Reductase Operon Research Articles

The novel regulator HdrR controls the transcription of the heterodisulfide reductase operon hdrBCA in Methanosarcina barkeri.

Methanogenic archaea play a key role in the global carbon cycle because these microorganisms remineralize organic compounds in various anaerobic environments. The microorganism Methanosarcina barkeri is a metabolically versatile methanogen, which can utilize acetate, methanol, and H2/CO2 to synthesize methane. However, the regulatory mechanisms underlying methanogenesis for different substrates remain unknown. In this study, RNA-seq analysis was used to investigate M. barkeri growth and gene transcription under different substrate regimes. According to the results, M. barkeri showed the best growth under methanol, followed by H2/CO2 and acetate, and these findings corresponded well with the observed variations in genes transcription abundance for different substrates. In addition, we identified a novel regulator, MSBRM_RS03855 (designated as HdrR), which specifically activates the transcription of the heterodisulfide reductase hdrBCA operon in M. barkeri. HdrR was able to bind to the hdrBCA operon promoter to regulate transcription. Furthermore, the structural model analyses revealed a helix-turn-helix domain, which is likely involved in DNA binding. Taken together, HdrR serves as a model to reveal how certain regulatory factors control the expression of key enzymes in the methanogenic pathway.IMPORTANCEThe microorganism Methanosarcina barkeri has a pivotal role in the global carbon cycle and contributes to global temperature homeostasis. The consequences of biological methanogenesis are far-reaching, including impacts on atmospheric methane and CO2 concentrations, agriculture, energy production, waste treatment, and human health. As such, reducing methane emissions is crucial to meeting set climate goals. The methanogenic activity of certain microorganisms can be drastically reduced by inhibiting the transcription of the hdrBCA operon, which encodes heterodisulfide reductases. Here, we provide novel insight into the mechanisms regulating hdrBCA operon transcription in the model methanogen M. barkeri. The results clarified that HdrR serves as a regulator of heterodisulfide reductase hdrBCA operon transcription during methanogenesis, which expands our understanding of the unique regulatory mechanisms that govern methanogenesis. The findings presented in this study can further our understanding of how genetic regulation can effectively reduce the methane emissions caused by methanogens.

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least 2 decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

The horizontal gene transfer of perchlorate reduction genomic island in three bacteria from an ecological niche.

Three new strains of dissimilatory perchlorate-reducing bacteria (DPRB), QD19-16, QD1-5, and P3-1, were isolated from an active sludge. Phylogenetic trees based on 16S rRNA genes indicated that QD19-16, QD1-5, and P3-1 belonged to Brucella, Acidovorax, and Citrobacter, respectively, expanding the distribution of DPRB in the Proteobacteria. The three strains were gram-negative and facultative anaerobes with rod-shaped cells without flagella, which were 1.0-1.6μm long and 0.5-0.6μm wide. The three DPRB strains utilized similar broad spectrum of electron donors and acceptors and demonstrated a similar capability to reduce perchlorate within 6days. The enzyme activity of perchlorate reductase in QD19-16 toward chlorate was higher than that toward perchlorate. The high sequence similarity of the perchlorate reductase operon and chlorite dismutase genes in the perchlorate reduction genomic islands (PRI) of the three strains implied that they were monophyletic origin from a common ancestral PRI. Two transposase genes (tnp1 and tnp2) were found in the PRIs of strain QD19-16 and QD1-5, but were absent in the strain P3-1 PRI. The presence of fragments of IR sequences in the P3-1 PRI suggested that P3-1 PRI had previously contained these two tnp genes. Therefore, it is plausible to suggest that a common ancestral PRI transferred across the strains Brucella sp. QD19-16, Acidovorax sp. QD1-5, and Citrobacter sp. P3-1 through horizontal gene transfer, facilitated by transposases. These results provided a direct evidence of horizontal gene transfer of PRI that could jump across phylogenetically unrelated bacteria through transposase. KEY POINTS: • Three new DPRB strains can effectively remove high concentration of perchlorate. • The PRIs of three DPRB strains are acquired from a single ancestral PRI. • PRIs are incorporated into different bacteria genome through HGT by transposase.

Probing the Reactivity of [4Fe-4S] Fumarate and Nitrate Reduction (FNR) Regulator with O2 and NO: Increased O2 Resistance and Relative Specificity for NO of the [4Fe-4S] L28H FNR Cluster

The Escherichia coli fumarate and nitrate reduction (FNR) regulator acts as the cell’s master switch for the transition between anaerobic and aerobic respiration, controlling the expression of >300 genes in response to O2 availability. Oxygen is perceived through a reaction with FNR’s [4Fe-4S] cluster cofactor. In addition to its primary O2 signal, the FNR [4Fe-4S] cluster also reacts with nitric oxide (NO). In response to physiological concentrations of NO, FNR de-represses the transcription of hmp, which encodes a principal NO-detoxifying enzyme, and fails to activate the expression of the nitrate reductase (nar) operon, a significant source of endogenous cellular NO. Here, we show that the L28H variant of FNR, which is much less reactive towards O2 than wild-type FNR, remains highly reactive towards NO. A high resolution structure and molecular dynamics (MD) simulations of the closely related L28H-FNR from Aliivibrio fischeri revealed decreased conformational flexibility of the Cys20-Cys29 cluster-binding loop that is suggested to inhibit outer-sphere O2 reactivity, but only partially impair inner-sphere NO reactivity. Our data provide new insights into the mechanistic basis for how iron–sulfur cluster regulators can distinguish between O2 and NO.

Characterisation of an Escherichia coli line that completely lacks ribonucleotide reduction yields insights into the evolution of parasitism and endosymbiosis.

Life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. As ribonucleotide reduction has on occasion been lost in parasites and endosymbionts, which are instead dependent on their host for deoxyribonucleotide synthesis, it should in principle be possible to knock this process out if growth media are supplemented with deoxyribonucleosides. We report the creation of a strain of Escherichia coli where all three ribonucleotide reductase operons have been deleted following introduction of a broad spectrum deoxyribonucleoside kinase from Mycoplasma mycoides. Our strain shows slowed but substantial growth in the presence of deoxyribonucleosides. Under limiting deoxyribonucleoside levels, we observe a distinctive filamentous cell morphology, where cells grow but do not appear to divide regularly. Finally, we examined whether our lines can adapt to limited supplies of deoxyribonucleosides, as might occur in the switch from de novo synthesis to dependence on host production during the evolution of parasitism or endosymbiosis. Over the course of an evolution experiment, we observe a 25-fold reduction in the minimum concentration of exogenous deoxyribonucleosides necessary for growth. Genome analysis reveals that several replicate lines carry mutations in deoB and cdd. deoB codes for phosphopentomutase, a key part of the deoxyriboaldolase pathway, which has been hypothesised as an alternative to ribonucleotide reduction for deoxyribonucleotide synthesis. Rather than complementing the loss of ribonucleotide reduction, our experiments reveal that mutations appear that reduce or eliminate the capacity for this pathway to catabolise deoxyribonucleotides, thus preventing their loss via central metabolism. Mutational inactivation of both deoB and cdd is also observed in a number of obligate intracellular bacteria that have lost ribonucleotide reduction. We conclude that our experiments recapitulate key evolutionary steps in the adaptation to life without ribonucleotide reduction.

The emergence of the tetrathionate reductase operon in the Escherichia coli/Shigella pan-genome.

Escherichia coli pathogenic variants (pathovars) are generally characterized by defined virulence traits and are susceptible to the evolution of hybridized identities due to the considerable plasticity of the E. coli genome. We have isolated a strain from a purified diet intended for research animals that further demonstrates the ability of E. coli to acquire novel genetic elements leading potentially to emergent new pathovars. Utilizing next generation sequencing to obtain a whole genome profile, we report an atypical strain of E. coli, EcoFA807-17, possessing a tetrathionate reductase (ttr) operon, which enables the utilization of tetrathionate as an electron acceptor, thus facilitating respiration in anaerobic environments such as the mammalian gut. The ttr operon is a potent virulence factor for several enteric pathogens, most prominently Salmonella enterica. However, the presence of chromosomally integrated tetrathionate reductase genes does not appear to have been previously reported in wild-type E. coli or Shigella. Accordingly, it is possible that the appearance of this virulence factor may signal the evolution of new mechanisms of pathogenicity in E. coli and Shigella and may potentially alter the effectiveness of existing assays using tetrathionate reductase as a unique marker for the detection of Salmonella enterica.

Hydroxyurea Induces a Stress Response That Alters DNA Replication and Nucleotide Metabolism in Bacillus subtilis.

Hydroxyurea (HU) is classified as a ribonucleotide reductase (RNR) inhibitor and has been widely used to stall DNA replication by depleting deoxyribonucleoside triphosphate (dNTP) pools. Recent evidence in Escherichia coli shows that HU readily forms breakdown products that damage DNA directly, indicating that toxicity is a result of secondary effects. Because HU is so widely used in the laboratory and as a clinical therapeutic, it is important to understand its biological effects. To determine how Bacillus subtilis responds to HU-induced stress, we performed saturating transposon insertion mutagenesis followed by deep sequencing (Tn-seq), transcriptome sequencing (RNA-seq) analysis, and measurement of replication fork progression. Our data show that B. subtilis cells elongate, and replication fork progression is slowed, following HU challenge. The transcriptomic data show that B. subtilis cells initially mount a metabolic response likely caused by dNTP pool depletion before inducing the DNA damage response (SOS) after prolonged exposure. To compensate for reduced nucleotide pools, B. subtilis upregulates the purine and pyrimidine biosynthetic machinery and downregulates the enzymes producing ribose 5-phosphate. We show that overexpression of the RNR genes nrdEF suppresses the growth interference caused by HU, suggesting that RNR is an important target of HU in B. subtilis. Although genes involved in nucleotide and carbon metabolism showed considerable differential expression, we also find that genes of unknown function (y-genes) represent the largest class of differentially expressed genes. Deletion of individual y-genes caused moderate growth interference in the presence of HU, suggesting that cells have several ways of coping with HU-induced metabolic stress. IMPORTANCE Hydroxyurea (HU) has been widely used as a clinical therapeutic and an inhibitor of DNA replication. Some evidence suggests that HU inhibits ribonucleotide reductase, depleting dNTP pools, while other evidence shows that toxic HU breakdown products are responsible for growth inhibition and genotoxic stress. Here, we use multiple, complementary approaches to characterize the response of Bacillus subtilis to HU. B. subtilis responds by upregulating the expression of purine and pyrimidine biosynthesis. We show that HU challenge reduced DNA replication and that overexpression of the ribonucleotide reductase operon suppressed growth interference by HU. Our results demonstrate that HU targets RNR and several other metabolic enzymes contributing to toxicity in bacteria.

Reduction of alternative electron acceptors drives biofilm formation in Shewanella algae

Shewanella spp. possess a broad respiratory versatility, which contributes to the occupation of hypoxic and anoxic environmental or host-associated niches. Here, we observe a strain-specific induction of biofilm formation in response to supplementation with the anaerobic electron acceptors dimethyl sulfoxide (DMSO) and nitrate in a panel of Shewanella algae isolates. The respiration-driven biofilm response is not observed in DMSO and nitrate reductase deletion mutants of the type strain S. algae CECT 5071, and can be restored upon complementation with the corresponding reductase operon(s) but not by an operon containing a catalytically inactive nitrate reductase. The distinct transcriptional changes, proportional to the effect of these compounds on biofilm formation, include cyclic di-GMP (c-di-GMP) turnover genes. In support, ectopic expression of the c-di-GMP phosphodiesterase YhjH of Salmonella Typhimurium but not its catalytically inactive variant decreased biofilm formation. The respiration-dependent biofilm response of S. algae may permit differential colonization of environmental or host niches.

Mycobacterium tuberculosis and M. bovis BCG Moreau Fumarate Reductase Operons Produce Different Polypeptides That May Be Related to Non-canonical Functions.

Tuberculosis is a world widespread disease, caused by Mycobacterium tuberculosis (M.tb). Although considered an obligate aerobe, this organism can resist life-limiting conditions such as microaerophily mainly due to its set of enzymes responsible for energy production and coenzyme restoration under these conditions. One of these enzymes is fumarate reductase, an heterotetrameric complex composed of a catalytic (FrdA), an iron-sulfur cluster (FrdB) and two transmembrane (FrdC and FrdD) subunits involved in anaerobic respiration and important for the maintenance of membrane potential. In this work, aiming to further characterize this enzyme function in mycobacteria, we analyzed the expression of FrdB-containing proteins in M.tb and Mycobacterium bovis Bacillus Calmette–Guérin (BCG) Moreau, the Brazilian vaccine strain against tuberculosis. We identified three isoforms in both mycobacteria, two of them corresponding to the predicted encoded polypeptides of M.tb (27 kDa) and BCG Moreau (40 kDa) frd sequences, as due to an insertion on the latter’s operon a fused FrdBC protein is expected. The third 52 kDa band can be explained by a transcriptional slippage event, typically occurring when mutation arises in a repetitive region within a coding sequence, thought to reduce its impact allowing the production of both native and variant forms. Comparative modeling of the M.tb and BCG Moreau predicted protein complexes allowed the detection of subtle overall differences, showing a high degree of structure and maybe functional resemblance among them. Axenic growth and macrophage infection assays show that the frd locus is important for proper bacterial development in both scenarios, and that both M.tb’s and BCG Moreau’s alleles can partially revert the hampered phenotype of the knockout strain. Altogether, our results show that the frdABCD operon of Mycobacteria may have evolved to possess other yet non-described functions, such as those necessary during aerobic logarithmic growth and early stage steps of infection.

Comparative Genomics of Haemophilus ducreyi; Insight into genes related to Virulence, Disease and Defense

Haemophilus ducreyi, a fastidious, gram-negative, coccobacillus bacteria of Pasteurellaceae family causes sexually transmitteddisease known as Chancroid which results genital ulcer disease globally characterized by painful sores on the genitalia and also have been associated particularly with facilitating the transmission of human immunodeficiency virus (HIV). Currently, only asingle reference strain (AE017143) are available. This study presents comparative analysis of ten draft genomes isolated fromChildren with Cutaneous Lesions in Vanuatu and Ghana. Computational analysis of draft sequences have triggered the genesrelated to virulence, disease and diagnosis. This overall function based analysis based on virulence, disease and diagnosis finallyshowed that during comparison between ten isolates with reference strain, four genes are common in all additional isolates except reference strain. Random analysis among additional ten isolates except reference strain revealed two genes which are common in three isolates but absolutely absent in all other isolates. Those genes were characterized and found to be functioned as Mercuric ion reductase and Mercury resistance operon regulatory protein. Gene gain/loss was also observed among different isolates in association of CRISPRs and prophage due to their prospective contribution in evolutionary change. This study on multiple strains of Haemophilus ducreyi revealed the biological system of this bacteria including the genes related to virulence and causation of disease which can be effective novel drug/vaccine targets in future prospects.

Comparative genomic analysis between Corynebacterium pseudotuberculosis strains isolated from buffalo.

Corynebacterium pseudotuberculosis is a Gram-positive, pleomorphic, facultative intracellular pathogen that causes Oedematous Skin Disease (OSD) in buffalo. To better understand the pathogenic mechanisms of OSD, we performed a comparative genomic analysis of 11 strains of C. pseudotuberculosis isolated from different buffalo found to be infected in Egypt during an outbreak that occurred in 2008. Sixteen previously described pathogenicity islands (PiCp) were present in all of the new buffalo strains, but one of them, PiCp12, had an insertion that contained both a corynephage and a diphtheria toxin gene, both of which may play a role in the adaptation of C. pseudotuberculosis to this new host. Synteny analysis showed variations in the site of insertion of the corynephage during the same outbreak. A gene functional comparison showed the presence of a nitrate reductase operon that included genes involved in molybdenum cofactor biosynthesis, which is necessary for a positive nitrate reductase phenotype and is a possible adaptation for intracellular survival. Genomes from the buffalo strains also had fusions in minor pilin genes in the spaA and spaD gene cluster (spaCX and spaYEF), which could suggest either an adaptation to this particular host, or mutation events in the immediate ancestor before this particular epidemic. A phylogenomic analysis confirmed a clear separation between the Ovis and Equi biovars, but also showed what appears to be a clustering by host species within the Equi strains.

Genomic Analysis of the Extremely Halophilic Archaeon Halobacterium noricense CBA1132 Isolated from Solar Salt That Is an Essential Material for Fermented Foods.

The extremely halophilic archaeon Halobacterium noricense is a member of the genus Halobacterium. Strain CBA1132 (= KCCM 43183, JCM 31150) was isolated from solar salt. The genome of strain CBA1132 assembled with 4 contigs, including three rRNA genes, 44 tRNA genes, and 3,208 open reading frames. Strain CBA1132 had nine putative CRISPRs and the genome contained genes encoding metal resistance determinants: copper-translocating P-type ATPase (CtpA), arsenical pump-driving ATPase (ArsA), arsenate reductase (ArsC), and arsenical resistance operon repressor (ArsR). Strain CBA1132 was related to Halobacterium noricense, with 99.2% 16S rRNA gene sequence similarity. Based on the comparative genomic analysis, strain CBA1132 has distinctly evolved; moreover, essential genes related to nitrogen metabolism were only detected in the genome of strain CBA1132 among the reported genomes in the genus Halobacterium. This genome sequence of Halobacterium noricense CBA1132 may be of use in future molecular biological studies.

Fumarate-Mediated Persistence of Escherichia coli against Antibiotics.

Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) inEscherichia coliled to a higher frequency of persister formation. The persister frequency ofE. coliwas increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-relatedhipA7mutation indicated that surplus fumarate markedly elevated theE. colipersister frequency. AnE. colistrain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ∼1,000-fold more effectively than the wild-type strain in the stationary phase. It appears thatSDHandFRDrepresent a paired system that gives rise to and maintainsE. colipersisters by producing and utilizing fumarate, respectively.

NarJ subfamily system specific chaperone diversity and evolution is directed by respiratory enzyme associations.

BackgroundRedox enzyme maturation proteins (REMPs) describe a diverse family of prokaryotic chaperones involved in the biogenesis of anaerobic complex iron sulfur molybdoenzyme (CISM) respiratory systems. Many REMP family studies have focused on NarJ subfamily members from Escherichia coli: NarJ, NarW, DmsD, TorD and YcdY. The aim of this bioinformatics study was to expand upon the evolution, distribution and genetic association of these 5 REMP members within 130 genome sequenced taxonomically diverse species representing 324 Prokaryotic sequences. NarJ subfamily member diversity was examined at the phylum-species level and at the amino acid/nucleotide level to determine how close their genetic associations were between their respective CISM systems within phyla.ResultsThis study revealed that NarJ members possessed unique motifs that distinguished Gram-negative from Gram-positive/Archaeal species and identified a strict genetic association with its nitrate reductase complex (narGHI) operon compared to all other members. NarW appears to be found specifically in Gammaproteobacteria. DmsD also showed close associations with the dimethylsulfoxide reductase (dmsABC) operon compared to TorD. Phylogenetic analysis revealed that YcdY has recently evolved from DmsD and that YcdY has likely diverged into 2 subfamilies linked to Zn- dependent alkaline phosphatase (ycdX) operons and a newly identified operon containing part of Zn-metallopeptidase FtsH complex component (hflC) and NADH-quinone dehydrogenase (mdaB). TorD demonstrated the greatest diversity in operon association. TorD was identifed within operons from either trimethylamine-N-oxide reductase (torAC) or formate dehydrogenase (fdhGHI), where each type of TorD had a unique motif. Additionally a subgroup of dmsD and torD members were also linked to operons with biotin sulfoxide (bisC) and polysulfide reductase (nrfD) indicating a potential role in the maturation of diverse CISM.ConclusionExamination of diverse prokaryotic NarJ subfamily members demonstrates that the evolution and genetic association of each member is uniquely biased by its CISM operon association.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0412-3) contains supplementary material, which is available to authorized users.

Translational coupling of nasST expression in Azotobacter vinelandii prevents overexpression of the nasT gene.

The nasST operon encodes the transcriptional regulators of assimilatory nitrate reductase operons in phylogenetically diverse bacteria. NasT is a RNA-binding antiterminator and helps RNA polymerase read through the regulatory terminator sequences upstream of the structural genes. NasS senses nitrate and nitrite and regulates the activity of NasT through stoichiometric interaction. In this study, we analyzed the nasST sequence in Azotobacter vinelandii and revealed that the nasS and nasT genes overlap by 19 nucleotides. Our genetic analyses suggested that translational initiation of NasT was coupled with NasS translation, a regulatory mechanism that prevents overproduction of NasT. The significance of tight control of nasT expression was demonstrated in a nasT-overexpression strain, where expression of the assimilatory nitrate reductase operon was deregulated.

The Molecular Basis for Selenate Reduction in Citrobacter freundii

The redox cycling of selenium in aquatic environments is strongly affected by microbial activity. Citrobacter freudii is a facultative anaerobic bacterium found in marine and freshwater settings that is known to reduce selenate. In this study, we conducted selenate reduction experiments with C. freundii to investigate the mechanisms involved the selenate reduction process. The results indicate that C. freundii catalyzes the reduction of selenate to elemental selenium in the absence of oxygen. We detected the functional selenate reductase gene ynfE, which is predicted to encode for a molybdenum-binding Tat-secreted protein. A FNR binding site was located immediately upstream of the ynfE gene suggesting the expression of the selenate reductase occurs under anaerobic conditions and is regulated by oxygen-sensing transcription factors. To gain a more complete understanding of the selenate reduction process, we sequenced the genome of C. freundii. The genome analysis revealed the complete selenate reductase operon ynfEGHdmsD, showing high sequence identity to selenate reductases found in related gammaproteobacteria. Genes for molybdate uptake, molybdopterin guanine dinucleotide biosynthesis, twin arginine translocation, and FNR regulation were also identified in the genome sequence. Based on the experimental results and genome sequence, we developed a model to describe anaerobic selenate reduction in C. freundii. The results of this work provide new insights into the molecular basis of selenate reduction activity in a geochemically relevant facultative anaerobic bacterium.

The Clostridium difficile proline racemase is not essential for early logarithmic growth and infection.

Proline racemase (PrdF), which is important for energy metabolism via the Stickland pathway and is unique to certain clostridia, was investigated as a potential anti-Clostridium difficile target by examining its effects on the growth and virulence of C. difficile. Inactivation of PrdF by insertional mutagenesis did not affect early logarithmic growth but only attenuated growth in the mid- and late logarithmic phases. There was no effect on virulence in vivo, suggesting that PrdF is also not required for C. difficile infection. These findings indicate that PrdF as well as other enzymes encoded by the proline reductase operon are all nonessential and are unsuitable targets for anti-C. difficile drug discovery.

The ResD Response Regulator, through Functional Interaction with NsrR and Fur, Plays Three Distinct Roles in Bacillus subtilis Transcriptional Control

The ResD response regulator activates transcription of diverse genes in Bacillus subtilis in response to oxygen limitation. ResD regulon genes that are the most highly induced during nitrate respiration include the nitrite reductase operon (nasDEF) and the flavohemoglobin gene (hmp), whose products function in nitric oxide (NO) metabolism. Transcription of these genes is also under the negative control of the NO-sensitive NsrR repressor. Recent studies showed that the NsrR regulon contains genes with no apparent relevance to NO metabolism and that the ResD response regulator and NsrR coordinately regulate transcription. To determine whether these genes are direct targets of NsrR and ResD, we used chromatin affinity precipitation coupled with tiling chip (ChAP-chip) and ChAP followed by quantitative PCR (ChAP-qPCR) analyses. The study showed that ResD and NsrR directly control transcription of the ykuNOP operon in the Fur regulon. ResD functions as an activator at the nasD and hmp promoters, whereas it functions at the ykuN promoter as an antirepressor of Fur and a corepressor for NsrR. This mechanism likely participates in fine-tuning of transcript levels in response to different sources of stress, such as oxygen limitation, iron limitation, and exposure to NO.

Corynebacterium glutamicum ArnR Controls Expression of Nitrate Reductase Operon narKGHJI and Nitric Oxide (NO)-Detoxifying Enzyme Gene hmp in an NO-Responsive Manner

Corynebacterium glutamicum ArnR is a novel transcriptional regulator that represses expression of the nitrate reductase operon narKGHJI and the nitric oxide (NO)-detoxifying flavohemoglobin gene hmp under aerobic conditions. In a previous study, we showed that ArnR-mediated repression is relieved during anaerobic nitrate respiration, but we could not pinpoint the specific signal that ArnR senses. In this study, we show that in the absence of nitrate, ArnR-mediated repression is maintained under anaerobic conditions. The derepression in response to nitrate is eliminated by disruption of narG, suggesting that ArnR senses nitrate derivatives generated during nitrate respiration. Specifically, the hmp gene is upregulated in the presence of nitrite or nitric oxide (NO) in an ArnR-dependent manner, although the response of narK appears to be greatly affected by ArnR-independent regulation. In vitro binding of ArnR to the narK and hmp promoter regions is more strongly inhibited by NO than by nitrite. We previously showed that the UV-visible spectrum of ArnR is typical of a Fe-S cluster-containing protein. Site-directed mutagenesis of each of three cysteine residues, which are possibly involved in coordination of the cofactor in the ArnR protein, results in loss of the binding of this protein to its target promoters in vitro and eliminates the repression of the target genes in vivo under aerobic conditions. These observations suggest that the cofactor coordinated by these three cysteine residues in the ArnR protein plays a critical role in the NO-responsive expression of the narKGHJI operon and the hmp gene.

Identification of a possible respiratory arsenate reductase in Denitrovibrio acetiphilus, a member of the phylum Deferribacteres

Denitrovibrio acetiphilus N2460(T) is one of the few members of the phylum Deferribacteres with a sequenced genome. N2460(T) was capable of growing with dimethyl sulfoxide, selenate, orarsenate provided as a terminal electron acceptor, and we identified 15 genes that could possibly encode respiratory reductases for these compounds. The protein encoded by one of these genes, YP_003504839, clustered with respiratory arsenate reductases on a phylogenetic tree. Transcription of the gene for YP_003504839, Dacet_2121, was highly induced when arsenate was provided as a terminal electron acceptor. Dacet_2121 exists in a possible operon that is distinct from the previously characterized respiratory arsenate reductase operon in Shewanella sp. ANA-3.