Distinct Interaction Mechanism of RNAP and ResD and Distal Subsites for Transcription Activation of Nitrite Reductase in Bacillus subtilis ψ

The ResD-ResE signal transduction system plays a pivotal role in anaerobic nitrate respiration in Bacillus subtilis . The nasD operon encoding nitrite reductase is essential for nitrate respiration and is tightly controlled by the ResD response regulator. To understand the mechanism of ResD-dependent transcription activation of the nasD operon, we explored ResD-RNA polymerase (RNAP), ResD-DNA, and RNAP-DNA interactions required for nasD transcription. Full transcriptional activation requires the upstream promoter region where five molecules of ResD bind. The distal ResD-binding subsite at −87 to −84 partially overlaps a sequence similar to the consensus distal subsite of the upstream (UP) element with which the Escherichia coli C-terminal domain of the α subunit (αCTD) of RNAP interacts to stimulate transcription. We propose that interaction between αCTD and ResD at the promoter-distal site is essential for stimulating nasD transcription. Although nasD has an extended −10 promoter, it lacks a reasonable −35 element. Genetic analysis and structural simulations predicted that the absence of the −35 element might be compensated by interactions between σ A and αCTD, and between αCTD and ResD at the promoter-proximal ResD-binding subsite. Thus, our work suggested that ResD likely participates in nasD transcription activation by binding to two αCTD subunits at the proximal and distal promoter sites, representing a unique configuration for transcription activation. IMPORTANCE A significant number of ResD-controlled genes have been identified and transcription regulatory pathways in which ResD participates have emerged. Nevertheless, the mechanism of how ResD activates transcription of different genes in a nucleotide sequence-specific manner has been less explored. This study suggested that among the five ResD-binding subsites in the promoter of the nasD operon, the promoter-proximal and -distal ResD-binding subsites play important roles in nasD activation by adapting different modes of protein-protein and protein-DNA interactions. The finding of a new-type of protein-promoter architecture provides insight into the understanding of transcription activation mechanisms controlled by transcription factors including the ubiquitous response regulators of two-component regulatory systems particularly in Gram-positive bacteria.

The MarR family regulator OsbR controls oxidative stress response, anaerobic nitrate respiration, and biofilm formation in Chromobacterium violaceum

Background Chromobacterium violaceum is an environmental opportunistic pathogen that causes rare but deadly infections in humans. The transcriptional regulators that C. violaceum uses to sense and respond to environmental cues remain largely unknown. Results Here, we described a novel transcriptional regulator in C. violaceum belonging to the MarR family that we named OsbR (oxidative stress response and biofilm formation regulator). Transcriptome profiling by DNA microarray using strains with deletion or overexpression of osbR showed that OsbR exerts a global regulatory role in C. violaceum, regulating genes involved in oxidative stress response, nitrate reduction, biofilm formation, and several metabolic pathways. EMSA assays showed that OsbR binds to the promoter regions of several OsbR-regulated genes, and the in vitro DNA binding activity was inhibited by oxidants. We demonstrated that the overexpression of osbR caused activation of ohrA even in the presence of the repressor OhrR, which resulted in improved growth under organic hydroperoxide treatment, as seem by growth curve assays. We showed that the proper regulation of the nar genes by OsbR ensures optimal growth of C. violaceum under anaerobic conditions by tuning the reduction of nitrate to nitrite. Finally, the osbR overexpressing strain showed a reduction in biofilm formation, and this phenotype correlated with the OsbR-mediated repression of two gene clusters encoding putative adhesins. Conclusions Together, our data indicated that OsbR is a MarR-type regulator that controls the expression of a large number of genes in C. violaceum, thereby contributing to oxidative stress defense (ohrA/ohrR), anaerobic respiration (narK1K2 and narGHJI), and biofilm formation (putative RTX adhesins).

Nitrite as a causal factor for nitrate-dependent anaerobic corrosion of metallic iron induced by Prolixibacter strains

Microbially influenced corrosion (MIC) may contribute significantly to overall corrosion risks, especially in the gas and petroleum industries. In this study, we isolated four Prolixibacter strains, which belong to the phylum Bacteroidetes, and examined their nitrate-respiration- and Fe0-corroding activities, together with two previously isolated Prolixibacter strains. Four of the six Prolixibacter strains reduced nitrate under anaerobic conditions, while the other two strains did not. The anaerobic growth of the four nitrate-reducing strains was enhanced by nitrate, which was not observed in the two nitrate-non-reducing strains. When the nitrate-reducing strains were grown anaerobically in the presence of Fe0 or carbon steel, the corrosion of the materials was enhanced by more than 20-fold compared to that in aseptic controls. This enhancement was not observed in cultures of the nitrate-non-reducing strains. The oxidation of Fe0 in the anaerobic cultures of nitrate-reducing strains occurred concomitantly with the reduction of nitrite. Since nitrite chemically oxidized Fe0 under anaerobic and aseptic conditions, the corrosion of Fe0- and carbon-steel by the nitrate-reducing Prolixibacter strains was deduced to be mainly enhanced via the biological reduction of nitrate to nitrite, followed by the chemical oxidation of Fe0 to Fe2+ and Fe3+ coupled to the reduction of nitrite.

Transcriptional and Post-transcriptional Control of the Nitrate Respiration in Bacteria

In oxygen (O2) limiting environments, numerous aerobic bacteria have the ability to shift from aerobic to anaerobic respiration to release energy. This process requires alternative electron acceptor to replace O2 such as nitrate (NO3–), which has the next best reduction potential after O2. Depending on the organism, nitrate respiration involves different enzymes to convert NO3– to ammonium (NH4+) or dinitrogen (N2). The expression of these enzymes is tightly controlled by transcription factors (TFs). More recently, bacterial small regulatory RNAs (sRNAs), which are important regulators of the rapid adaptation of microorganisms to extremely diverse environments, have also been shown to control the expression of genes encoding enzymes or TFs related to nitrate respiration. In turn, these TFs control the synthesis of multiple sRNAs. These results suggest that sRNAs play a central role in the control of these metabolic pathways. Here we review the complex interplay between the transcriptional and the post-transcriptional regulators to efficiently control the respiration on nitrate.

The MarR Family Regulator OsbR Controls Oxidative Stress Response, anaerobic Nitrate Respiration, and Biofilm Formation in Chromobacterium Violaceum

Background: Chromobacterium violaceum is an environmental opportunistic pathogen that causes rare but deadly infections in humans. The transcriptional regulators that C. violaceum uses to sense and respond to environmental cues remain largely unknown. Results: Here, we described a novel transcriptional regulator in C. violaceum belonging to the MarR family that we named OsbR (oxidative stress response and biofilm formation regulator). Transcriptome profiling by DNA microarray using strains with deletion or overexpression of osbR showed that OsbR exerts a global regulatory role in C. violaceum, regulating genes involved in oxidative stress response, nitrate reduction, biofilm formation, and several metabolic pathways. EMSA assays showed that OsbR binds to the promoter regions of several OsbR-regulated genes, and the in vitro DNA binding activity was inhibited by oxidants. We demonstrated that the overexpression of osbR caused activation of ohrA even in the presence of the repressor OhrR, which resulted in improved growth under organic hydroperoxide treatment. We showed that the proper regulation of the nar genes by OsbR ensures optimal growth of C. violaceum under anaerobic conditions by tuning the reduction of nitrate to nitrite. Finally, the osbR overexpressing strain showed a reduction in biofilm formation, and this phenotype correlated with the OsbR-mediated repression of two gene clusters encoding putative adhesins. Conclusions: Together, our data indicated that OsbR is a MarR-type regulator that controls the expression of a large number of genes in C. violaceum, thereby contributing to oxidative stress defense, anaerobic respiration, and biofilm formation.

Metaproteogenomic Profiling of Chemosynthetic Microbial Biofilms Reveals Metabolic Flexibility During Colonization of a Shallow-Water Gas Vent

Tor Caldara is a shallow-water gas vent located in the Mediterranean Sea, with active venting of CO2 and H2S. At Tor Caldara, filamentous microbial biofilms, mainly composed of Epsilon- and Gammaproteobacteria, grow on substrates exposed to the gas venting. In this study, we took a metaproteogenomic approach to identify the metabolic potential and in situ expression of central metabolic pathways at two stages of biofilm maturation. Our findings indicate that inorganic reduced sulfur species are the main electron donors and CO2 the main carbon source for the filamentous biofilms, which conserve energy by oxygen and nitrate respiration, fix dinitrogen gas and detoxify heavy metals. Three metagenome-assembled genomes (MAGs), representative of key members in the biofilm community, were also recovered. Metaproteomic data show that metabolically active chemoautotrophic sulfide-oxidizing members of the Epsilonproteobacteria dominated the young microbial biofilms, while Gammaproteobacteria become prevalent in the established community. The co-expression of different pathways for sulfide oxidation by these two classes of bacteria suggests exposure to different sulfide concentrations within the biofilms, as well as fine-tuned adaptations of the enzymatic complexes. Taken together, our findings demonstrate a shift in the taxonomic composition and associated metabolic activity of these biofilms in the course of the colonization process.

Nitrate-responsive suppression of DMSO respiration in a facultative anaerobic haloarchaeon Haloferax volcanii

Haloferax volcanii is a facultative anaerobic haloarchaeon that can grow using nitrate or dimethyl sulfoxide (DMSO) as respiratory substrates in an anaerobic condition. Comparative transcriptome analysis of denitrifying and aerobic cells of H. volcanii indicated extensive changes in the gene expression involving activation of denitrification, suppression of DMSO respiration, and conversion of the heme biosynthetic pathway under denitrifying condition. Anaerobic growth of H. volcanii by DMSO respiration was inhibited at nitrate concentrations lower than 1 mM, whereas the nitrate-responsive growth inhibition was not observed in the ΔnarO mutant. A reporter assay experiment demonstrated that transcription of the dms operon was suppressed by nitrate. In contrast, anaerobic growth of the ΔdmsR mutant by denitrification was little affected by addition of DMSO. NarO has been identified as an activator of the denitrification-related genes in response to anaerobic conditions, and here we found that NarO is also involved in nitrate-responsive suppression of the dms operon. Nitrate-responsive suppression of DMSO respiration is known in several bacteria, such as Escherichia coli and photosynthetic Rhodobacter sp. This is the first report to show that a regulatory mechanism that suppresses DMSO respiration in response to nitrate exists not only in bacteria but also in the haloarchaea. IMPORTANCE Haloferax volcanii can grow anaerobically by denitrification (nitrate respiration) or DMSO respiration. In the facultative anaerobic bacteria that can grow by both nitrate respiration and DMSO respiration, nitrate respiration is preferentially induced when both nitrate and DMSO are available as respiratory substrates. The results of transcriptome analysis, growth phenotyping, and reporter assay indicated that DMSO respiration is suppressed in response to nitrate in H. volcanii. The haloarchaea-specific regulator NarO, which activates denitrification under anaerobic conditions, is suggested to be involved in the nitrate-responsive suppression of DMSO respiration.

A-type carrier proteins are involved in [4Fe-4S] cluster insertion into the radical SAM protein MoaA for the synthesis of active molybdoenzymes

Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them were characterized in detail in Escherichia coli, namely IscA, SufA and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA involved in [4Fe-4S] cluster insertion into the S-adenosyl-methionine dependent radical SAM protein MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth of nitrate respiration, based on low gene expression levels. IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics and gene regulation. Still remaining critical gaps in our knowledge are how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SusA and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions.