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19200.9863N-Succinyltransferase Encoded by a Cryptic Siderophore Biosynthesis Gene Cluster in Streptomyces Modifies Structurally Distinct Antibiotics. The antibiotic desertomycin A and its previously undescribed inactive N-succinylated analogue, desertomycin X, were isolated from Streptomyces sp. strain YIM 121038. Genome sequencing and analysis readily identified the desertomycin biosynthetic gene cluster (BGC), which lacked genes encoding acyltransferases that would account for desertomycin X formation. Scouting the genome for putative N-acyltransferase genes led to the identification of a candidate within a cryptic siderophore BGC (csb) encoding a putative homologue of the N6'-hydroxylysine acetyltransferase IucB. Expression of the codon-optimized gene designated csbC in Escherichia coli yielded the recombinant protein that was able to N-succinylate desertomycin A as well as several other structurally distinct antibiotics harboring amino groups. Some antibiotics were rendered antibiotically inactive due to the CsbC-catalyzed succinylation in vitro. Unlike many known N-acyltransferases involved in antibiotic resistance, CsbC could not efficiently acetylate the same antibiotics. When expressed in E. coli, CsbC provided low-level resistance to kanamycin and ampicillin, suggesting that it may play a role in antibiotic resistance in natural habitats, where the concentration of antibiotics is usually low. IMPORTANCE In their natural habitats, bacteria encounter a plethora of organic compounds, some of which may be represented by antibiotics produced by certain members of the microbial community. A number of antibiotic resistance mechanisms have been described, including those specified by distinct genes encoding proteins that degrade, modify, or expel antibiotics. In this study, we report identification and characterization of an enzyme apparently involved in the biosynthesis of a siderophore, but also having the ability of modify and thereby inactivate a wide variety of structurally diverse antibiotics. This discovery sheds light on additional capabilities of bacteria to withstand antibiotic treatment and suggests that enzymes involved in secondary metabolism may have an additional function in the natural environment.202236040031
80610.9855A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans. The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.200717464069
19420.9851Possible Role of CHAD Proteins in Copper Resistance. Conserved Histidine Alpha-helical Domain (CHAD) proteins attached to the surface of polyphosphate (PolyP) have been studied in some bacteria and one archaeon. However, the activity of CHAD proteins is unknown beyond their interaction with PolyP granules. By using bioinformatic analysis, we report that several species of the biomining acidophilic bacteria contain orthologs of CHAD proteins with high sequence identity. Furthermore, the gene coding for the CHAD protein is in the same genetic context of the enzyme polyphosphate kinase (PPK), which is in charge of PolyP synthesis. Particularly, the group of ppk and CHAD genes is highly conserved. Metallosphaera sedula and other acidophilic archaea used in biomining also contain CHAD proteins. These archaea show high levels of identity in genes coding for a cluster having the same organization. Amongst these genes are chad and ppx. In general, both biomining bacteria and archaea contain high PolyP levels and are highly resistant to heavy metals. Therefore, the presence of this conserved genetic organization suggests a high relevance for their metabolism. It has been formerly reported that a crystallized CHAD protein contains a copper-binding site. Based on this previous knowledge, in the present report, it was determined that all analyzed CHAD proteins are very conserved at their structural level. In addition, it was found that the lack of YgiF, an Escherichia coli CHAD-containing protein, decreases copper resistance in this bacterium. This phenotype was not only complemented by transforming E. coli with YgiF but also by expressing CHAD from Acidithiobacillus ferrooxidans in it. Interestingly, the strains in which the possible copper-binding sites were mutated were also more metal sensitive. Based on these results, we propose that CHAD proteins are involved in copper resistance in microorganisms. These findings are very interesting and may eventually improve biomining operations in the future.202438399813
12530.9850ROD1, a novel gene conferring multiple resistance phenotypes in Saccharomyces cerevisiae. Glutathione-dependent detoxification reactions are catalyzed by the enzyme glutathione S-transferase and are important in drug resistance in organisms ranging from bacteria to humans. The yeast Issatchenkia orientalis expresses a glutathione S-transferase (GST) protein that is induced when the GST substrate o-dinitrobenzene (o-DNB) is added to the culture. In this study, we show that overproduction of the I. orientalis GST in Saccharomyces cerevisiae leads to an increase in o-dinitrobenzene resistance in S. cerevisiae cells. To recover genes that influence o-DNB resistance in S. cerevisiae, a high copy plasmid library was screened for loci that elevate o-DNB tolerance. One gene was recovered and designated ROD1 (resistance to o-dinitrobenzene). This locus was found to encode a novel protein with no significant sequence similarity with proteins of known function in the data base. An epitope-tagged version of Rod1p was produced in S. cerevisiae and shown to function properly. Subcellular fractionation experiments indicated that this factor was found in the particulate fraction by differential centrifugation. Overproduction of Rod1p leads to resistance to not only o-DNB but also zinc and calcium. Strains that lack the ROD1 gene are hypersensitive to these same compounds. Rod1p represents a new type of molecule influencing drug tolerance in eukaryotes.19968621680
54940.9849Extracytoplasmic function sigma factor σ(D) confers resistance to environmental stress by enhancing mycolate synthesis and modifying peptidoglycan structures in Corynebacterium glutamicum. Mycolates are α-branched, β-hydroxylated, long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. They form an outer membrane, which functions as a permeability barrier and confers pathogenic mycobacteria to resistance to antibiotics. Although the mycolate biosynthetic pathway has been intensively studied, knowledge of transcriptional regulation of genes involved in this pathway is limited. Here, we report that the extracytoplasmic function sigma factor σ(D) is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σ(D) -binding regions in the genome, establishing a consensus promoter sequence for σ(D) recognition. The σ(D) regulon comprised acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase and mycolyltransferases; they were involved in mycolate synthesis. Indeed, deletion or overexpression of sigD encoding σ(D) modified the extractable mycolate amount. Immediately downstream of sigD, rsdA encoded anti-σ(D) and was under the control of a σ(D) -dependent promoter. Another σ(D) regulon member, l,d-transpeptidase, conferred lysozyme resistance. Thus, σ(D) modifies peptidoglycan cross-linking and enhances mycolate synthesis to provide resistance to environmental stress.201829148103
17850.9844Molecular basis of bacterial resistance to organomercurial and inorganic mercuric salts. Bacteria mediate resistance to organomercurial and inorganic mercuric salts by metabolic conversion to nontoxic elemental mercury, Hg(0). The genes responsible for mercury resistance are organized in the mer operon, and such operons are often found in plasmids that also bear drug resistance determinants. We have subcloned three of these mer genes, merR, merB, and merA, and have studied their protein products via protein overproduction and purification, and structural and functional characterization. MeR is a metalloregulatory DNA-binding protein that acts as a repressor of both its own and structural gene transcription in the absence of Hg(II); in addition it acts as a positive effector of structural gene transcription when Hg(II) is present. MerB, organomercury lyase, catalyzes the protonolytic fragmentation of organomercurials to the parent hydrocarbon and Hg(II) by an apparent SE2 mechanism. MerA, mercuric ion reductase, is an FAD-containing and redox-active disulfide-containing enzyme with homology to glutathione reductase. It has evolved the unique catalytic capacity to reduce Hg(II) to Hg(0) and thereby complete the detoxification scheme.19883277886
60960.9843A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. In nutrient-starved bacteria, RelA and SpoT proteins have key roles in reducing cell growth and overcoming stresses. Here we identify functional SpoT orthologs in metazoa (named Mesh1, encoded by HDDC3 in human and Q9VAM9 in Drosophila melanogaster) and reveal their structures and functions. Like the bacterial enzyme, Mesh1 proteins contain an active site for ppGpp hydrolysis and a conserved His-Asp-box motif for Mn(2+) binding. Consistent with these structural data, Mesh1 efficiently catalyzes hydrolysis of guanosine 3',5'-diphosphate (ppGpp) both in vitro and in vivo. Mesh1 also suppresses SpoT-deficient lethality and RelA-induced delayed cell growth in bacteria. Notably, deletion of Mesh1 (Q9VAM9) in Drosophila induces retarded body growth and impaired starvation resistance. Microarray analyses reveal that the amino acid-starved Mesh1 null mutant has highly downregulated DNA and protein synthesis-related genes and upregulated stress-responsible genes. These data suggest that metazoan SpoT orthologs have an evolutionarily conserved function in starvation responses.201020818390
54570.9841Characterization of the organic hydroperoxide resistance system of Brucella abortus 2308. The organic hydroperoxide resistance protein Ohr has been identified in numerous bacteria where it functions in the detoxification of organic hydroperoxides, and expression of ohr is often regulated by a MarR-type regulator called OhrR. The genes annotated as BAB2_0350 and BAB2_0351 in the Brucella abortus 2308 genome sequence are predicted to encode OhrR and Ohr orthologs, respectively. Using isogenic ohr and ohrR mutants and lacZ promoter fusions, it was determined that Ohr contributes to resistance to organic hydroperoxide, but not hydrogen peroxide, in B. abortus 2308 and that OhrR represses the transcription of both ohr and ohrR in this strain. Moreover, electrophoretic mobility shift assays and DNase I footprinting revealed that OhrR binds directly to a specific region in the intergenic region between ohr and ohrR that shares extensive nucleotide sequence similarity with so-called "OhrR boxes" described in other bacteria. While Ohr plays a prominent role in protecting B. abortus 2308 from organic hydroperoxide stress in in vitro assays, this protein is not required for the wild-type virulence of this strain in cultured murine macrophages or experimentally infected mice.201222821968
55780.9841Identification of a MarR Subfamily That Regulates Arsenic Resistance Genes. In this study, comprehensive analyses were performed to determine the function of an atypical MarR homolog in Achromobacter sp. strain As-55. Genomic analyses of Achromobacter sp. As-55 showed that this marR is located adjacent to an arsV gene. ArsV is a flavin-dependent monooxygenase that confers resistance to the antibiotic methylarsenite [MAs(III)], the organoarsenic compound roxarsone(III) [Rox(III)], and the inorganic antimonite [Sb(III)]. Similar marR genes are widely distributed in arsenic-resistant bacteria. Phylogenetic analyses showed that these MarRs are found in operons predicted to be involved in resistance to inorganic and organic arsenic species, so the subfamily was named MarR(ars). MarR(ars) orthologs have three conserved cysteine residues, which are Cys36, Cys37, and Cys157 in Achromobacter sp. As-55, mutation of which compromises the response to MAs(III)/Sb(III). GFP-fluorescent biosensor assays show that AdMarR(ars) (MarR protein of Achromobacter deleyi As-55) responds to trivalent As(III) and Sb(III) but not to pentavalent As(V) or Sb(V). The results of RT-qPCR assays show that arsV is expressed constitutively in a marR deletion mutant, indicating that marR represses transcription of arsV. Moreover, electrophoretic mobility shift assays (EMSAs) demonstrate that AdMarR(ars) binds to the promoters of both marR and arsV in the absence of ligands and that DNA binding is relieved upon binding of As(III) and Sb(III). Our results demonstrate that AdMarR(ars) is a novel As(III)/Sb(III)-responsive transcriptional repressor that controls expression of arsV, which confers resistance to MAs(III), Rox(III), and Sb(III). AdMarR(ars) and its orthologs form a subfamily of MarR proteins that regulate genes conferring resistance to arsenic-containing antibiotics. IMPORTANCE In this study, a MarR family member, AdMarR(ars) was shown to regulate the arsV gene, which confers resistance to arsenic-containing antibiotics. It is a founding member of a distinct subfamily that we refer to as MarR(ars), regulating genes conferring resistance to arsenic and antimony antibiotic compounds. AdMarR(ars) was shown to be a repressor containing conserved cysteine residues that are required to bind As(III) and Sb(III), leading to a conformational change and subsequent derepression. Here we show that members of the MarR family are involved in regulating arsenic-containing compounds.202134613763
634990.9841High-level chromate resistance in Arthrobacter sp. strain FB24 requires previously uncharacterized accessory genes. BACKGROUND: The genome of Arthrobacter sp. strain FB24 contains a chromate resistance determinant (CRD), consisting of a cluster of 8 genes located on a 10.6 kb fragment of a 96 kb plasmid. The CRD includes chrA, which encodes a putative chromate efflux protein, and three genes with amino acid similarities to the amino and carboxy termini of ChrB, a putative regulatory protein. There are also three novel genes that have not been previously associated with chromate resistance in other bacteria; they encode an oxidoreductase (most similar to malate:quinone oxidoreductase), a functionally unknown protein with a WD40 repeat domain and a lipoprotein. To delineate the contribution of the CRD genes to the FB24 chromate [Cr(VI)] response, we evaluated the growth of mutant strains bearing regions of the CRD and transcript expression levels in response to Cr(VI) challenge. RESULTS: A chromate-sensitive mutant (strain D11) was generated by curing FB24 of its 96-kb plasmid. Elemental analysis indicated that chromate-exposed cells of strain D11 accumulated three times more chromium than strain FB24. Introduction of the CRD into strain D11 conferred chromate resistance comparable to wild-type levels, whereas deletion of specific regions of the CRD led to decreased resistance. Using real-time reverse transcriptase PCR, we show that expression of each gene within the CRD is specifically induced in response to chromate but not by lead, hydrogen peroxide or arsenate. Higher levels of chrA expression were achieved when the chrB orthologs and the WD40 repeat domain genes were present, suggesting their possible regulatory roles. CONCLUSION: Our findings indicate that chromate resistance in Arthrobacter sp. strain FB24 is due to chromate efflux through the ChrA transport protein. More importantly, new genes have been identified as having significant roles in chromate resistance. Collectively, the functional predictions of these additional genes suggest the involvement of a signal transduction system in the regulation of chromate efflux and warrants further study.200919758450
339100.9840Multiple mechanisms of resistance to cisplatin toxicity in an Escherichia coli K12 mutant. The mechanisms underlying cellular resistance to the antitumor drug cis-diamminedichloro-platinum(II) (CDDP) were studied in Escherichia coli K12. A bacterial strain (MC4100/DDP) was selected from the MC4100 wild-type strain after growth for four cycles in CDDP. MC4100/DDP bacteria showed a high level of resistance and exhibited various modifications including (1) a decrease in drug uptake and platinum/DNA binding which only partly contributed to resistance, (2) an increase in glutathione content not involved in the resistant phenotype, (3) an increase in DNA repair capacity. Resistance was unmodified by introducing a uvrA mutation which neutralizes the excision-repair pathway. In contrast, it was abolished by deletion of the recA gene which abolishes recombination and SOS repair but also by a mutation in the recA gene leading to RecA co-protease minus (no SOS induction). RecA protein was unchanged in MC4100/DDP but the expression of RecA-dependent gene(s) was required for CDDP resistance. The regulation of genes belonging to the SOS regulon was analysed in MC4100/DDP by monitoring the expression of sfiA and recA::lacZ gene fusions after UV irradiation. These gene fusions were derepressed faster and the optimal expression was obtained for a lower number of UV lesions in MC4100/DDP, suggesting a role of RecA co-protease activity in the mechanism of resistance to CDDP in this E. coli strain.19947974517
126110.9840Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum. Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite D,L-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of L-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.200515668756
196120.9840A specialized citric acid cycle requiring succinyl-coenzyme A (CoA):acetate CoA-transferase (AarC) confers acetic acid resistance on the acidophile Acetobacter aceti. Microbes tailor macromolecules and metabolism to overcome specific environmental challenges. Acetic acid bacteria perform the aerobic oxidation of ethanol to acetic acid and are generally resistant to high levels of these two membrane-permeable poisons. The citric acid cycle (CAC) is linked to acetic acid resistance in Acetobacter aceti by several observations, among them the oxidation of acetate to CO2 by highly resistant acetic acid bacteria and the previously unexplained role of A. aceti citrate synthase (AarA) in acetic acid resistance at a low pH. Here we assign specific biochemical roles to the other components of the A. aceti strain 1023 aarABC region. AarC is succinyl-coenzyme A (CoA):acetate CoA-transferase, which replaces succinyl-CoA synthetase in a variant CAC. This new bypass appears to reduce metabolic demand for free CoA, reliance upon nucleotide pools, and the likely effect of variable cytoplasmic pH upon CAC flux. The putative aarB gene is reassigned to SixA, a known activator of CAC flux. Carbon overflow pathways are triggered in many bacteria during metabolic limitation, which typically leads to the production and diffusive loss of acetate. Since acetate overflow is not feasible for A. aceti, a CO(2) loss strategy that allows acetic acid removal without substrate-level (de)phosphorylation may instead be employed. All three aar genes, therefore, support flux through a complete but unorthodox CAC that is needed to lower cytoplasmic acetate levels.200818502856
131130.9839Characterization of Two Highly Arsenic-Resistant Caulobacteraceae Strains of Brevundimonas nasdae: Discovery of a New Arsenic Resistance Determinant. Arsenic (As), distributed widely in the natural environment, is a toxic substance which can severely impair the normal functions in living cells. Research on the genetic determinants conferring functions in arsenic resistance and metabolism is of great importance for remediating arsenic-contaminated environments. Many organisms, including bacteria, have developed various strategies to tolerate arsenic, by either detoxifying this harmful element or utilizing it for energy generation. More and more new arsenic resistance (ars) determinants have been identified to be conferring resistance to diverse arsenic compounds and encoded in ars operons. There is a hazard in mobilizing arsenic during gold-mining activities due to gold- and arsenic-bearing minerals coexisting. In this study, we isolated 8 gold enrichment strains from the Zijin gold and copper mine (Longyan, Fujian Province, China) wastewater treatment site soil, at an altitude of 192 m. We identified two Brevundimonas nasdae strains, Au-Bre29 and Au-Bre30, among these eight strains, having a high minimum inhibitory concentration (MIC) for As(III). These two strains contained the same ars operons but displayed differences regarding secretion of extra-polymeric substances (EPS) upon arsenite (As(III)) stress. B. nasdae Au-Bre29 contained one extra plasmid but without harboring any additional ars genes compared to B. nasdae Au-Bre30. We optimized the growth conditions for strains Au-Bre29 and Au-Bre30. Au-Bre30 was able to tolerate both a lower pH and slightly higher concentrations of NaCl. We also identified folE, a folate synthesis gene, in the ars operon of these two strains. In most organisms, folate synthesis begins with a FolE (GTP-Cyclohydrolase I)-type enzyme, and the corresponding gene is typically designated folE (in bacteria) or gch1 (in mammals). Heterologous expression of folE, cloned from B. nasdae Au-Bre30, in the arsenic-hypersensitive strain Escherichia coli AW3110, conferred resistance to As(III), arsenate (As(V)), trivalent roxarsone (Rox(III)), pentavalent roxarsone (Rox(V)), trivalent antimonite (Sb(III)), and pentavalent antimonate (Sb(V)), indicating that folate biosynthesis is a target of arsenite toxicity and increased production of folate confers increased resistance to oxyanions. Genes encoding Acr3 and ArsH were shown to confer resistance to As(III), Rox(III), Sb(III), and Sb(V), and ArsH also conferred resistance to As(V). Acr3 did not confer resistance to As(V) and Rox(V), while ArsH did not confer resistance to Rox(V).202235628430
546140.9839Resistance to organic hydroperoxides requires ohr and ohrR genes in Sinorhizobium meliloti. BACKGROUND: Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Medicago sativa. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H2O2 and superoxides has been extensively studied in S. meliloti. In contrast resistance to organic peroxides has not been investigated while S. meliloti genome encodes putative organic peroxidases. Organic peroxides are produced by plants and are highly toxic. The resistance to these oxygen radicals has been studied in various bacteria but never in plant nodulating bacteria. RESULTS: In this study we report the characterisation of organic hydroperoxide resistance gene ohr and its regulator ohrR in S. meliloti. The inactivation of ohr affects resistance to cumene and ter-butyl hydroperoxides but not to hydrogen peroxide or menadione in vitro. The expression of ohr and ohrR genes is specifically induced by organic peroxides. OhrR binds to the intergenic region between the divergent genes ohr and ohrR. Two binding sites were characterised. Binding to the operator is prevented by OhrR oxidation that promotes OhrR dimerisation. The inactivation of ohr did not affect symbiosis and nitrogen fixation, suggesting that redundant enzymatic activity exists in this strain. Both ohr and ohrR are expressed in nodules suggesting that they play a role during nitrogen fixation. CONCLUSIONS: This report demonstrates the significant role Ohr and OhrR proteins play in bacterial stress resistance against organic peroxides in S. meliloti. The ohr and ohrR genes are expressed in nodule-inhabiting bacteroids suggesting a role during nodulation.201121569462
574150.9839Pyrroloquinoline quinone and a quinoprotein kinase support γ-radiation resistance in Deinococcus radiodurans and regulate gene expression. Deinococcus radiodurans is known for its extraordinary resistance to various DNA damaging agents including γ-radiation and desiccation. The pqqE:cat and Δdr2518 mutants making these cells devoid of pyrroloquinoline quinone (PQQ) and a PQQ inducible Ser/Thr protein kinase, respectively, became sensitive to γ-radiation. Transcriptome analysis of these mutants showed differential expression of the genes including those play roles in oxidative stress tolerance and (DSB) repair in D. radiodurans and in genome maintenance and stress response in other bacteria. Escherichia coli cells expressing DR2518 and PQQ showed improved resistance to γ-radiation, which increased further when both DR2518 and PQQ were present together. Although, profiles of genes getting affected in these mutants were different, there were still a few common genes showing similar expression trends in both the mutants and some others as reported earlier in oxyR and pprI mutant of this bacterium. These results suggested that PQQ and DR2518 have independent roles in γ-radiation resistance of D. radiodurans but their co-existence improves radioresistance further, possibly by regulating differential expression of the genes important for bacterial response to oxidative stress and DNA damage.201322961447
544160.9839Organic Hydroperoxide Induces Prodigiosin Biosynthesis in Serratia sp. ATCC 39006 in an OhrR-Dependent Manner. The biosynthesis of prodigiosin in the model prodigiosin-producing strain, Serratia sp. ATCC 39006, is significantly influenced by environmental and cellular signals. However, a comprehensive regulatory mechanism for this process has not been well established. In the present study, we demonstrate that organic hydroperoxide activates prodigiosin biosynthesis in an OhrR-dependent manner. Specifically, the MarR-family transcriptional repressor OhrR (Ser39006_RS05455) binds to its operator located far upstream of the promoter region of the prodigiosin biosynthesis operon (319 to 286 nucleotides [nt] upstream of the transcription start site) and negatively regulates the expression of prodigiosin biosynthesis genes. Organic hydroperoxide disassociates the binding between OhrR and its operator, thereby promoting the prodigiosin production. Moreover, OhrR modulates the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide by regulating the transcription of its own gene and the downstream cotranscribed ohr gene. These results demonstrate that OhrR is a pleiotropic repressor that modulates the prodigiosin production and the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide stress. IMPORTANCE Bacteria naturally encounter various environmental and cellular stresses. Organic hydroperoxides generated from the oxidation of polyunsaturated fatty acids are widely distributed and usually cause lethal oxidative stress by damaging cellular components. OhrR is known as a regulator that modulates the resistance of bacteria to organic hydroperoxide stress. In the current study, organic hydroperoxide disassociates OhrR from the promoter of prodigiosin biosynthesis gene cluster, thus promoting transcription of pigA to -O genes. In this model, organic hydroperoxide acts as an inducer of prodigiosin synthesis in Serratia sp. ATCC 39006. These results improve our understanding of the regulatory network of prodigiosin synthesis and serve as an example for identifying the cross talk between the stress responses and the regulation of secondary metabolism.202235044847
582170.9838Sulfane 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.202134829649
124180.9838A bacterial view of the periodic table: genes and proteins for toxic inorganic ions. Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite.200516133099
374190.9838Simultaneous detection and removal of organomercurial compounds by using the genetic expression system of an organomercury lyase from the transposon Tn MERI1. Using a newly identified organomercury lyase gene (merB3) expression system from Tn MERI1, the mercury resistance transposon first found in Gram-positive bacteria, a dual-purpose system to detect and remove organomercurial contamination was developed. A plasmid was constructed by fusing the promoterless luxAB genes as bioluminescence reporter genes downstream of the merB3 gene and its operator/promoter region. Another plasmid, encoding mer operon genes from merR1 to merA, was also constructed to generate an expression regulatory protein, MerR1, and a mercury reductase enzyme, MerA. These two plasmids were transformed into Escherichia coli cells to produce a biological system that can detect and remove environmental organomercury contamination. Organomercurial compounds, such as neurotoxic methylmercury at nanomolar levels, were detected using the biomonitoring system within a few minutes and were removed during the next few hours.200212073137