Multiple Antibiotic Resistance Regulator Research Articles

Crystal structure of a Clostridioides difficile multiple antibiotic resistance regulator (MarR) CD0473 suggests a potential redox-regulated function

Clostridioides difficile may constitute a small part of normal gut microbiota in humans without causing any symptoms, but an uncontrolled growth common to hospitalized patients can cause Clostridioides difficile infection (CDI) leading to severe colonic symptoms. As the bacteria are attaining resistance to various antibiotics worldwide, CDI is becoming a serious public health problem. Although a family of transcription factors called MarR (Multiple antibiotic resistance Regulator) plays a key role in the bacterial response to various environmental stresses including antibiotics, most of the 14 MarRs predicted to exist in the C. difficile genome lack structural or functional studies. In this respect, X-ray crystal structure of a C. difficile MarR CD0473 with a yet unknown function has been determined using a Hg-soaked crystal. In the structure, two closely located flexible conformations of Hg-bound cysteines suggested a possibility of intra-subunit disulfide bridge formation. By searching the neighboring intergenic regions of CD0473, two pseudo-palindromic DNA sites were found and shown to bind the protein. MarR CD0473 binding stronger to the DNA in an oxidizing condition supported further that it may function as a redox regulated switch likely via its oxidized disulfide formation.

Regulation of bacterial virulence genes by PecS family transcription factors.

Bacterial plant pathogens adjust their gene expression programs in response to environmental signals and host-derived compounds. This ensures that virulence genes or genes encoding proteins, which promote bacterial fitness in a host environment, are expressed only when needed. Such regulation is in the purview of transcription factors, many of which belong to the ubiquitous multiple antibiotic resistance regulator (MarR) protein family. PecS proteins constitute a subset of this large protein family. PecS has likely been distributed by horizontal gene transfer, along with the divergently encoded efflux pump PecM, suggesting its integration into existing gene regulatory networks. Here, we discuss the roles of PecS in the regulation of genes associated with virulence and fitness of bacterial plant pathogens. A comparison of phenotypes and differential gene expression associated with the disruption of pecS shows that functional consequences of PecS integration into existing transcriptional networks are highly variable, resulting in distinct PecS regulons. Although PecS universally binds to the pecS-pecM intergenic region to repress the expression of both genes, binding modes differ. A particularly relaxed sequence preference appears to apply for Dickeya dadantii PecS, perhaps to optimize its integration as a global regulator and regulate genes ancestral to the acquisition of pecS-pecM. Even inducing ligands for PecS are not universally conserved. It appears that PecS function has been optimized to match the unique regulatory needs of individual bacterial species and that its roles must be appreciated in the context of the regulatory networks into which it was recruited.

MarR family proteins sense sulfane sulfur in bacteria.

Members of the multiple antibiotic resistance regulator (MarR) protein family are ubiquitous in bacteria and play critical roles in regulating cellular metabolism and antibiotic resistance. MarR family proteins function as repressors, and their interactions with modulators induce the expression of controlled genes. The previously characterized modulators are insufficient to explain the activities of certain MarR family proteins. However, recently, several MarR family proteins have been reported to sense sulfane sulfur, including zero-valent sulfur, persulfide (R-SSH), and polysulfide (R-SnH, n ≥ 2). Sulfane sulfur is a common cellular component in bacteria whose levels vary during bacterial growth. The changing levels of sulfane sulfur affect the expression of many MarR-controlled genes. Sulfane sulfur reacts with the cysteine thiols of MarR family proteins, causing the formation of protein thiol persulfide, disulfide bonds, and other modifications. Several MarR family proteins that respond to reactive oxygen species (ROS) also sense sulfane sulfur, as both sulfane sulfur and ROS induce the formation of disulfide bonds. This review focused on MarR family proteins that sense sulfane sulfur. However, the sensing mechanisms reviewed here may also apply to other proteins that detect sulfane sulfur, which is emerging as a modulator of gene regulation.

A novel Escherichia coli cell–based bioreporter for quantification of salicylic acid in cosmetics

Transcription factor–based bioreporters have been extensively studied for monitoring and detecting environmental toxicants. In Escherichia coli, the multiple antibiotic resistance regulator (MarR) induces transcription upon binding to salicylic acid (SA). We generated SA-specific E. coli cell–based bioreporters utilizing the operator region of the mar operon and MarR as components of the reporter and sensing domains, respectively. Although bioreporters based on endogenous MarR and wild-type E. coli cells responded to SA, their sensitivity and selectivity were insufficient for practical sample monitoring. To improve these parameters, we genetically engineered host strains for optimal MarR expression, which enhanced the sensitivity of the biosensor to micromolar quantities of SA with increased selectivity. Under the optimized experimental conditions, the biosensor could quantify SA in environmental samples. For validation, the SA concentration in artificially contaminated SA-containing cosmetic samples was determined using the developed biosensor. Reliability assessment by comparing the concentrations determined using LC–MS/MS revealed > 90% accuracy of the bioreporters. Although bioreporters are not considered standard tools for environmental monitoring, bacterial cell–based bioreporters may serve as alternative tools owing to their affordability and simplicity. The SA biosensor developed in this study can potentially be a valuable tool for monitoring SA in environmental systems.Key points• SA-responsive bioreporter is generated by employing mar operon system in E. coli• SA specificity and selectivity were enhanced by genetic/biochemical engineering• The novel bioreporter would be valuable for SA monitoring in environmental systems

Thiol-Based Modification of MarR Protein VnrR Regulates Resistance Toward Nitrofuran in Vibrio cholerae By Promoting the Expression of a Novel Nitroreductase VnrA and of NO-Detoxifying Enzyme HmpA.

Aims: Epidemiological investigations have indicated low resistance toward nitrofuran in clinical isolates, suggesting its potential application in the treatment of multidrug-resistant bacteria. Therefore, it is valuable to explore the mechanism of bacterial resistance to nitrofuran. Results: Through phenotypic screening of ten multiple antibiotic resistance regulator (MarR) proteins in Vibrio cholerae, we discovered that the regulator VnrR (VCA1058) plays a crucial role in defending against nitrofuran, specifically furazolidone (FZ). Our findings demonstrate that VnrR responds to FZ metabolites, such as hydroxylamine, methylglyoxal, hydrogen peroxide (H2O2), β-hydroxyethylhydrazine. Notably, VnrR exhibits reversible responses to the addition of H2O2 through three cysteine residues (Cys180, Cys223, Cys247), leading to the derepression of its upstream gene, vnrA (vca1057). Gene vnrA encodes a novel nitroreductase, which directly contributes to the degradation of FZ. Our study reveals that V. cholerae metabolizes FZ via the vnrR-vnrA system and achieves resistance to FZ with the assistance of the classical reactive oxygen/nitrogen species scavenging pathway. Innovation and Conclusion: This study represents a significant advancement in understanding the antibiotic resistance mechanisms of V. cholerae and other pathogens. Our findings demonstrate that the MarR family regulator, VnrR, responds to the FZ metabolite H2O2, facilitating the degradation and detoxification of this antibiotic in a thiol-dependent manner. These insights not only enrich our knowledge of antibiotic resistance but also provide new perspectives for the control and prevention of multidrug-resistant bacteria.

The virulence regulator AbsR in avian pathogenic Escherichia coli has pleiotropic effects on bacterial physiology

Avian pathogenic Escherichia coli (APEC) belonging to extraintestinal pathogenic E. coli (ExPEC) can cause severe infections in extraintestinal tissues in birds and humans, such as the lungs and blood. MprA (microcin production regulation, locus A, herein renamed AbsR, a blood survival regulator), a member of the MarR (multiple antibiotic resistance regulator) transcriptional regulator family, governs the expression of capsule biosynthetic genes in human ExPEC and represents a promising druggable target for antimicrobials. However, a deep understanding of the AbsR regulatory mechanism as well as its regulon is lacking. In this study, we present a systems-level analysis of the APEC AbsR regulon using ChIP-Seq (chromatin immunoprecipitation sequencing) and RNA-Seq (RNA sequencing) methods. We found that AbsR directly regulates 99 genes and indirectly regulates 667 genes. Furthermore, we showed that: 1) AbsR contributes to antiphagocytotic effects by macrophages and virulence in a mouse model for systemic infection by directly activating the capsular gene cluster; 2) AbsR positively impacts biofilm formation via direct regulation of the T2SS (type II secretion system) but plays a marginal role in virulence; and 3) AbsR directly upregulates the acid tolerance signaling system EvgAS to withstand acid stress but is dispensable in ExPEC virulence. Finally, our data indicate that the role of AbsR in virulence gene regulation is relatively conserved in ExPEC strains. Altogether, this study provides a comprehensive analysis of the AbsR regulon and regulatory mechanism, and our data suggest that AbsR likely influences virulence primarily through the control of capsule production. Interestingly, we found that AbsR severely represses the expression of the type I-F CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) systems, which could have implications in CRISPR biology and application.

The DmeRF System Is Involved in Maintaining Cobalt Homeostasis in Vibrio parahaemolyticus

Although cobalt (Co) is indispensable for life, it is toxic to cells when accumulated in excess. The DmeRF system is a well-characterized metal-response system that contributes to Co and nickel resistance in certain bacterial species. The Vibrio parahaemolyticus RIMD 2210633 genome also harbors a dmeRF operon that encodes a multiple antibiotic resistance regulator family transcriptional regulator and a cation diffusion facilitator family protein. Quantitative real-time PCR, growth curves analysis, inductively coupled plasma-mass spectrometry, β-galactosidase activity assays, electrophoretic mobility shift assays, and a mouse infection experiment were performed to characterize the function of the DmeRF system in V. parahaemolyticus. Zinc, copper, and Co significantly increase dmeF expression, with Co inducing the greatest increase. DmeF promotes V. parahaemolyticus growth under high-Co conditions. Additionally, increased accumulation of cellular Co in the ΔdmeF mutant indicates that DmeF is potentially involved in Co efflux. Moreover, DmeR represses the dmeRF operon by binding directly to its promoter in the absence of Co. Finally, the DmeRF system was not required for V. parahaemolyticus virulence in mice. Collectively, our data indicate that the DmeRF system is involved in maintaining Co homeostasis in V. parahaemolyticus and DmeR functioning as a repressor of the operon.

Understanding the regulatory effects of a biofilm regulator (BifR) and the implication of its interactions in Burkholderia thailandensis

Biofilm formation plays a key role in the pathogenesis of chronic infections. In pathogenic Burkholderia species, biofilm formation is a severe complication, and it makes bacteria resistant to host defenses and antibiotics. BifR (Biofilm regulator) is a recently described redox‐sensitive member of the multiple antibiotic resistance regulator (MarR) protein family that has been found to repress biofilm formation in Burkholderia thailandensis. BifR appears to regulate the expression of other proteins critical to biofilm formation, but its overall functions are poorly understood. BifR is encoded in the same operon with an efflux pump (emrB) and divergently oriented to a gene (ecsC) that codes for a protease (LasA) that has been shown to be implicated in virulence by its contribution to elastin degradation in Pseudomonas aeruginosa. Previously, BifR was shown to repress the expression of emrB‐bifRand ecsCand to repress the expression of these genes further under oxidizing conditions. However, the substrate of the efflux pump (EmrB) is unknown. To determine the substrate of EmrB, we carried out antibiotic sensitivity tests on the Wild Type (WT), emrB‐bifRmutant and bifRmutant strains. Following a 24‐hour incubation at 37 oC, our results showed that the bifR mutant was resistant to novobiocin at concentrations of 50 ug/ml, 75 ug/ml and slightly resistant at 100 ug/ml. The bifRstrain was also more resistant to ampicillin at concentrations of 50 ug/ml, 75 ug/ml and 100 ug/ml. Similar results were obtained for the related β‐lactam, penicillin G. This indicates that novobiocin and β‐lactam antibiotics are substrates for EmrB. We also conducted onion tissue maceration and tested for the survival of Caenorhabditis elegans after infection with the three strains. Our results showed that after 72 hours of incubation of the onions at 30 oC, the bifRmutant strain was more virulent compared to the WT.Meanwhile, with a 70 % survival of the C. elegansafter 12 hours of incubation at room temperature, the emrB‐bifRmutant strain was more virulent. This study reveals that BifR plays a role in virulence and that the role may be host specific.

Sulfane Sulfur Is a Strong Inducer of the Multiple Antibiotic Resistance Regulator MarR in Escherichia coli.

Sulfane sulfur, including persulfide and polysulfide, is produced from the metabolism of sulfur-containing organic compounds or from sulfide oxidation. It is a normal cellular component, participating in signaling. In bacteria, it modifies gene regulators to activate the expression of genes involved in sulfur metabolism. However, to determine whether sulfane sulfur is a common signal in bacteria, additional evidence is required. The ubiquitous multiple antibiotic resistance regulator (MarR) family of regulators controls the expression of numerous genes, but the intrinsic inducers are often elusive. Recently, two MarR family members, Pseudomonas aeruginosa MexR and Staphylococcus aureus MgrA, have been reported to sense sulfane sulfur. Here, we report that Escherichia coli MarR, the prototypical member of the family, also senses sulfane sulfur to form one or two disulfide or trisulfide bonds between two dimers. Although the tetramer with two disulfide bonds does not bind to its target DNA, our results suggest that the tetramer with one disulfide bond does bind to its target DNA, with reduced affinity. An MarR-repressed mKate reporter is strongly induced by polysulfide in E. coli. Further investigation is needed to determine whether sulfane sulfur is a common signal of the family members, but three members sense cellular sulfane sulfur to turn on antibiotic resistance genes. The findings offer additional support for a general signaling role of sulfane sulfur in bacteria.

The Adc regulon mediates zinc homeostasis in Streptococcus mutans.

Zinc (Zn2+ ) is an essential divalent trace metal for living cells. Intracellular zinc homeostasis is critical to the survival and virulence of bacteria. Thus, the frequent fluctuations of salivary zinc, caused by the low physiological level and the frequent exogenous zinc introduction, present a serious challenge for bacteria colonizing the oral cavity. However, the regulation strategies to keep intracellular Zn2+ homeostasis in Streptococcus mutans, an important causative pathogen of dental caries, are unknown. Because zinc uptake is primarily mediated by an ATP-binding ABC transporter AdcABC in Streptococcus strains, we examined the function of AdcABC and transcription factor AdcR in S. mutans in this study. The results demonstrated that deletion of either adcA or adcCB gene impaired the growth but enhanced the extracellular polymeric matrix production in S. mutans, both of which could be relieved after excessive Zn2+ supplementation. Using RNA sequencing analysis, quantitative reverse transcription polymerase chain reaction examination, LacZ-reporter studies, and electrophoretic mobility shift assay, we showed that a MarR (multiple antibiotic resistance regulator) family transcription factor, AdcR, negatively regulates the expression of the genes adcR, adcC, adcB, and adcA by acting on the adcRCB and adcA promoters in response to Zn2+ concentration in their environmental niches. The deletion of adcR increases the sensitivity of S. mutans to excessive Zn2+ supply. Taken together, our findings suggest that Adc regulon, which consists of a Zn2+ uptake transporter AdcCBA and a Zn2+ -responsive repressor AdcR, plays a prominent role in the maintenance of intracellular zinc homeostasis of S. mutans.

PaeR is a Copper‐Sensing MarR Homolog from Clostridium difficile that Binds Cooperatively to its Promoter DNA Region

Multiple antibiotic resistance regulator (MarR) proteins are widely found in prokaryotes and archaea. Most characterized MarR homologs function as transcriptional repressors of genes involved in metabolic and stress response pathways. MarR proteins regulate transcription through sequence-specific DNA binding interactions and many respond allosterically to small molecule ligands or metal ions. In this report, we describe the DNA and ligand binding properties of PaeR (putative antibiotic efflux regulator), a previously uncharacterized MarR homolog from the pathogen, Clostridium difficile. Biophysical characterization of PaeR indicates that it exists predominantly as a homodimer with a thermal unfolding temperature of 59.3°C. Electrophoretic mobility shift assays (EMSAs) reveal PaeR to be a sequence-specific DNA binding protein that associates with DNA as a homodimer with very high binding affinity (Kd = 3.2 ± 0.6 nM) at a pseudo-palindromic site in its promoter region. Interestingly, our data indicates that PaeR binds to at least one additional site in its promoter/operator region with positive binding cooperativity. Initial screens to identify potential effectors of PaeR binding to DNA indicate that this protein is responsive to low concentrations of copper ions. Homology modeling of the PaeR homodimer suggests a possible mechanism for copper-induced effects on DNA binding. These data, in conjunction with the genetic context of the paeR gene, suggest a possible role of PaeR in regulating antibiotic resistance in C. difficile.

EpsR Negatively Regulates Streptococcus mutans Exopolysaccharide Synthesis

Streptococcus mutans is considered the primary etiological agent of human dental caries. Glucosyltransferases (Gtfs) from S. mutans play important roles in the formation of biofilm matrix and the development of cariogenic oral biofilm. Therefore, Gtfs are considered an important target to prevent the development of dental caries. However, the role of transcription factors in regulating gtf expression is not yet clear. Here, we identify a MarR (multiple antibiotic resistance regulator) family transcription factor named EpsR (exopolysaccharide synthesis regulator), which negatively regulates gtfB expression and exopolysaccharide (EPS) production in S. mutans. The epsR in-frame deletion strain grew slowly, aggregated more easily in the presence of dextran, and displayed different colony morphology and biofilm structure. Notably, epsR deletion resulted in altered 3-dimensional biofilm architecture, increased water-insoluble EPS production, and upregulated GtfB protein content and activity. In addition, global gene expression profiling revealed differences in the expression levels of 69 genes in which gtfB was markedly upregulated. The conserved DNA motif for EpsR binding was determined by electrophoretic mobility shift assay and DNase I footprinting assays. Moreover, analysis of β-galactosidase activity suggested that EpsR acted as a repressor and inhibited gtfB expression. Taken together, our findings indicate that EpsR is an important transcription factor that regulates gtfB expression and EPS production in S. mutans. These results add new aspects to the complexity of regulating the expression of genes involved in the cariogenicity of S. mutans, which might lead to novel strategies to prevent the formation of cariogenic biofilm that may favor diseases.

Discovery of New Antibacterial Accramycins from a Genetic Variant of the Soil Bacterium, Streptomyces sp. MA37.

Continued mining of natural products from the strain Streptomyces sp. MA37 in our laboratory led to the discovery of a minor specialized metabolite (SM) called accramycin A. Owing to its low yield (0.2 mg/L) in the wild type strain, we investigated the roles of regulatory genes in the corresponding biosynthetic gene cluster (acc BGC) through gene inactivation with the aim of improving the titer of this compound. One of the resulting mutants (∆accJ) dramatically upregulated the production of accramycin A 1 by 330-fold (66 mg/L). Furthermore, ten new metabolites, accramycins B–K 2–11, were discovered, together with two known compounds, naphthacemycin B1 12 and fasamycin C 13 from the mutant extract. This suggested that accJ, annotated as multiple antibiotic resistance regulator (MarR), is a negative regulator gene in the accramycin biosynthesis. Compounds 1–13 inhibited the Gram-positive pathogens (Staphylococcus aureus, Enterococcus faecalis) and clinical isolates Enterococcus faecium (K59-68 and K60-39) and Staphylococcus haemolyticus with minimal inhibitory concentration (MIC) values in the range of 1.5–12.5 µg/mL. Remarkably, compounds 1–13 displayed superior activity against K60-39 (MIC = 3.1–6.3 µg/mL) compared to ampicillin (MIC = 25 µg/mL), and offered promising potential for the development of accramycin-based antibiotics that target multidrug-resistant Enterococcus clinical isolates. Our results highlight the importance of identifying the roles of regulatory genes in natural product discovery.

PaeR, a Multiple Antibiotic Resistance Regulator Homolog from Clostridium difficile is a Sequence‐Specific DNA Binding Protein that Responds to Small Molecule Effectors

Proteins belonging to the multiple antibiotic resistance regulator (MarR) family are ubiquitous amongst species in the bacterial and archaeal domains. These proteins function as transcriptional regulators of a wide variety of cellular processes including catabolic pathways, oxidative stress response mechanisms, efflux or degradation of toxins, and virulence factor expression. DNA binding by MarR proteins is mediated by a characteristic winged helix‐turn‐helix (wHTH) motif which makes sequence‐specific contacts with both the DNA major and minor grooves. For a number of MarR homologs, DNA binding is abrogated by ligand binding. The natural ligands of MarR proteins are structurally diverse but generally fall into three main categories: small aromatic compounds, metals and reactive oxygen species. In this study, we report the first biochemical investigation of a MarR homolog from the pathogenic bacterium Clostridium difficile which we have named PaeR (putative antibiotic efflux regulator). Biophysical investigations of the structural properties of PaeR indicate that this protein exists predominantly as a homodimer in solution and unfolds in a manner consistent with a simple, two‐state transition. PaeR binds at two sites in its own promoter/operator region, suggesting autoregulation. Analysis of binding at a single site indicates a high‐affinity interaction with a microscopic dissociation constant of 3.3 ±0.4 nM. Competitive electrophoretic mobility shift assays (EMASs) indicate that PaeR interacts with DNA in a highly sequence‐specific manner. Ligand binding studies indicate that PaeR binding to its cognate DNA binding sequence is attenuated by small aromatic compounds. These findings, in conjunction with the genetic context of paeR in the C. difficile genome, suggests a possible role of this MarR homolog in regulating antibiotic resistance in this pathogen.Support or Funding InformationBill and Linda Frost Fund

CtcS, a MarR family regulator, regulates chlortetracycline biosynthesis

BackgroundChlortetracycline (CTC) is one of the commercially important tetracyclines (TCs) family product and is mainly produced by Streptomyces. CTC is still in a great demand due to its broad-spectrum activity against pathogens. Engineering transcriptional control allows the cell to allocate its valuable resources towards protein production and provides an important method for the build-up of desired metabolites. Despite extensive efforts concerning transcriptional regulation for increasing the productivities of TCs, the regulatory mechanisms of the CTC biosynthesis remain poorly understood.ResultsIn this study, the possible regulatory function of CtcS, a potential member of MarR (multiple antibiotic resistance regulator) family of transcriptional regulators in S. aureofaciens F3, was demonstrated. Knockdown of ctcS altered the transcription of several biosynthesis-related genes and reduced the production of tetracycline (TC) and CTC, without obvious effect on morphological differentiation and cell growth. Especially, CtcS directly repressed the transcription of the adjacent divergent gene ctcR (which encodes a putative TC resistance efflux protein). A CtcS-binding site was identified within the promoter region of ctcR by DNase I footprinting and an inverted repeat (5′-CTTGTC-3′) composed of two 6-nt half sites in the protected region was found. Moreover, both CTC and TC could attenuate the binding activity of CtcS with target DNA.ConclusionctcS regulated the production of TC and CTC in S. aureofaciens F3 and the overexpression of it could be used as a simple approach for the construction of engineering strain with higher productivity. Meanwhile, CtcS was characterized as a TC- and CTC-responsive MarR family regulator. This study provides a previously unrecognized function of CtcS and will benefit the research on the regulatory machinery of the MarR family regulators.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance.

MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)-uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882-ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR-uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Characterization of a Bi-directional Promoter OtrRp Involved in Oxytetracycline Biosynthesis

Previous studies identified a MarR (multiple antibiotic resistance regulator) family transcription factor OtrR in the oxytetracycline biosynthetic gene cluster, which regulated the expression of an efflux pump OtrB. The genes otrB and otrR were divergent arranged and the inter-ORF (open reading frame) region between the two genes contained the promoter otrBp. In this study, we demonstrated that the reverse complementary sequence of otrBp contained the promoter of otrR, and its activity was also repressed by OtrR by sharing the same operator otrO within otrBp, and allosteric regulated by oxytetracycline. Our findings offered a solid base for the synthetic biological application of the bi-direction promoter in controlling two elements at the same time using only one signal molecule.

OsmC in Corynebacterium glutamicum was a thiol-dependent organic hydroperoxide reductase

Bacterial antioxidants play a vital role in the detoxification of exogenous peroxides. Several antioxidant defenses including low-molecular-weight thiols (LMWTs) and protective enzymes were developed to help the bacterium withstand the adverse stress. Although osmotically induced bacterial protein C (OsmC), classified as the organic hydroperoxide reductase (Ohr)/OsmC superfamily, has been demonstrated in some mycobacterial species, including M. tuberculosis and M. smegmatis, its physiological and biochemical functions in C. glutamicum remained elusive. Here we found the lack of C. glutamicum osmC gene resulted in decreased cell viability and increased intracellular reactive oxygen species accumulation under organic hydroperoxides (OHPs) stress conditions. The osmC expression was induced in the multiple antibiotic resistance regulator MarR-dependent manner by OHPs, and not by other oxidants or osmotic stress. Peroxide reductase activity showed that OsmC had a narrow range of substrates-only degrading OHPs, and detoxified OHPs mainly by linking the alkyl hydroperoxide reductase (AhpD) system (AhpD/dihydrolipoamide dehydrogenase (Lpd)/dihydrolipoamide acyltransferase (SucB)). Site-directed mutagenesis confirmed Cys48 was the peroxidatic cysteine, while Cys114 was the resolving Cys residue that formed an intramolecular disulfide bond with oxidized Cys48. Therefore, C. glutamicum OsmC was a thiol-dependent OHP reductase and played important role of protection against OHPs together with Ohr.

Annotation of a hypothetical protein (WP_002969292.1) from Brucella abortus.

Brucellosis is a zoonotic disease caused mainly by the bacteria belonging to the genus Brucella, most common of them is Brucella abortus. Genome sequencing of Brucella was completed in 2005. While majority of the proteins were assigned function, a large number of the peptides remained un-annotated and were referred as 'hypothetical'. These hypothetical proteins may contain crucial information about the biology and pathogenesis of the B. abortus. Therefore, it is of interest to annotate one such hypothetical protein as a multiple antibiotic resistance regulator protein, MarR. The physiological parameters, localization and the structural features were predicted for this protein which corroborated as the winged-helix type DNA-binding domain superfamily of transcription factors.

Transcriptional analysis reveals the metabolic state of Burkholderia zhejiangensis CEIB S4-3 during methyl parathion degradation.

Burkholderia zhejiangensis CEIB S4-3 has the ability to degrade methyl parathion (MP) and its main hydrolysis byproduct p-nitrophenol (PNP). According to genomic data, several genes related with metabolism of MP and PNP were identified in this strain. However, the metabolic state of the strain during the MP degradation has not been evaluated. In the present study, we analyzed gene expression changes during MP hydrolysis and PNP degradation through a transcriptomic approach. The transcriptional analysis revealed differential changes in the expression of genes involved in important cellular processes, such as energy production and conversion, transcription, amino acid transport and metabolism, translation, ribosomal structure and biogenesis, among others. Transcriptomic data also exhibited the overexpression of both PNP-catabolic gene clusters (pnpABA′E1E2FDC and pnpE1E2FDC) present in the strain. We found and validated by quantitative reverse transcription polymerase chain reaction the expression of the methyl parathion degrading gene, as well as the genes responsible for PNP degradation contained in two clusters. This proves the MP degradation pathway by the strain tested in this work. The exposure to PNP activates, in the first instance, the expression of the transcriptional regulators multiple antibiotic resistance regulator and Isocitrate Lyase Regulator (IclR), which are important in the regulation of genes from aromatic compound catabolism, as well as the expression of genes that encode transporters, permeases, efflux pumps, and porins related to the resistance to multidrugs and other xenobiotics. In the presence of the pesticide, 997 differentially expressed genes grouped in 104 metabolic pathways were observed. This report is the first to describe the transcriptomic analysis of a strain of B. zhejiangensis during the biodegradation of PNP.