H2 Is a Major Intermediate in Desulfovibrio vulgaris Corrosion of Iron.

Desulfovibrio vulgaris has been a primary pure culture sulfate reducer for developing microbial corrosion concepts. Multiple mechanisms for how it accepts electrons from Fe0 have been proposed. We investigated Fe0 oxidation with a mutant of D. vulgaris in which hydrogenase genes were deleted. The hydrogenase mutant grew as well as the parental strain with lactate as the electron donor, but unlike the parental strain, it was not able to grow on H2. The parental strain reduced sulfate with Fe0 as the sole electron donor, but the hydrogenase mutant did not. H2 accumulated over time in Fe0 cultures of the hydrogenase mutant and sterile controls but not in parental strain cultures. Sulfide stimulated H2 production in uninoculated controls apparently by both reacting with Fe0 to generate H2 and facilitating electron transfer from Fe0 to H+. Parental strain supernatants did not accelerate H2 production from Fe0, ruling out a role for extracellular hydrogenases. Previously proposed electron transfer between Fe0 and D. vulgaris via soluble electron shuttles was not evident. The hydrogenase mutant did not reduce sulfate in the presence of Fe0 and either riboflavin or anthraquinone-2,6-disulfonate, and these potential electron shuttles did not stimulate parental strain sulfate reduction with Fe0 as the electron donor. The results demonstrate that D. vulgaris primarily accepts electrons from Fe0 via H2 as an intermediary electron carrier. These findings clarify the interpretation of previous D. vulgaris corrosion studies and suggest that H2-mediated electron transfer is an important mechanism for iron corrosion under sulfate-reducing conditions. IMPORTANCE Microbial corrosion of iron in the presence of sulfate-reducing microorganisms is economically significant. There is substantial debate over how microbes accelerate iron corrosion. Tools for genetic manipulation have only been developed for a few Fe(III)-reducing and methanogenic microorganisms known to corrode iron and in each case those microbes were found to accept electrons from Fe0 via direct electron transfer. However, iron corrosion is often most intense in the presence of sulfate-reducing microbes. The finding that Desulfovibrio vulgaris relies on H2 to shuttle electrons between Fe0 and cells revives the concept, developed in some of the earliest studies on microbial corrosion, that sulfate reducers consumption of H2 is a major microbial corrosion mechanism. The results further emphasize that direct Fe0-to-microbe electron transfer has yet to be rigorously demonstrated in sulfate-reducing microbes.

DsrC is involved in fermentative growth and interacts directly with the FlxABCD-HdrABC complex in Desulfovibrio vulgaris Hildenborough.

DsrC is a key protein in dissimilatory sulfur metabolism, where it works as co-substrate of the dissimilatory sulfite reductase DsrAB. DsrC has two conserved cysteines in a C-terminal arm that are converted to a trisulfide upon reduction of sulfite. In sulfate-reducing bacteria, DsrC is essential and previous works suggested additional functions beyond sulfite reduction. Here, we studied whether DsrC also plays a role during fermentative growth of Desulfovibrio vulgaris Hildenborough, by studying two strains where the functionality of DsrC is impaired by a lower level of expression (IPFG07) and additionally by the absence of one conserved Cys (IPFG09). Growth studies coupled with metabolite and proteomic analyses reveal that fermentation leads to lower levels of DsrC, but impairment of its function results in reduced growth by fermentation and a shift towards more fermentative metabolism during sulfate respiration. In both respiratory and fermentative conditions, there is increased abundance of the FlxABCD-HdrABC complex and Adh alcohol dehydrogenase in IPFG09 versus the wild type, which is reflected in higher production of ethanol. Pull-down experiments confirmed a direct interaction between DsrC and the FlxABCD-HdrABC complex, through the HdrB subunit. Dissimilatory sulfur metabolism, where sulfur compounds are used for energy generation, is a key process in the ecology of anoxic environments, and is more widespread among bacteria than previously believed. Two central proteins for this type of metabolism are DsrAB dissimilatory sulfite reductase and its co-substrate DsrC. Using physiological, proteomic and biochemical studies of Desulfovibrio vulgaris Hildenborough and mutants affected in DsrC functionality, we show that DsrC is also relevant for fermentative growth of this model organism and that it interacts directly with the soluble FlxABCD-HdrABC complex that links the NAD(H) pool with dissimilatory sulfite reduction.

Norepinephrine induces growth of Desulfovibrio vulgaris in an iron dependent manner.

Desulfovibrio spp. is a commensal sulfate reducing bacterium that is present in small numbers in the gastrointestinal tract. Increased concentrations of Desulfovibrio spp. (blooms) have been reported in patients with inflammatory bowel disease and irritable bowel syndrome. Since stress has been reported to exacerbate symptoms of these chronic diseases, this study examined whether the stress catecholamine norepinephrine (NE) promotes Desulfovibrio growth. Norepinephrine-stimulated growth has been reported in other bacterial taxa, and this effect may depend on the availability of the micronutrient iron. OBJECTIVES: This study tested whether norepinephrine exposure affects the in vitro growth of Desulfovibrio vulgaris in an iron dependent manner. METHODS: DSV was incubated in a growth medium with and without 1 µm of norepinephrine. An additional growth assay added the iron chelator deferoxamine in NE exposed DSV. Iron regulatory genes were assessed with and without the treatment of NE and Deferoxamine. RESULTS: We found that norepinephrine significantly increased growth of D. vulgaris. Norepinephrine also increased bacterial production of hydrogen sulfide. Additionally, norepinephrine significantly increased bacterial expression in three of the four tested iron regulatory genes. The iron chelator deferoxamine inhibited growth of D. vulgaris in a dose-dependent manner and reversed the effect of norepinephrine on proliferation of D. vulgaris and on bacterial expression of iron regulatory genes. CONCLUSION: The data presented in this work suggests that promotion of D. vulgaris growth by norepinephrine is iron dependent.

Extracellular proteins of Desulfovibrio vulgaris as adsorbents and redox shuttles promote biomineralization of antimony.

Biomineralization is the key process governing the biogeochemical cycling of multivalent metals in the environment. Although some sulfate-reducing bacteria (SRB) are recently recognized to respire metal ions, the role of their extracellular proteins in the immobilization and redox transformation of antimony (Sb) remains elusive. Here, a model strain Desulfovibrio vulgaris Hildenborough (DvH) was used to study microbial extracellular proteins of functions and possible mechanisms in Sb(V) biomineralization. We found that the functional groups (N-H, CO, O-CO, NH2-R and RCOH/RCNH2) of extracellular proteins could adsorb and fix Sb(V) through electrostatic attraction and chelation. DvH could rapidly reduce Sb(V) adsorbed on the cell surface and form amorphous nanometer-sized stibnite and/or antimony trioxide, respectively with sulfur and oxygen. Proteomic analysis indicated that some extracellular proteins involved in electron transfer increased significantly (p < 0.05) at 1.8 mM Sb(V). The upregulated flavoproteins could serve as a redox shuttle to transfer electrons from c-type cytochrome networks to reduce Sb(V). Also, the upregulated extracellular proteins involved in sulfur reduction, amino acid transport and protein synthesis processes, and the downregulated flagellar proteins would contribute to a better adaption under 1.8 mM Sb(V). This study advances our understanding of how microbial extracellular proteins promote Sb biomineralization in DvH.

Expression, Purification and Characterization of a ZIP Family Transporter from Desulfovibrio vulgaris.

The ZIP family transport zinc ions from the extracellular medium across the plasma membrane or from the intracellular compartments across endomembranes, which play fundamental roles in metal homeostasis and are broadly involved in physiological and pathological processes. Desulfovibrio is the predominant sulphate-reducing bacteria in human colonic microbiota, but also a potential choice for metal bioremediation. while, there are no published studies describing the zinc transporters from Desulfovibrio up to now. In this study, we obtained for the first time a heterologously expressed ZIP homolog from Desulfovibrio vulgaris, termed dvZip. The purified dvZip was reconstituted into proteoliposomes, and confirmed its zinc transport ability in vitro. By combining topology prediction, homology modeling and phylogenetic approaches, we also noticed that dvZip belongs to the GufA and probably have 8 transmembrane α-helical segments (TM 1-8) in which both termini are located on the extracellular, with TM2, 4, 5 and 7 create an inner bundle. We believe that purification and characterization of zinc (probably also cadmium) transporters from Desulfovibrio vulgaris such as dvZip could shed light on understanding of metal homeostasis of Desulfovibrio and provided protein products for future detailed function and structural studies.

Comparative metabolic modeling of multiple sulfate-reducing prokaryotes reveals versatile energy conservation mechanisms.

Sulfate-reducing prokaryotes (SRPs) are crucial participants in the cycling of sulfur, carbon, and various metals in the natural environment and in engineered systems. Despite recent advances in genetics and molecular biology bringing a huge amount of information about the energy metabolism of SRPs, little effort has been made to link this important information with their biotechnological studies. This study aims to construct multiple metabolic models of SRPs that systematically compile genomic, genetic, biochemical, and molecular information about SRPs to study their energy metabolism. Pan-genome analysis was conducted to compare the genomes of SRPs, from which a list of orthologous genes related to central and energy metabolism was obtained. Twenty-four SRP metabolic models via the inference of pan-genome analysis were efficiently constructed. The metabolic model of the well-studied model SRP Desulfovibrio vulgaris Hildenborough (DvH) was validated via flux balance analysis (FBA). The DvH model predictions matched reported experimental growth and energy yields, which demonstrated that the core metabolic model worked successfully. Further, steady-state simulation of SRP metabolic models under different growth conditions showed how the use of different electron transfer pathways leads to energy generation. Three energy conservation mechanisms were identified, including menaquinone-based redox loop, hydrogen cycling, and proton pumping. Flavin-based electron bifurcation (FBEB) was also demonstrated to be an essential mechanism for supporting energy conservation. The developed models can be easily extended to other species of SRPs not examined in this study. More importantly, the present work develops an accurate and efficient approach for constructing metabolic models of multiple organisms, which can be applied to other critical microbes in environmental and industrial systems, thereby enabling the quantitative prediction of their metabolic behaviors to benefit relevant applications.

OrpR is a σ54 -dependent activator using an iron-sulfur cluster for redox sensing in Desulfovibrio vulgaris Hildenborough.

Enhancer binding proteins (EBPs) are key players of σ54 -regulation that control transcription in response to environmental signals. In the anaerobic microorganism Desulfovibrio vulgaris Hildenborough (DvH), orp operons have been previously shown to be coregulated by σ54 -RNA polymerase, the integration host factor IHF and a cognate EBP, OrpR. In this study, ChIP-seq experiments indicated that the OrpR regulon consists of only the two divergent orp operons. In vivo data revealed that (i) OrpR is absolutely required for orp operons transcription, (ii) under anaerobic conditions, OrpR binds on the two dedicated DNA binding sites and leads to high expression levels of the orp operons, (iii) increasing the redox potential of the medium leads to a drastic down-regulation of the orp operons expression. Moreover, combining functional and biophysical studies on the anaerobically purified OrpR leads us to propose that OrpR senses redox potential variations via a redox-sensitive [4Fe-4S]2+ cluster in the sensory PAS domain. Overall, the study herein presents the first characterization of a new Fe-S redox regulator belonging to the σ54 -dependent transcriptional regulator family probably advantageously selected by cells adapted to the anaerobic lifestyle to monitor redox stress conditions.

Metabolic Exchange and Energetic Coupling between Nutritionally Stressed Bacterial Species: Role of Quorum-Sensing Molecules.

Formation of multispecies communities allows nearly every niche on earth to be colonized, and the exchange of molecular information among neighboring bacteria in such communities is key for bacterial success. To clarify the principles controlling interspecies interactions, we previously developed a coculture model with two anaerobic bacteria, Clostridium acetobutylicum (Gram positive) and Desulfovibrio vulgaris Hildenborough (Gram negative, sulfate reducing). Under conditions of nutritional stress for D. vulgaris, the existence of tight cell-cell interactions between the two bacteria induced emergent properties. Here, we show that the direct exchange of carbon metabolites produced by C. acetobutylicum allows D vulgaris to duplicate its DNA and to be energetically viable even without its substrates. We identify the molecular basis of the physical interactions and how autoinducer-2 (AI-2) molecules control the interactions and metabolite exchanges between C. acetobutylicum and D. vulgaris (or Escherichia coli and D. vulgaris). With nutrients, D. vulgaris produces a small molecule that inhibits in vitro the AI-2 activity and could act as an antagonist in vivo Sensing of AI-2 by D. vulgaris could induce formation of an intercellular structure that allows directly or indirectly metabolic exchange and energetic coupling between the two bacteria.IMPORTANCE Bacteria have usually been studied in single culture in rich media or under specific starvation conditions. However, in nature they coexist with other microorganisms and build an advanced society. The molecular bases of the interactions controlling this society are poorly understood. Use of a synthetic consortium and reducing complexity allow us to shed light on the bacterial communication at the molecular level. This study presents evidence that quorum-sensing molecule AI-2 allows physical and metabolic interactions in the synthetic consortium and provides new insights into the link between metabolism and bacterial communication.

Structural basis for assembly of non-canonical small subunits into type I-C Cascade.

Bacteria and archaea employ CRISPR (clustered, regularly, interspaced, short palindromic repeats)-Cas (CRISPR-associated) systems as a type of adaptive immunity to target and degrade foreign nucleic acids. While a myriad of CRISPR-Cas systems have been identified to date, type I-C is one of the most commonly found subtypes in nature. Interestingly, the type I-C system employs a minimal Cascade effector complex, which encodes only three unique subunits in its operon. Here, we present a 3.1 Å resolution cryo-EM structure of the Desulfovibrio vulgaris type I-C Cascade, revealing the molecular mechanisms that underlie RNA-directed complex assembly. We demonstrate how this minimal Cascade utilizes previously overlooked, non-canonical small subunits to stabilize R-loop formation. Furthermore, we describe putative PAM and Cas3 binding sites. These findings provide the structural basis for harnessing the type I-C Cascade as a genome-engineering tool.

Experimental evolution reveals nitrate tolerance mechanisms in Desulfovibrio vulgaris.

Elevated nitrate in the environment inhibits sulfate reduction by important microorganisms of sulfate-reducing bacteria (SRB). Several SRB may respire nitrate to survive under elevated nitrate, but how SRB that lack nitrate reductase survive to elevated nitrate remains elusive. To understand nitrate adaptation mechanisms, we evolved 12 populations of a model SRB (i.e., Desulfovibrio vulgaris Hildenborough, DvH) under elevated NaNO3 for 1000 generations, analyzed growth and acquired mutations, and linked their genotypes with phenotypes. Nitrate-evolved (EN) populations significantly (p < 0.05) increased nitrate tolerance, and whole-genome resequencing identified 119 new mutations in 44 genes of 12 EN populations, among which six functional gene groups were discovered with high mutation frequencies at the population level. We observed a high frequency of nonsense or frameshift mutations in nitrosative stress response genes (NSR: DVU2543, DVU2547, and DVU2548), nitrogen regulatory protein C family genes (NRC: DVU2394-2396, DVU2402, and DVU2405), and nitrate cluster (DVU0246-0249 and DVU0251). Mutagenesis analysis confirmed that loss-of-functions of NRC and NSR increased nitrate tolerance. Also, functional gene groups involved in fatty acid synthesis, iron regulation, and two-component system (LytR/LytS) known to be responsive to multiple stresses, had a high frequency of missense mutations. Mutations in those gene groups could increase nitrate tolerance through regulating energy metabolism, barring entry of nitrate into cells, altering cell membrane characteristics, or conferring growth advantages at the stationary phase. This study advances our understanding of nitrate tolerance mechanisms and has important implications for linking genotypes with phenotypes in DvH.

Effects of Genetic and Physiological Divergence on the Evolution of a Sulfate-Reducing Bacterium under Conditions of Elevated Temperature.

Adaptation via natural selection is an important driver of evolution, and repeatable adaptations of replicate populations, under conditions of a constant environment, have been extensively reported. However, isolated groups of populations in nature tend to harbor both genetic and physiological divergence due to multiple selective pressures that they have encountered. How this divergence affects adaptation of these populations to a new common environment remains unclear. To determine the impact of prior genetic and physiological divergence in shaping adaptive evolution to accommodate a new common environment, an experimental evolution study with the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) was conducted. Two groups of replicate populations with genetic and physiological divergence, derived from a previous evolution study, were propagated in an elevated-temperature environment for 1,000 generations. Ancestor populations without prior experimental evolution were also propagated in the same environment as a control. After 1,000 generations, all the populations had increased growth rates and all but one had greater fitness in the new environment than the ancestor population. Moreover, improvements in growth rate were moderately affected by the divergence in the starting populations, while changes in fitness were not significantly affected. The mutations acquired at the gene level in each group of populations were quite different, indicating that the observed phenotypic changes were achieved by evolutionary responses that differed between the groups. Overall, our work demonstrated that the initial differences in fitness between the starting populations were eliminated by adaptation and that phenotypic convergence was achieved by acquisition of mutations in different genes.IMPORTANCE Improving our understanding of how previous adaptation influences evolution has been a long-standing goal in evolutionary biology. Natural selection tends to drive populations to find similar adaptive solutions for the same selective conditions. However, variations in historical environments can lead to both physiological and genetic divergence that can make evolution unpredictable. Here, we assessed the influence of divergence on the evolution of a model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, in response to elevated temperature and found a significant effect at the genetic but not the phenotypic level. Understanding how these influences drive evolution will allow us to better predict how bacteria will adapt to various ecological constraints.

Transcriptome Analysis of the Acid Stress Response of Desulfovibrio vulgaris ATCC 7757.

The application of sulfate-reducing bacteria (SRB) shows great potential in the anaerobic biological treatment of acid mine wastewater; therefore, it has attracted much attention. The low pH in acidic wastewater affects the growth and reducing power of SRB. To uncover the mechanism underlying the reduction efficiency of SRB under acidic conditions, in this study, transcriptomic analysis was performed with Desulfovibrio vulgaris ATCC 7757 under three different pH conditions (pH 4.0, 5.5 and 7.0) and in the initial inoculation, logarithmic growth and plateau phases. Our results showed that ATCC 7757 still had biological activity at pH 4.0 and exhibited gene expression patterns at pH 4.0 that were different from those at pH 5.5 and pH 7. Importantly, the gene expression pattern was similar between pH 5.5 and pH 7. Transcriptomic analysis identified differentially expressed genes that affected the growth of ATCC 7757 under pH 7.0 at 22 h compared to 15 h; 196 of these genes were upregulated and 575 were downregulated. These differentially expressed genes were mainly enriched in genetic information processing and metabolism. Additionally, we identified 57 candidate genes associated with low-pH tolerance. Adaptation to low pH was reflected by an increase in the expression of genes involved in cell membrane structure and proton transport. The expression of genes involved in the reduction process decreased, including the genes DVU0499 and sat, which encode proteins that affect the sulfate reduction process. Both gene activities were validated by qPCR. Our results will contribute to further promoting the reducing power of SRB in acid mine wastewater and the development of successful bioremediation strategies.

Characterization of a butyrate kinase from Desulfovibrio vulgaris str. Hildenborough.

Short and branched chain fatty acid kinases participate in both bacterial anabolic and catabolic processes, including fermentation, through the reversible, ATP-dependent synthesis of acyl phosphates. This study reports biochemical properties of a predicted butyrate kinase from Desulfovibrio vulgaris str. Hildenborough (DvBuk) expressed heterologously and purified from Escherichia coli. Gel filtration chromatography indicates purified DvBuk is active as a dimer. The optimum temperature and pH for DvBuk activity is 44°C and 7.5, respectively. The enzyme displays enhanced thermal stability in the presence of substrates as observed for similar enzymes. Measurement of kcat and KM for various substrates reveals DvBuk exhibits the highest catalytic efficiencies for butyrate, valerate and isobutyrate. In particular, these measurements reveal this enzyme's apparent high affinity for C4 fatty acids relative to other butyrate kinases. These results have implications on structure and function relationships within the ASKHA superfamily of phosphotransferases, particularly regarding the acyl binding pocket, as well as potential physiological roles for this enzyme in Desulfovibrio vulgaris str. Hildenborough.

Effect of Quorum Sensing on the Ability of Desulfovibrio vulgaris To Form Biofilms and To Biocorrode Carbon Steel in Saline Conditions.

Sulfate-reducing bacteria (SRB) are key contributors to microbe-induced corrosion (MIC), which can lead to serious economic and environmental impact. The presence of a biofilm significantly increases the MIC rate. Inhibition of the quorum-sensing (QS) system is a promising alternative approach to prevent biofilm formation in various industrial settings, especially considering the significant ecological impact of conventional chemical-based mitigation strategies. In this study, the effect of the QS stimulation and inhibition on Desulfovibrio vulgaris is described in terms of anaerobic respiration, cell activity, biofilm formation, and biocorrosion of carbon steel. All these traits were repressed when bacteria were in contact with QS inhibitors but enhanced upon exposure to QS signal molecules compared to the control. The difference in the treatments was confirmed by transcriptomic analysis performed at different time points after treatment application. Genes related to lactate and pyruvate metabolism, sulfate reduction, electron transfer, and biofilm formation were downregulated upon QS inhibition. In contrast, QS stimulation led to an upregulation of the above-mentioned genes compared to the control. In summary, these results reveal the impact of QS on the activity of D. vulgaris, paving the way toward the prevention of corrosive SRB biofilm formation via QS inhibition.IMPORTANCE Sulfate-reducing bacteria (SRB) are considered key contributors to biocorrosion, particularly in saline environments. Biocorrosion imposes tremendous economic costs, and common approaches to mitigate this problem involve the use of toxic and hazardous chemicals (e.g., chlorine), which raise health and environmental safety concerns. Quorum-sensing inhibitors (QSIs) can be used as an alternative approach to inhibit biofilm formation and biocorrosion. However, this approach would only be effective if SRB rely on QS for the pathways associated with biocorrosion. These pathways would include biofilm formation, electron transfer, and metabolism. This study demonstrates the role of QS in Desulfovibrio vulgaris on the above-mentioned pathways through both phenotypic measurements and transcriptomic approach. The results of this study suggest that QSIs can be used to mitigate SRB-induced corrosion problems in ecologically sensitive areas.

σ54 -Dependent regulator DVU2956 switches Desulfovibrio vulgaris from biofilm formation to planktonic growth and regulates hydrogen sulfide production.

Microbiologically influenced corrosion causes $100 billion in damage per year, and biofilms formed by sulfate-reducing bacteria (SRB) are the major culprit. However, little is known about the regulation of SRB biofilm formation. Using Desulfovibrio vulgaris as a model SRB organism, we compared the transcriptomes of biofilm and planktonic cells and identified that the gene for σ54 -dependent regulator DVU2956 is repressed in biofilms. Utilizing a novel promoter that is primarily transcribed in biofilms (Pdvu0304 ), we found production of DVU2956 inhibits biofilm formation by 70%. Corroborating this result, deleting dvu2956 increased biofilm formation, and this biofilm phenotype could be complemented. By producing proteins in biofilms from genes controlled by DVU2956 (dvu2960 and dvu2962), biofilm formation was inhibited almost completely. A second round of RNA-seq for the production of DVU2956 revealed DVU2956 influences electron transport via an Hmc complex (high-molecular-weight cytochrome c encoded by dvu0531-dvu0536) and the Fe-only hydrogenase (encoded by dvu1769, hydA and dvu1770, hydB) to control H2 S production. Corroborating these results, producing DVU2956 in biofilms decreased H2 S production by half, deleting dvu2956 increased H2 S production by 131 ± 5%, and producing DVU2956 in the dvu2956 strain reduced H2 S production. Therefore, DVU2956 maintains SRB in the planktonic state and reduces H2 S formation.

LurR is a regulator of the central lactate oxidation pathway in sulfate-reducing Desulfovibrio species.

The central carbon/lactate utilization pathway in the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, is encoded by the highly conserved operon DVU3025-3033. Our earlier in vitro genome-wide study had suggested a network of four two-component system regulators that target this large operon; however, how these four regulators control this operon was not known. Here, we probe the regulation of the lactate utilization operon with mutant strains and DNA-protein binding assays. We show that the LurR response regulator is required for optimal growth and complete lactate utilization, and that it activates the DVU3025-3033 lactate oxidation operon as well as DVU2451, a lactate permease gene, in the presence of lactate. We show by electrophoretic mobility shift assays that LurR binds to three sites in the upstream region of DVU3025, the first gene of the operon. NrfR, a response regulator that is activated under nitrite stress, and LurR share similar binding site motifs and bind the same sites upstream of DVU3025. The DVU3025 promoter also has a binding site motif (Pho box) that is bound by PhoB, a two-component response regulator activated under phosphate limitation. The lactate utilization operon, the regulator LurR, and LurR binding sites are conserved across the order Desulfovibrionales whereas possible modulation of the lactate utilization genes by additional regulators such as NrfR and PhoB appears to be limited to D. vulgaris.

Physiological and transcriptomic analyses reveal CuO nanoparticle inhibition of anabolic and catabolic activities of sulfate-reducing bacterium.

The widespread use of CuO nanoparticles (NPs) results in their continuous release into the environment, which could pose risks to public health and to microbial ecosystems. Following consumption, NPs will initially enter into sewer systems and interact with and potentially influence sewer microbial communities. An understanding of the response of microbes in sewers, particularly sulfate-reducing bacteria (SRB), to the CuO NPs induced stress is important as hydrogen sulfide produced by SRB can cause sewer corrosion and odour emissions. In this study, we elucidated how the anabolic and catabolic processes of a model SRB, Desulfovibrio vulgaris Hidenborough (D. vulgaris), respond to CuO NPs. Physiological analyses indicated that the exposure of the culture to CuO NPs at elevated concentrations (>50 mg/L) inhibited both its anabolic and catabolic activities, as revealed by lowered cell proliferation and sulfate reduction rate. The antibacterial effects of CuO NPs were mainly attributed to the overproduction of reactive oxygen species. Transcriptomic analysis indicated that genes encoding for flagellar assembly and some genes involved in electron transfer and respiration were down-regulated, while genes for the ferric uptake regulator (Fur) were up-regulated. Moreover, the CuO NPs exposure significantly up-regulated genes involved in protein synthesis and ATP synthesis. These results suggest that CuO NPs inhibited energy conversion, cell mobility, and iron starvation to D. vulgaris. Meanwhile, D. vulgaris attempted to respond to the stress of CuO NPs by increasing protein and ATP synthesis. These findings offer new insights into the bacterial-nanoparticles interaction at the transcriptional level, and advance our understanding of impacts of CuO NPs on SRB in the environment.

Adaptation of Desulfovibrio alaskensis G20 to perchlorate, a specific inhibitor of sulfate reduction.

Hydrogen sulfide produced by sulfate-reducing microorganisms (SRM) poses significant health and economic risks, particularly during oil recovery. Previous studies identified perchlorate as a specific inhibitor of SRM. However, constant inhibitor addition to natural systems results in new selective pressures. Consequently, we investigated the ability of Desulfovibrio alaskensis G20 to evolve perchlorate resistance. Serial transfers in increasing concentrations of perchlorate led to robust growth in the presence of 100 mM inhibitor. Isolated adapted strains demonstrated a threefold increase in perchlorate resistance compared to the wild-type ancestor. Whole genome sequencing revealed a single base substitution in Dde_2265, the sulfate adenylyltransferase (sat). We purified and biochemically characterized the Sat from both wild-type and adapted strains, and showed that the adapted Sat was approximately threefold more resistant to perchlorate inhibition, mirroring whole cell results. The ability of this mutation to confer resistance across other inhibitors of sulfidogenesis was also assayed. The generalizability of this mutation was confirmed in multiple evolving G20 cultures and in another SRM, D. vulgaris Hildenborough. This work demonstrates that a single nucleotide polymorphism in Sat can have a significant impact on developing perchlorate resistance and emphasizes the value of adaptive laboratory evolution for understanding microbial responses to environmental perturbations.

A new family of transcriptional regulators of tungstoenzymes and molybdate/tungstate transport.

Bacterial genes for molybdenum-containing and tungsten-containing enzymes are often differentially regulated depending on the metal availability in the environment. Here, we describe a new family of transcription factors with an unusual DNA-binding domain related to excisionases of bacteriophages. These transcription factors are associated with genes for various molybdate and tungstate-specific transporting systems as well as molybdo/tungsto-enzymes in a wide range of bacterial genomes. We used a combination of computational and experimental techniques to study a member of the TF family, named TaoR (for tungsten-containing aldehyde oxidoreductase regulator). In Desulfovibrio vulgaris Hildenborough, a model bacterium for sulfate reduction studies, TaoR activates expression of aldehyde oxidoreductase aor and represses tungsten-specific ABC-type transporter tupABC genes under tungsten-replete conditions. TaoR binding sites at aor promoter were identified by electrophoretic mobility shift assay and DNase I footprinting. We also reconstructed TaoR regulons in 45 Deltaproteobacteria by comparative genomics approach and predicted target genes for TaoR family members in other Proteobacteria and Firmicutes.

Growth of an anaerobic sulfate-reducing bacterium sustained by oxygen respiratory energy conservation after O2 -driven experimental evolution.

Desulfovibrio species are representatives of microorganisms at the boundary between anaerobic and aerobic lifestyles, since they contain the enzymatic systems required for both sulfate and oxygen reduction. However, the latter has been shown to be solely a protective mechanism. By implementing the oxygen-driven experimental evolution of Desulfovibrio vulgaris Hildenborough, we have obtained strains that have evolved to grow with energy derived from oxidative phosphorylation linked to oxygen reduction. We show that a few mutations are sufficient for the emergence of this phenotype and reveal two routes of evolution primarily involving either inactivation or overexpression of the gene encoding heterodisulfide reductase. We propose that the oxygen respiration for energy conservation that sustains the growth of the O2 -evolved strains is associated with a rearrangement of metabolite fluxes, especially NAD+ /NADH, leading to an optimized O2 reduction. These evolved strains are the first sulfate-reducing bacteria that exhibit a demonstrated oxygen respiratory process that enables growth.