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Understanding the extracellular electron transfer mechanisms of electroactive bacteria could help determine their potential in microbial fuel cells (MFCs) and their microbial syntrophy with redox-active minerals in natural environments. However, the mechanisms of extracellular electron transfer to electrodes by sulfate-reducing bacteria (SRB) remain underexplored. Here, we utilized double-chamber MFCs with carbon cloth electrodes to investigate the extracellular electron transfer mechanisms of Desulfovibrio vulgaris Hildenborough ( Dv H), a model SRB, under varying lactate and sulfate concentrations using different Dv H mutants. Our MFC setup indicated that Dv H can harvest electrons from lactate at the anode and transfer them to cathode, where Dv H could further utilize these electrons. Patterns in current production compared with variations of electron donor/acceptor ratios in the anode and cathode suggested that attachment of Dv H to the electrode and biofilm density were critical for effective electricity generation. Electron microscopy analysis of Dv H biofilms indicated Dv H utilized filaments that resemble pili to attach to electrodes and facilitate extracellular electron transfer from cell to cell and to the electrode. Proteomics profiling indicated that Dv H adapted to electroactive respiration by presenting more pili- and flagellar-related proteins. The mutant with a deletion of the major pilus-producing gene yielded less voltage and far less attachment to both anodic and catholic electrodes, suggesting the importance of pili in extracellular electron transfer. The mutant with a deficiency in biofilm formation, however, did not eliminate current production indicating the existence of indirect extracellular electron transfer. Untargeted metabolomics profiling showed flavin-based metabolites, potential electron shuttles.IMPORTANCEWe explored the application of Desulfovibrio vulgaris Hildenborough in microbial fuel cells (MFCs) and investigated its potential extracellular electron transfer (EET) mechanism. We also conducted untargeted proteomics and metabolomics profiling, offering insights into how DvH adapts metabolically to different electron donors and acceptors. An understanding of the EET mechanism and metabolic flexibility of Dv H holds promise for future uses including bioremediation or enhancing efficacy in MFCs for wastewater treatment applications., Competing Interests: The authors declare no conflict of interest.

CRISPR-Cas tools for mammalian genome editing typically rely on single Cas9 or Cas12a proteins. While type I CRISPR systems in Class I may offer greater specificity and versatility, they are not well-developed for genome editing. Here, we present an alternative type I-C CRISPR system from Desulfovibrio vulgaris (Dvu) for efficient and precise genome editing in mammalian cells and animals. We optimized the Dvu type I-C editing complex to generate precise deletions at multiple loci in various cell lines and pig primary fibroblast cells using a paired PAM-in crRNA strategy. These edited pig cells can serve as donors for generating transgenic cloned piglets. The Dvu type I-C editor also enabled precise large fragment replacements with homology-directed repair. Additionally, we adapted the Dvu-Cascade effector for cytosine and adenine base editing, developing Dvu-CBE and Dvu-ABE systems. These systems efficiently induced C-to-T and A-to-G substitutions in human genes without double-strand breaks. Off-target analysis confirmed the high specificity of the Dvu type I-C editor. Our findings demonstrate the Dvu type I-C editor's potential for diverse mammalian genome editing applications, including deletions, fragment replacement, and base editing, with high efficiency and specificity for biomedicine and agriculture., (© 2024. Science China Press.)

Resource partitioning is central to the incredible productivity of microbial communities, including gigatons in annual methane emissions through syntrophic interactions. Previous work revealed how a sulfate reducer (Desulfovibrio vulgaris, Dv) and a methanogen (Methanococcus maripaludis, Mm) underwent evolutionary diversification in a planktonic context, improving stability, cooperativity, and productivity within 300-1000 generations. Here, we show that mutations in just 15 Dv and 7 Mm genes within a minimal assemblage of this evolved community gave rise to co-existing ecotypes that were spatially enriched within a few days of culturing in a fluidized bed reactor. The spatially segregated communities partitioned resources in the simulated subsurface environment, with greater lactate utilization by attached Dv but partial utilization of resulting H 2 by low affinity hydrogenases of Mm in the same phase. The unutilized H 2 was scavenged by high affinity hydrogenases of planktonic Mm, producing copious amounts of methane. Our findings show how a few mutations can drive resource partitioning amongst niche-differentiated ecotypes, whose interplay synergistically improves productivity of the entire mutualistic community., (© 2024. The Author(s).)

Molybdenum- or tungsten-dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO 2 to formate, with biotechnological applications envisaged in climate-change mitigation. The role of Met405 in the active site of Desulfovibrio vulgaris formate dehydrogenase AB (DvFdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active-site geometry in the absence of methionine S δ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed. This variant displayed reduced activity in both formate oxidation and CO 2 reduction, together with an increased sensitivity to oxygen inactivation., (open access.)

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., (© 2023 Applied Microbiology International and John Wiley & Sons Ltd.)

Desulfovibrio vulgaris has been a primary pure culture sulfate reducer for developing microbial corrosion concepts. Multiple mechanisms for how it accepts electrons from Fe 0 have been proposed. We investigated Fe 0 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 H 2 . The parental strain reduced sulfate with Fe 0 as the sole electron donor, but the hydrogenase mutant did not. H 2 accumulated over time in Fe 0 cultures of the hydrogenase mutant and sterile controls but not in parental strain cultures. Sulfide stimulated H 2 production in uninoculated controls apparently by both reacting with Fe 0 to generate H 2 and facilitating electron transfer from Fe 0 to H + . Parental strain supernatants did not accelerate H 2 production from Fe 0 , ruling out a role for extracellular hydrogenases. Previously proposed electron transfer between Fe 0 and D. vulgaris via soluble electron shuttles was not evident. The hydrogenase mutant did not reduce sulfate in the presence of Fe 0 and either riboflavin or anthraquinone-2,6-disulfonate, and these potential electron shuttles did not stimulate parental strain sulfate reduction with Fe 0 as the electron donor. The results demonstrate that D. vulgaris primarily accepts electrons from Fe 0 via H 2 as an intermediary electron carrier. These findings clarify the interpretation of previous D. vulgaris corrosion studies and suggest that H 2 -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 Fe 0 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 H 2 to shuttle electrons between Fe 0 and cells revives the concept, developed in some of the earliest studies on microbial corrosion, that sulfate reducers consumption of H 2 is a major microbial corrosion mechanism. The results further emphasize that direct Fe 0 -to-microbe electron transfer has yet to be rigorously demonstrated in sulfate-reducing microbes.

Background: The σ54 subunit controls a unique class of promoters in bacteria. Such promoters, without exception, require enhancer binding proteins (EBPs) for transcription initiation. Desulfovibrio vulgaris Hildenborough, a model bacterium for sulfate reduction studies, has a high number of EBPs, more than most sequenced bacteria. The cellular processes regulated by many of these EBPs remain unknown. Results: To characterize the σ54-dependent regulome of D. vulgaris Hildenborough, we identified EBP binding motifs and regulated genes by a combination of computational and experimental techniques. These predictions were supported by our reconstruction of σ54-dependent promoters by comparative genomics. We reassessed and refined the results of earlier studies on regulation in D. vulgaris Hildenborough and consolidated them with our new findings. It allowed us to reconstruct the σ54 regulome in D. vulgaris Hildenborough. This regulome includes 36 regulons that consist of 201 coding genes and 4 non-coding RNAs, and is involved in nitrogen, carbon and energy metabolism, regulation, transmembrane transport and various extracellular functions. To the best of our knowledge, this is the first report of direct regulation of alanine dehydrogenase, pyruvate metabolism genes and type III secretion system by σ54-dependent regulators. Conclusions: The σ54-dependent regulome is an important component of transcriptional regulatory network in D. vulgaris Hildenborough and related free-living Deltaproteobacteria. Our study provides a representative collection of σ54-dependent regulons that can be used for regulation prediction in Deltaproteobacteria and other taxa. [ABSTRACT FROM AUTHOR]

Many species have evolved to function as specialized mutualists, often to the detriment of their ability to survive independently. However, there are few, if any, well-controlled observations of the evolutionary processes underlying the genesis of new mutualisms. Here, we show that within the first 1,000 generations of initiating independent syntrophic interactions between a sulfate reducer (Desulfovibrio vulgaris) and a hydrogenotrophic methanogen (Methanococcus maripaludis), D. vulgaris frequently lost the capacity to grow by sulfate respiration, thus losing the primary physiological attribute of the genus. The loss of sulfate respiration was a consequence of mutations in one or more of three key genes in the pathway for sulfate respiration, required for sulfate activation (sat) and sulfate reduction to sulfite (apsA or apsB). Because loss-of-function mutations arose rapidly and independently in replicated experiments, and because these mutations were correlated with enhanced growth rate and productivity, gene loss could be attributed to natural selection, even though these mutations should significantly restrict the independence of the evolved D. vulgaris. Together, these data present an empirical demonstration that specialization for a mutualistic interaction can evolve by natural selection shortly after its origin. They also demonstrate that a sulfate-reducing bacterium can readily evolve to become a specialized syntroph, a situation that may have often occurred in nature. [ABSTRACT FROM AUTHOR]

Sulfate-reducing bacteria (SRB) cause souring and their biofilms are often the culprit in Microbiologically Influenced Corrosion (MIC). The two most common green biocides for SRB treatment are tetrakis-hydroxymethylphosphonium sulfate (THPS) and glutaraldehyde. It is unlikely that there will be another equally effective green biocide in the market any time soon. This means more effective biocide treatment probably will rely on biocide cocktails. In this work a triple biocide cocktail consisting of glutaraldehyde or THPS, ethylenediaminedisuccinate (EDDS) and methanol was used to treat planktonic SRB and to remove established SRB biofilms. Desulfovibrio vulgaris (ATCC 7757), a corrosive SRB was used as an example in the tests. Laboratory results indicated that with the addition of 10-15% (v/v) methanol to the glutaraldehyde and EDDS double combination, mitigation of planktonic SRB growth in ATCC 1249 medium and a diluted medium turned from inhibition to a kill effect while the chelator dosage was cut from 2,000 to 1,000 ppm. Biofilm removal was achieved when 50 ppm glutaraldehyde combined with 15% methanol and 1,000 ppm EDDS was used. THPS showed similar effects when it was used to replace glutaraldehyde in the triple biocide cocktail to treat planktonic SRB. [ABSTRACT FROM AUTHOR]

The ability to conduct advanced functional genonhic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosornal manipulations in a systematk manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal nmdification at designated loci. l'hese constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chroniosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation. [ABSTRACT FROM AUTHOR]

The pathway of electrons required for the reduction of sulfate in sulfate-reducing bacteria (SRB) is not yet fully characterized. In order to determine the role of a transmembrane protein complex suggested to be involved in this process, a deletion in Desulfovibrio vulgaris Hildenborough was created by marker exchange mutagenesis that eliminated four genes putatively encoding the QmoABC complex and a hypothetical protein (DVU0851). The Qmo (quinone-interacting membrane-bound oxidoreductase) complex is proposed to be responsible for transporting electrons to the dissimilatory adenosine-5'-phosphosulfate reductase in SRB. In support of the predicted role of this complex, the deletion mutant was unable to grow using sulfate as its sole electron acceptor with a range of electron donors. To explore a possible role for the hypothetical protein in sulfate reduction, a second mutant was constructed that had lost only the gene that codes for the DVU0851 protein. The second constructed mutant grew with sulfate as the sole electron acceptor; however, there was a lag that was not present with the wild-type or complemented strain. Neither deletion strain was significantly impaired for growth with sulfite or thiosulfate as the terminal electron acceptor. Complementation of the Δ(qmoABC-DVU0851) mutant with all four genes or only the qmoABC genes restored its ability to grow by sulfate respiration. These results confirmed the prediction that the Qmo complex is in the electron pathway for sulfate reduction and revealed that no other transmembrane complex could compensate when Qmo was lacking. [ABSTRACT FROM AUTHOR]

Although sulfate-reducing bacteria (SRB), such as Desulfovibrio vulgaris Hildenborough (DvH) are often eradicated in oil and gas operations with biocides, such as glutaraldehyde (Glut), tetrakis (hydroxymethyl) phosphonium sulfate (THPS), and benzalkonium chloride (BAC), their response to these agents is not well known. Whole genome microarrays of D. vulgaris treated with biocides well below the minimum inhibitory concentration showed that 256, 96, and 198 genes were responsive to Glut, THPS, and BAC, respectively, and that these three commonly used biocides affect the physiology of the cell quite differently. Glut induces expression of genes required to degrade or refold proteins inactivated by either chemical modification or heat shock, whereas BAC appears to target ribosomal structure. THPS appears to primarily affect energy metabolism of SRB. Mutants constructed for genes strongly up-regulated by Glut, were killed by Glut to a similar degree as the wild type. Hence, it is difficult to achieve increased sensitivity to this biocide by single gene mutations, because Glut affects so many targets. Our results increase understanding of the biocide's mode of action, allowing a more intelligent combination of mechanistically different agents. This can reduce stress on budgets for chemicals and on the environment. [ABSTRACT FROM AUTHOR]

Background: Communities of microorganisms control the rates of key biogeochemical cycles, and are important for biotechnology, bioremediation, and industrial microbiological processes. For this reason, we constructed a model microbial community comprised of three species dependent on trophic interactions. The three species microbial community was comprised of Clostridium cellulolyticum, Desulfovibrio vulgaris Hildenborough, and Geobacter sulfurreducens and was grown under continuous culture conditions. Cellobiose served as the carbon and energy source for C. cellulolyticum, whereas D. vulgaris and G. sulfurreducens derived carbon and energy from the metabolic products of cellobiose fermentation and were provided with sulfate and fumarate respectively as electron acceptors. Results: qPCR monitoring of the culture revealed C. cellulolyticum to be dominant as expected and confirmed the presence of D. vulgaris and G. sulfurreducens. Proposed metabolic modeling of carbon and electron flow of the threespecies community indicated that the growth of C. cellulolyticum and D. vulgaris were electron donor limited whereas G. sulfurreducens was electron acceptor limited. Conclusions: The results demonstrate that C. cellulolyticum, D. vulgaris, and G. sulfurreducens can be grown in coculture in a continuous culture system in which D. vulgaris and G. sulfurreducens are dependent upon the metabolic byproducts of C. cellulolyticum for nutrients. This represents a step towards developing a tractable model ecosystem comprised of members representing the functional groups of a trophic network. [ABSTRACT FROM AUTHOR]

In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to be inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3')-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1,000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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, NH 2 -R and RCOH/RCNH 2 ) 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Enhancer binding proteins (EBPs) are key players of σ 54 -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 σ 54 -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 σ 54 -dependent transcriptional regulator family probably advantageously selected by cells adapted to the anaerobic lifestyle to monitor redox stress conditions., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

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., (© 2021 Wiley Periodicals LLC.)

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., (Copyright © 2021 Ranava et al.)

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.

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 NaNO 3 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.

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.

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., (Copyright © 2020 Kempher et al.)

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., (© FEMS 2020.)

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., (Copyright © 2019 American Society for Microbiology.)

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 σ 54 -dependent regulator DVU2956 is repressed in biofilms. Utilizing a novel promoter that is primarily transcribed in biofilms (P dvu0304 ), 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 H 2 S production. Corroborating these results, producing DVU2956 in biofilms decreased H 2 S production by half, deleting dvu2956 increased H 2 S production by 131 ± 5%, and producing DVU2956 in the dvu2956 strain reduced H 2 S production. Therefore, DVU2956 maintains SRB in the planktonic state and reduces H 2 S formation., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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&#95;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., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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 O 2 -evolved strains is associated with a rearrangement of metabolite fluxes, especially NAD + /NADH, leading to an optimized O 2 reduction. These evolved strains are the first sulfate-reducing bacteria that exhibit a demonstrated oxygen respiratory process that enables growth., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The C-cluster of the enzyme carbon monoxide dehydrogenase (CODH) is a structurally distinctive Ni-Fe-S cluster employed to catalyze the reduction of CO 2 to CO as part of the Wood-Ljungdahl carbon fixation pathway. Using X-ray crystallography, we have observed unprecedented conformational dynamics in the C-cluster of the CODH from Desulfovibrio vulgaris , providing the first view of an oxidized state of the cluster. Combined with supporting spectroscopic data, our structures reveal that this novel, oxidized cluster arrangement plays a role in avoiding irreversible oxidative degradation at the C-cluster. Furthermore, mutagenesis of a conserved cysteine residue that binds the C-cluster in the oxidized state but not in the reduced state suggests that the oxidized conformation could be important for proper cluster assembly, in particular Ni incorporation. Together, these results lay a foundation for future investigations of C-cluster activation and assembly, and contribute to an emerging paradigm of metallocluster plasticity., Competing Interests: EW, MM, CU, LF, CL, VF, MP, SD, CD No competing interests declared, (© 2018, Wittenborn et al.)

A refined Desulfovibrio vulgaris Hildenborough flux balance analysis (FBA) model (iJF744) was developed, incorporating 1016 reactions that include 744 genes and 951 metabolites. A draft model was first developed through automatic model reconstruction using the ModelSeed Server and then curated based on existing literature. The curated model was further refined by incorporating three recently proposed redox reactions involving the Hdr-Flx and Qmo complexes and a lactate dehydrogenase (LdhAB, DVU 3027-3028) indicated by mutation and transcript analyses to serve electron transfer reactions central to syntrophic and respiratory growth. Eight different variations of this model were evaluated by comparing model predictions to experimental data determined for four different growth conditions - three for sulfate respiration (with lactate, pyruvate or H 2 /CO 2 -acetate) and one for fermentation in syntrophic coculture. The final general model supports (i) a role for Hdr-Flx in the oxidation of DsrC and ferredoxin, and reduction of NAD + in a flavin-based electron confurcating reaction sequence, (ii) a function of the Qmo complex in receiving electrons from the menaquinone pool and potentially from ferredoxin to reduce APS and (iii) a reduction of the soluble DsrC by LdhAB and a function of DsrC in electron transfer reactions other than sulfite reduction., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Biofilms of sulfate-reducing bacteria (SRB) are often the major cause of microbiologically influenced corrosion. The representative SRB Desulfovibrio vulgaris has previously been shown to have a biofilm that consists primarily of protein. In this study, by utilizing lectin staining, we identified that the biofilm of D. vulgaris also consists of the matrix components mannose, fucose and N-acetylgalactosamine (GalNAc), with mannose predominating. Based on these results, we found that the addition of mannose and the nonmetabolizable mannose analog 2-deoxy-d-glucose inhibits the biofilm formation of D. vulgaris as well as that of D. desulfuricans; both compounds also dispersed the SRB biofilms. In addition, the enzyme N-acetylgalactosaminidase, which degrades GalNAc, was effective in dispersing D. vulgaris biofilms. Therefore, by determining composition of the SRB biofilm, effective biofilm control methods may be devised., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Rapid genetic and phenotypic adaptation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough to salt stress was observed during experimental evolution. In order to identify key metabolites important for salt tolerance, a clone, ES10-5, which was isolated from population ES10 and allowed to experimentally evolve under salt stress for 5,000 generations, was analyzed and compared to clone ES9-11, which was isolated from population ES9 and had evolved under the same conditions for 1,200 generations. These two clones were chosen because they represented the best-adapted clones among six independently evolved populations. ES10-5 acquired new mutations in genes potentially involved in salt tolerance, in addition to the preexisting mutations and different mutations in the same genes as in ES9-11. Most basal abundance changes of metabolites and phospholipid fatty acids (PLFAs) were lower in ES10-5 than ES9-11, but an increase of glutamate and branched PLFA i17:1ω9c under high-salinity conditions was persistent. ES9-11 had decreased cell motility compared to the ancestor; in contrast, ES10-5 showed higher cell motility under both nonstress and high-salinity conditions. Both genotypes displayed better growth energy efficiencies than the ancestor under nonstress or high-salinity conditions. Consistently, ES10-5 did not display most of the basal transcriptional changes observed in ES9-11, but it showed increased expression of genes involved in glutamate biosynthesis, cation efflux, and energy metabolism under high salinity. These results demonstrated the role of glutamate as a key osmolyte and i17:1ω9c as the major PLFA for salt tolerance in D. vulgaris The mechanistic changes in evolved genotypes suggested that growth energy efficiency might be a key factor for selection. IMPORTANCE High salinity (e.g., elevated NaCl) is a stressor that affects many organisms. Salt tolerance, a complex trait involving multiple cellular pathways, is attractive for experimental evolutionary studies. Desulfovibrio vulgaris Hildenborough is a model sulfate-reducing bacterium (SRB) that is important in biogeochemical cycling of sulfur, carbon, and nitrogen, potentially for bio-corrosion, and for bioremediation of toxic heavy metals and radionuclides. The coexistence of SRB and high salinity in natural habitats and heavy metal-contaminated field sites laid the foundation for the study of salt adaptation of D. vulgaris Hildenborough with experimental evolution. Here, we analyzed a clone that evolved under salt stress for 5,000 generations and compared it to a clone evolved under the same condition for 1,200 generations. The results indicated the key roles of glutamate for osmoprotection and of i17:1ω9c for increasing membrane fluidity during salt adaptation. The findings provide valuable insights about the salt adaptation mechanism changes during long-term experimental evolution., (Copyright © 2017 Zhou et al.)

Biofilms of sulfate-reducing bacteria (SRB) are of particular interest as members of this group are culprits in corrosion of industrial metal and concrete pipelines as well as being key players in subsurface metal cycling. Yet the mechanism of biofilm formation by these bacteria has not been determined. Here we show that two supposedly identical wild-type cultures of the SRB Desulfovibrio vulgaris Hildenborough maintained in different laboratories have diverged in biofilm formation. From genome resequencing and subsequent mutant analyses, we discovered that a single nucleotide change within DVU1017, the ABC transporter of a type I secretion system (T1SS), was sufficient to eliminate biofilm formation in D. vulgaris Hildenborough. Two T1SS cargo proteins were identified as likely biofilm structural proteins, and the presence of at least one (with either being sufficient) was shown to be required for biofilm formation. Antibodies specific to these biofilm structural proteins confirmed that DVU1017, and thus the T1SS, is essential for localization of these adhesion proteins on the cell surface. We propose that DVU1017 is a member of the lapB category of microbial surface proteins because of its phenotypic similarity to the adhesin export system described for biofilm formation in the environmental pseudomonads. These findings have led to the identification of two functions required for biofilm formation in D. vulgaris Hildenborough and focus attention on the importance of monitoring laboratory-driven evolution, as phenotypes as fundamental as biofilm formation can be altered. IMPORTANCE The growth of bacteria attached to a surface (i.e., biofilm), specifically biofilms of sulfate-reducing bacteria, has a profound impact on the economy of developed nations due to steel and concrete corrosion in industrial pipelines and processing facilities. Furthermore, the presence of sulfate-reducing bacteria in oil wells causes oil souring from sulfide production, resulting in product loss, a health hazard to workers, and ultimately abandonment of wells. Identification of the required genes is a critical step for determining the mechanism of biofilm formation by sulfate reducers. Here, the transporter by which putative biofilm structural proteins are exported from sulfate-reducing Desulfovibrio vulgaris Hildenborough cells was discovered, and a single nucleotide change within the gene coding for this transporter was found to be sufficient to completely stop formation of biofilm., (Copyright © 2017 De León et al.)

Microbial populations can withstand, overcome and persist in the face of environmental fluctuation. Previously, we demonstrated how conditional gene regulation in a fluctuating environment drives dilution of condition-specific transcripts, causing a population of Desulfovibrio vulgaris Hildenborough (DvH) to collapse after repeatedly transitioning from sulfate respiration to syntrophic conditions with the methanogen Methanococcus maripaludis. Failure of the DvH to successfully transition contributed to the collapse of this model community. We investigated the mechanistic basis for loss of robustness by examining whether conditional gene regulation altered heterogeneity in gene expression across individual DvH cells. We discovered that robustness of a microbial population across environmental transitions was attributable to the retention of cells in two states that exhibited different condition-specific gene expression patterns. In our experiments, a population with disrupted conditional regulation successfully alternated between cell states. Meanwhile, a population with intact conditional regulation successfully switched between cell states initially, but collapsed after repeated transitions, possibly due to the high energy requirements of regulation. These results demonstrate that the survival of this entire model microbial community is dependent on the regulatory system's influence on the distribution of distinct cell states among individual cells within a clonal population., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Managing trade-offs through gene regulation is believed to confer resilience to a microbial community in a fluctuating resource environment. To investigate this hypothesis, we imposed a fluctuating environment that required the sulfate-reducer Desulfovibrio vulgaris to undergo repeated ecologically relevant shifts between retaining metabolic independence (active capacity for sulfate respiration) and becoming metabolically specialized to a mutualistic association with the hydrogen-consuming Methanococcus maripaludis Strikingly, the microbial community became progressively less proficient at restoring the environmentally relevant physiological state after each perturbation and most cultures collapsed within 3-7 shifts. Counterintuitively, the collapse phenomenon was prevented by a single regulatory mutation. We have characterized the mechanism for collapse by conducting RNA-seq analysis, proteomics, microcalorimetry, and single-cell transcriptome analysis. We demonstrate that the collapse was caused by conditional gene regulation, which drove precipitous decline in intracellular abundance of essential transcripts and proteins, imposing greater energetic burden of regulation to restore function in a fluctuating environment., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

The sulfate-reducing bacteria of the Desulfovibrio genus make three distinct modified tetrapyrroles, haem, sirohaem and adenosylcobamide, where sirohydrochlorin acts as the last common biosynthetic intermediate along the branched tetrapyrrole pathway. Intriguingly, D. vulgaris encodes two sirohydrochlorin chelatases, CbiK P and CbiK C , that insert cobalt/iron into the tetrapyrrole macrocycle but are thought to be distinctly located in the periplasm and cytoplasm respectively. Fusing GFP onto the C-terminus of CbiK P confirmed that the protein is transported to the periplasm. The structure-function relationship of CbiK P was studied by constructing eleven site-directed mutants and determining their chelatase activities, oligomeric status and haem binding abilities. Residues His154 and His216 were identified as essential for metal-chelation of sirohydrochlorin. The tetrameric form of the protein is stabilized by Arg54 and Glu76, which form hydrogen bonds between two subunits. His96 is responsible for the binding of two haem groups within the main central cavity of the tetramer. Unexpectedly, CbiK P is shown to bind two additional haem groups through interaction with His103. Thus, although still retaining cobaltochelatase activity, the presence of His96 and His103 in CbiK P , which are absent from all other known bacterial cobaltochelatases, has evolved CbiK P a new function as a haem binding protein permitting it to act as a potential haem chaperone or transporter., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Syntrophies are metabolic cooperations, whereby two organisms co-metabolize a substrate in an interdependent manner. Many of the observed natural syntrophic interactions are mandatory in the absence of strong electron acceptors, such that one species in the syntrophy has to assume the role of electron sink for the other. While this presents an ecological setting for syntrophy to be beneficial, the potential genetic drivers of syntrophy remain unknown to date. Here, we show that the syntrophic sulfate-reducing species Desulfovibrio vulgaris displays a stable genetic polymorphism, where only a specific genotype is able to engage in syntrophy with the hydrogenotrophic methanogen Methanococcus maripaludis. This 'syntrophic' genotype is characterized by two genetic alterations, one of which is an in-frame deletion in the gene encoding for the ion-translocating subunit cooK of the membrane-bound COO hydrogenase. We show that this genotype presents a specific physiology, in which reshaping of energy conservation in the lactate oxidation pathway enables it to produce sufficient intermediate hydrogen for sustained M. maripaludis growth and thus, syntrophy. To our knowledge, these findings provide for the first time a genetic basis for syntrophy in nature and bring us closer to the rational engineering of syntrophy in synthetic microbial communities., Competing Interests: The authors declare no conflict of interests.

Bacteria employ surveillance complexes guided by CRISPR (clustered, regularly interspaced, short palindromic repeats) RNAs (crRNAs) to target foreign nucleic acids for destruction. Although most type I and type III CRISPR systems require four or more distinct proteins to form multi-subunit surveillance complexes, the type I-C systems use just three proteins to achieve crRNA maturation and double-stranded DNA target recognition. We show that each protein plays multiple functional and structural roles: Cas5c cleaves pre-crRNAs and recruits Cas7 to position the RNA guide for DNA binding and unwinding by Cas8c. Cryoelectron microscopy reconstructions of free and DNA-bound forms of the Cascade/I-C surveillance complex reveal conformational changes that enable R-loop formation with distinct positioning of each DNA strand. This streamlined type I-C system explains how CRISPR pathways can evolve compact structures that retain full functionality as RNA-guided DNA capture platforms., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hydrogen sulfide produced by sulfate-reducing bacteria (SRB) in sewers causes odor problems and asset deterioration due to the sulfide-induced concrete corrosion. Free nitrous acid (FNA) was recently demonstrated as a promising antimicrobial agent to alleviate hydrogen sulfide production in sewers. However, details of the antimicrobial mechanisms of FNA are largely unknown. Here, we report the multiple-targeted antimicrobial effects of FNA on the SRB Desulfovibrio vulgaris Hildenborough by determining the growth, physiological, and gene expression responses to FNA exposure. The activities of growth, respiration, and ATP generation were inhibited when exposed to FNA. These changes were reflected in the transcript levels detected during exposure. The removal of FNA was evident by nitrite reduction that likely involved nitrite reductase and the poorly characterized hybrid cluster protein, and the genes coding for these proteins were highly expressed. During FNA exposure, lowered ribosome activity and protein production were detected. Additionally, conditions within the cells were more oxidizing, and there was evidence of oxidative stress. Based on an interpretation of the measured responses, we present a model depicting the antimicrobial effects of FNA on D. vulgaris These findings provide new insight for understanding the responses of D. vulgaris to FNA and will provide a foundation for optimal application of this antimicrobial agent for improved control of sewer corrosion and odor management.IMPORTANCE Hydrogen sulfide produced by SRB in sewers causes odor problems and results in serious deterioration of sewer assets that requires very costly and demanding rehabilitation. Currently, there is successful application of the antimicrobial agent free nitrous acid (FNA), the protonated form of nitrite, for the control of sulfide levels in sewers (G. Jiang et al., Water Res 47:4331-4339, 2013, http://dx.doi.org/10.1016/j.watres.2013.05.024). However, the details of the antimicrobial mechanisms of FNA are largely unknown. In this study, we identified the key responses (decreased anaerobic respiration, reducing FNA, combating oxidative stress, and shutting down protein synthesis) of Desulfovibrio vulgaris Hildenborough, a model sewer corrosion bacterium, to FNA exposure by examining the growth, physiological, and gene expression changes. These findings provide new insight and underpinning knowledge for understanding the responses of D. vulgaris to FNA exposure, thereby benefiting the practical application of FNA for improved control of sewer corrosion and odor., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The initiation and progression of colorectal cancer (CRC) are intimately associated with genetic, environmental and biological factors. Desulfovibrio vulgaris (DSV), a sulfate-reducing bacterium, has been found excessive growth in CRC patients, suggesting a potential role in carcinogenesis. However, the precise mechanisms underlying this association remain incompletely understood. We have found Desulfovibrio was abundant in high-fat diet-induced Apc min/+ mice, and DSV, a member of Desulfovibrio , triggered colonocyte proliferation of germ-free mice. Furthermore, the level of DSV progressively rose from healthy individuals to CRC patients. Flagella are important accessory structures of bacteria, which can help them colonize and enhance their invasive ability. We found that D. vulgaris flagellin (DVF) drove the proliferation, migration, and invasion of CRC cells and fostered the growth of CRC xenografts. DVF enriched the epithelial-mesenchymal transition (EMT)-associated genes and characterized the facilitation of DVF on EMT. Mechanistically, DVF induced EMT through a functional transmembrane receptor called leucine-rich repeat containing 19 (LRRC19). DVF interacted with LRRC19 to modulate the ubiquitination of tumor necrosis factor receptor-associated factor (TRAF)6, rather than TRAF2. This interaction drove the ubiquitination of pivotal molecule TAK1, further enhancing its autophosphorylation and ultimately contributing to EMT. Collectively, DVF interacts with LRRC19 to activate the TRAF6/TAK1 signaling pathway, thereby promoting the EMT of CRC. These data shed new light on the role of gut microbiota in CRC and establish a potential clinical therapeutic target.

Sulfur metabolism is one of the oldest known redox geochemical cycles in our atmosphere. These redox processes utilize different sulfur anions and the reactions are performed by the gene products of dsr operon from phylogenetically diverse sets of microorganisms. The operon is involved in the maintenance of environmental sulfur balance. Interestingly, the dsr operon is found to be present in both sulfur anion oxidizing and reducing microorganisms and in both types of organisms DsrAB protein complex plays a vital role. Though there are various reports regarding the genetics of dsr operon there are practically no reports dealing with the structural aspects of sulfur metabolism by dsr operon. In our present study, we tried to compare the mechanisms of sulfur anion oxidation and reduction by Allochromatium vinosum and Desulfovibrio vulgaris respectively through DsrAB protein complex. We analyzed the modes of bindings of sulfur anions to the DsrAB protein complex and observed that for sulfur anion oxidizers, sulfide and thiosulfate are the best substrates whereas for reducers sulfate and sulfite have the best binding abilities. We analyzed the binding interaction pattern of the DsrA and DsrB proteins while forming the DsrAB protein complexes in Desulfovibrio vulgaris and Allochromatium vinosum. To our knowledge this is the first report that analyzes the differences in binding patterns of sulfur substrates with DsrAB protein from these two microorganisms. This study would therefore be essential to predict the biochemical mechanism of sulfur anion oxidation and reduction by these two microorganisms i.e., Desulfovibrio vulgaris (sulfur anion reducer) and Allochromatium vinosum (sulfur anion oxidizer). Our observations also highlight the mechanism of sulfur geochemical cycle which has important implications in future study of sulfur metabolism as it has a huge application in waste remediation and production of industrial bio-products viz. vitamins, bio-polyesters and bio-hydrogen., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The σ(54) subunit controls a unique class of promoters in bacteria. Such promoters, without exception, require enhancer binding proteins (EBPs) for transcription initiation. Desulfovibrio vulgaris Hildenborough, a model bacterium for sulfate reduction studies, has a high number of EBPs, more than most sequenced bacteria. The cellular processes regulated by many of these EBPs remain unknown., Results: To characterize the σ(54)-dependent regulome of D. vulgaris Hildenborough, we identified EBP binding motifs and regulated genes by a combination of computational and experimental techniques. These predictions were supported by our reconstruction of σ(54)-dependent promoters by comparative genomics. We reassessed and refined the results of earlier studies on regulation in D. vulgaris Hildenborough and consolidated them with our new findings. It allowed us to reconstruct the σ(54) regulome in D. vulgaris Hildenborough. This regulome includes 36 regulons that consist of 201 coding genes and 4 non-coding RNAs, and is involved in nitrogen, carbon and energy metabolism, regulation, transmembrane transport and various extracellular functions. To the best of our knowledge, this is the first report of direct regulation of alanine dehydrogenase, pyruvate metabolism genes and type III secretion system by σ(54)-dependent regulators., Conclusions: The σ(54)-dependent regulome is an important component of transcriptional regulatory network in D. vulgaris Hildenborough and related free-living Deltaproteobacteria. Our study provides a representative collection of σ(54)-dependent regulons that can be used for regulation prediction in Deltaproteobacteria and other taxa.

To investigate the genetic basis of microbial evolutionary adaptation to salt (NaCl) stress, populations of Desulfovibrio vulgaris Hildenborough (DvH), a sulfate-reducing bacterium important for the biogeochemical cycling of sulfur, carbon and nitrogen, and potentially the bioremediation of toxic heavy metals and radionuclides, were propagated under salt stress or non-stress conditions for 1200 generations. Whole-genome sequencing revealed 11 mutations in salt stress-evolved clone ES9-11 and 14 mutations in non-stress-evolved clone EC3-10. Whole-population sequencing data suggested the rapid selective sweep of the pre-existing polymorphisms under salt stress within the first 100 generations and the slow fixation of new mutations. Population genotyping data demonstrated that the rapid selective sweep of pre-existing polymorphisms was common in salt stress-evolved populations. In contrast, the selection of pre-existing polymorphisms was largely random in EC populations. Consistently, at 100 generations, stress-evolved population ES9 showed improved salt tolerance, namely increased growth rate (2.0-fold), higher biomass yield (1.8-fold) and shorter lag phase (0.7-fold) under higher salinity conditions. The beneficial nature of several mutations was confirmed by site-directed mutagenesis. All four tested mutations contributed to the shortened lag phases under higher salinity condition. In particular, compared with the salt tolerance improvement in ES9-11, a mutation in a histidine kinase protein gene lytS contributed 27% of the growth rate increase and 23% of the biomass yield increase while a mutation in hypothetical gene DVU2472 contributed 24% of the biomass yield increase. Our results suggested that a few beneficial mutations could lead to dramatic improvements in salt tolerance.

Unlabelled: Sulfate-reducing bacteria (SRB) are sensitive to low concentrations of nitrite, and nitrite has been used to control SRB-related biofouling in oil fields. Desulfovibrio vulgaris Hildenborough, a model SRB, carries a cytochrome c-type nitrite reductase (nrfHA) that confers resistance to low concentrations of nitrite. The regulation of this nitrite reductase has not been directly examined to date. In this study, we show that DVU0621 (NrfR), a sigma54-dependent two-component system response regulator, is the positive regulator for this operon. NrfR activates the expression of the nrfHA operon in response to nitrite stress. We also show that nrfR is needed for fitness at low cell densities in the presence of nitrite because inactivation of nrfR affects the rate of nitrite reduction. We also predict and validate the binding sites for NrfR upstream of the nrfHA operon using purified NrfR in gel shift assays. We discuss possible roles for NrfR in regulating nitrate reductase genes in nitrate-utilizing Desulfovibrio spp., Importance: The NrfA nitrite reductase is prevalent across several bacterial phyla and required for dissimilatory nitrite reduction. However, regulation of the nrfA gene has been studied in only a few nitrate-utilizing bacteria. Here, we show that in D. vulgaris, a bacterium that does not respire nitrate, the expression of nrfHA is induced by NrfR upon nitrite stress. This is the first report of regulation of nrfA by a sigma54-dependent two-component system. Our study increases our knowledge of nitrite stress responses and possibly of the regulation of nitrate reduction in SRB., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Flavin-based electron bifurcation (FBEB) is an important mechanism for the energy metabolism of anaerobes. A new family of NADH dehydrogenases, the flavin oxidoreductase (FlxABCD, previously called FloxABCD), was proposed to perform FBEB in sulphate-reducing organisms coupled with heterodisulfide reductase (HdrABC). We found that the hdrABC-flxABCD gene cluster is widespread among anaerobic bacteria, pointing to a general and important role in their bioenergetics. In this work, we studied FlxABCD of Desulfovibrio vulgaris Hildenborough. The hdr-flx genes are part of the same transcriptional unit and are increased in transcription during growth in ethanol-sulfate, and to a less extent during pyruvate fermentation. Two mutant strains were generated: one where expression of the hdr-flx genes was interrupted and another lacking the flxA gene. Both strains were unable to grow with ethanol-sulfate, whereas growth was restored in a flxA-complemented strain. The mutant strains also produced very reduced amounts of ethanol compared with the wild type during pyruvate fermentation. Our results show that in D. vulgaris, the FlxABCD-HdrABC proteins are essential for NADH oxidation during growth on ethanol, probably involving a FBEB mechanism that leads to reduction of ferredoxin and the small protein DsrC, while in fermentation they operate in reverse, reducing NAD(+) for ethanol production., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Objectives: To engineer a recombinant Escherichia coli to produce polyhydroxyalkanoate biopolymer beads displaying carbonic anhydrase (CA) from Desulfovibrio vulgaris str. "Miyazaki F" (DvCA)., Results: The highest measured specific activity of the immobilised CA was 211 U/mg DvCA. The immobilised CA was thermostable, retaining 114 U/mg DvCA of activity after incubation at 90 °C for 1 h. Additionally, the immobilised CA tolerated 30 min incubations in a variety of pH conditions and was especially tolerant of alkaline conditions, retaining 131 U/mg DvCA of activity after pH 12 incubation., Conclusion: CA has a potential role in atmospheric CO2 mitigation strategies and the stability of the functionalised beads indicates suitability for use in industrial settings such as coal-fired power plants.