfide. Here, we explore the effect of sulfide (produced by the cell or supplied exogenously) on Hg methylation. For this purpose, Pseudodesulfovibrio hydrargyri BerOc1 was cultivated in non-sulfidogenic conditions with addition of cysteine and sulfide as well as in sulfidogenic conditions. We report that Hg methylation depends on sulfide concentration in the culture and the sulfides produced by cysteine degradation or sulfate reduction could affect the Hg methylation pattern. Hg methylation was independent of hgcA expression. Interestingly, MeHg production was maximal at 0.1–0.5 mM of sulfides. Besides, a strong positive correlation between MeHg in the extracellular medium and the increase of sulfide concentrations was observed, suggesting a facilitated MeHg export with sulfide and/or higher desorption from the cell. We suggest that sulfides (exogenous or endogenous) play a key role in controlling mercury methylation and should be considered when investigating the impact of Hg in natural environments.

Park M., Kim Y.J., Park M., Yu J., Namirimu T., Roh Y., Kwon K.K.

Bacteria in the Desulfovibrionaceae family, which contribute to S element turnover as sulfate-reducing bacteria (SRB) and disproportionation of partially oxidized sulfoxy anions, have been extensively investigated since the importance of the sulfur cycle emerged. Novel species belonging to this taxon are frequently reported, because they exist in various environments and are easy to culture using established methods. Due to the rapid expansion of the taxon, correction and reclassification have been conducted. The development of high-throughput sequencing facilitated rapid expansion of genome sequence database. Genome-based criteria, based on these databases, proved to be potential classification standard by overcoming the limitations of 16S rRNA-based phylogeny. Although standards methods for taxogenomics are being established, the addition of a novel genus requires extensive calculations with taxa, including many species, such as Desulfovibrionaceae. Thus, the genome-based criteria for classification of Desulfovibrionaceae were established and validated in this study. The average amino-acid identity (AAI) cut-off value, 63.43 ± 0.01, was calculated to be an appropriate criterion for genus delineation of the family Desulfovibrionaceae. By applying the AAI cut-off value, 88 genomes of the Desulfovibrionaceae were divided into 27 genera, which follows the core gene phylogeny results. In this process, two novel genera (Alkalidesulfovibrio and Salidesulfovibrio) and one former invalid genus (“Psychrodesulfovibrio”) were officially proposed. Further, by applying the 95–96% average nucleotide identity (ANI) standard and the 70% digital DNA–DNA hybridization standard values for species delineation of strains that were classified as the same species, five strains have the potential to be newly classified. After verifying that the classification was appropriately performed through relative synonymous codon usage analysis, common characteristics were listed by group. In addition, by detecting metal resistance related genes via in silico analysis, it was confirmed that most strains display metal tolerance.

Takahashi A., Kojima H., Watanabe M., Fukui M.

A novel mesophilic and neutrophilic sulfate-reducing bacterium, strain SF6T, was isolated from sediment of a brackish lake in Japan. Cells of strain SF6T were motile and rod-shaped with length of 1.2–2.5 μm and width of 0.6–0.9 μm. Growth was observed at 10–37 °C with an optimum growth temperature of 28 °C. The pH range for growth was 5.8–8.2 with an optimum pH of 7.0. The most predominant fatty acid was anteiso-C15:0. Under sulfate-reducing conditions, strain SF6T utilized lactate, ethanol and glucose as growth substrate. Chemolithoautotrophic growth on H2 was not observed, although H2 was used as electron donor. Fermentative growth occurred on pyruvate. As electron acceptor, sulfate, sulfite, thiosulfate and nitrate supported heterotrophic growth of the strain. The complete genome of strain SF6T is composed of a circular chromosome with length of 3.8 Mbp and G+C content of 54 mol%. Analyses of the 16S rRNA gene and whole genome sequence indicated that strain SF6T belongs to the genus Pseudodesulfovibrio but distinct form all existing species in the genus. On the basis of its genomic and phenotypic properties, strain SF6T (= DSM111931T = NBRC 114895T) is proposed as the type strain of a new species, with name of Pseudodesulfovibrio sediminis sp. nov.

Isokpehi R.D., McInnis D.S., Destefano A.M., Johnson G.S., Walker A.D., Hall Y.A., Mapp B.W., Johnson M.O., Simmons S.S.

The presence of methylmercury in aquatic environments and marine food sources is of global concern. The chemical reaction for the addition of a methyl group to inorganic mercury occurs in diverse bacterial taxonomic groups including the Gram-negative, sulfate-reducing Desulfovibrionaceae family that inhabit extreme aquatic environments. The availability of whole-genome sequence datasets for members of the Desulfovibrionaceae presents opportunities to understand the microbial mechanisms that contribute to methylmercury production in extreme aquatic environments. We have applied bioinformatics resources and developed visual analytics resources to categorize a collection of 719 putative universal stress protein (USP) sequences predicted from 93 genomes of Desulfovibrionaceae. We have focused our bioinformatics investigations on protein sequence analytics by developing interactive visualizations to categorize Desulfovibrionaceae universal stress proteins by protein domain composition and functionally important amino acids. We identified 651 Desulfovibrionaceae universal stress protein sequences, of which 488 sequences had only one USP domain and 163 had two USP domains. The 488 single USP domain sequences were further categorized into 340 sequences with ATP-binding motif and 148 sequences without ATP-binding motif. The 163 double USP domain sequences were categorized into (1) both USP domains with ATP-binding motif (3 sequences); (2) both USP domains without ATP-binding motif (138 sequences); and (3) one USP domain with ATP-binding motif (21 sequences). We developed visual analytics resources to facilitate the investigation of these categories of datasets in the presence or absence of the mercury-methylating gene pair (hgcAB). Future research could utilize these functional categories to investigate the participation of universal stress proteins in the bacterial cellular uptake of inorganic mercury and methylmercury production, especially in anaerobic aquatic environments.

Frolova A.A., Merkel A.Y., Kuchierskaya A.A., Bonch-Osmolovskaya E.A., Slobodkin A.I.

The diversity of anaerobic microorganisms in terrestrial mud volcanoes is largely unexplored. Here we report the isolation of a novel sulfate-reducing alkaliphilic bacterium (strain F-1T) from a terrestrial mud volcano located at the Taman peninsula, Russia. Cells of strain F-1T were Gram-negative motile vibrios with a single polar flagellum; 2.0–4.0 µm in length and 0.5 µm in diameter. The temperature range for growth was 6–37 °C, with an optimum at 24 °C. The pH range for growth was 7.0–10.5, with an optimum at pH 9.5. Strain F-1T utilized lactate, pyruvate, and molecular hydrogen as electron donors and sulfate, sulfite, thiosulfate, elemental sulfur, fumarate or arsenate as electron acceptors. In the presence of sulfate, the end products of lactate oxidation were acetate, H2S and CO2. Lactate and pyruvate could also be fermented. The major product of lactate fermentation was acetate. The main cellular fatty acids were anteiso-C15:0, C16:0, C18:0, and iso-C17:1ω8. Phylogenetic analysis revealed that strain F-1T was most closely related to Pseudodesulfovibrio aespoeensis (98.05% similarity). The total size of the genome of the novel isolate was 3.23 Mb and the genomic DNA G + C content was 61.93 mol%. The genome contained all genes essential for dissimilatory sulfate reduction. We propose to assign strain F-1T to the genus Pseudodesulfovibrio, as a new species, Pseudodesulfovibrio alkaliphilus sp. nov. The type strain is F-1T (= KCTC 15918T = VKM B-3405T).

Galushko A., Kuever J.

Abstract De.sul.fo.vi.bri.o.na.ce'ae. N.L. masc. n. Desulfovibrio , type genus of the family; suff. ‐ aceae , ending to denote a family; N.L. fem. pl. n. Desulfovibrionaceae , the family of Desulfovibrio . Desulfobacterota / Desulfovibrionia / Desulfovibrionales / Desulfovibrionaceae Cells are vibrio or rod shaped and occur singly or in pairs. Gram‐stain‐negative. Endospores are not observed. Motile or nonmotile. Strict anaerobes with respiratory and fermentative types of metabolism. Chemoorganoheterotrophs (all members), chemolithoheterotrophs (most members) and chemolithoautotrophs (few members). Simple organic compounds such as lactate, pyruvate, fumarate (most members), succinate (most members), and malate (most members) can serve as sole electron donors and carbon sources and are oxidized to acetate. Some members use amino acids and sugars. Alcohols are used only as electron donors and oxidized to their corresponding carboxylic acids. H 2 and CO 2 and formate can be used in the presence of acetate or yeast extract as carbon sources (all members), the reductive glycine pathway can be used for autotrophic growth (a few members). The common electron acceptor for most members is sulfate, which is reduced to sulfide; thiosulfate, sulfite, or sulfur (often without growth) might be used as well. Lawsonia spp. are intracellular parasites that can grow only in complex media and cannot use inorganic sulfur compounds as terminal electron acceptors. Some members can grow by reduction of nitrate or fumarate to ammonia and succinate, respectively. Reduction of other electron acceptors, such as oxygen (not linked to growth), Fe 3+ , DMSO, or others, might occur. Fermentation of pyruvate, fumarate, or other simple organic compounds is common. Growth by disproportionation of oxidized sulfur compounds might occur (chemolithoorganotrophic growth) but has rarely been tested. All species are mesophilic and contain desulfoviridin with the only exception of Lawsonia . Neutrophiles (most species), moderate alkaliphiles, and acidophiles may occur. The family currently accommodates 25 genera. Members of this family are found in various anoxic habitats. DNA G+C content (mol%) : 33.1–70.0 (genome, LC, T m ). Type genus : Desulfovibrio Kluyver and van Niel 1936 AL .