Involvement of the leucine response transcription factor LeuO in regulation of the genes for sulfa drug efflux. - Related Documents




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619101.0000Involvement of the leucine response transcription factor LeuO in regulation of the genes for sulfa drug efflux. LeuO, a LysR family transcription factor, exists in a wide variety of bacteria of the family Enterobacteriaceae and is involved in the regulation of as yet unidentified genes affecting the stress response and pathogenesis expression. Using genomic screening by systematic evolution of ligands by exponential enrichment (SELEX) in vitro, a total of 106 DNA sequences were isolated from 12 different regions of the Escherichia coli genome. All of the SELEX fragments formed complexes in vitro with purified LeuO. After Northern blot analysis of the putative target genes located downstream of the respective LeuO-binding sequence, a total of nine genes were found to be activated by LeuO, while three genes were repressed by LeuO. The LeuO target gene collection included several multidrug resistance genes. A phenotype microarray assay was conducted to identify the gene(s) responsible for drug resistance and the drug species that are under the control of the LeuO target gene(s). The results described herein indicate that the yjcRQP operon, one of the LeuO targets, is involved in sensitivity control against sulfa drugs. We propose to rename the yjcRQP genes the sdsRQP genes (sulfa drug sensitivity determinant).200919429622
632410.9990Genetic and biochemical basis of tetracycline resistance. Properties of several, well characterized, tetracycline resistance determinants were compared. The determinants in Tn1721 and Tn10 (both from Gram-negative bacteria) each contain two genes; one encodes a repressor that regulates both its own transcription and that of a membrane protein that confers resistance by promoting efflux of the drug. Determinants from Gram-positive bacteria also encode efflux proteins, but expression of resistance is probably regulated by translational attenuation. The likely tetracycline binding site (a common dipeptide) in each efflux protein was predicted. The presence of the common binding site is consistent with the ability of an efflux protein originating in Bacillus species to be expressed in Escherichia coli.19863542941
620120.9990Overexpression of mfpA Gene Increases Ciprofloxacin Resistance in Mycobacterium smegmatis. Fluoroquinolones (FQs) are antibiotics useful in the treatment of drug-resistant tuberculosis, but FQ-resistant mutants can be selected rapidly. Although mutations in the DNA gyrase are the principal cause of this resistance, pentapeptide proteins have been found to confer low-level FQ resistance in Gram-negative bacteria. MfpA is a pentapeptide repeat protein conserved in mycobacterial chromosomes, where it is adjacent to a group of four highly conserved genes termed a conservon. We wished to characterize the transcriptional regulation of the mfpA gene and relate its expression to ciprofloxacin resistance in M. smegmatis. Reverse transcription PCR showed that mfpA gene is part of an operon containing the conservon genes. Using a transcriptional fusion, we showed that a promoter was located 5' to the mfpEA operon. We determined the promoter activity under different growth conditions and found that the expression of the operon increases slightly in late growth phases in basic pH and in subinhibitory concentrations of ciprofloxacin. Finally, by cloning the mfpA gene in an inducible vector, we showed that induced expression of mfpA increases the ciprofloxacin Minimal Inhibitory Concentration. These results confirm that increased expression of the mfpA gene, which is part of the mfpEA operon, increases ciprofloxacin resistance in M. smegmatis.202133824663
470730.9990Comparative transcriptome analyses of magainin I-susceptible and -resistant Escherichia coli strains. Antimicrobial peptides (AMPs) have attracted considerable attention because of their multiple and complex mechanisms of action toward resistant bacteria. However, reports have increasingly highlighted how bacteria can escape AMP administration. Here, the molecular mechanisms involved in Escherichia coli resistance to magainin I were investigated through comparative transcriptomics. Sub-inhibitory concentrations of magainin I were used to generate four experimental groups, including magainin I-susceptible E. coli, in the absence (C) and presence of magainin I (CM); and magainin I-resistant E. coli in the absence (R) and presence of magainin I (RM). The total RNA from each sample was extracted; cDNA libraries were constructed and further submitted for Illumina MiSeq sequencing. After RNA-seq data pre-processing and functional annotation, a total of 103 differentially expressed genes (DEGs) were identified, mainly related to bacterial metabolism. Moreover, down-regulation of cell motility and chaperone-related genes was observed in CM and RM, whereas cell communication, acid tolerance and multidrug efflux pump genes (ABC transporter, major facilitator and resistance-nodulation cell division superfamilies) were up-regulated in these same groups. DEGs from the C and R groups are related to basal levels of expression of homeostasis-related genes compared to CM and RM, suggesting that the presence of magainin I is required to change the transcriptomics panel in both C and R E. coli strains. These findings show the complexity of E. coli resistance to magainin I through the rearrangement of several metabolic pathways involved in bacterial physiology and drug response, also providing information on the development of novel antimicrobial strategies targeting resistance-related transcripts and proteins herein described.201830277857
632240.9990A soxRS-constitutive mutation contributing to antibiotic resistance in a clinical isolate of Salmonella enterica (Serovar typhimurium). The soxRS regulon is activated by redox-cycling drugs such as paraquat and by nitric oxide. The >15 genes of this system provide resistance to both oxidants and multiple antibiotics. An association between clinical quinolone resistance and elevated expression of the soxRS regulon has been observed in Escherichia coli, but this association has not been explored for other enteropathogenic bacteria. Here we describe a soxRS-constitutive mutation in a clinical strain of Salmonella enterica (serovar Typhimurium) that arose with the development of resistance to quinolones during treatment. The elevated quinolone resistance in this strain derived from a point mutation in the soxR gene and could be suppressed in trans by multicopy wild-type soxRS. Multiple-antibiotic resistance was also transferred to a laboratory strain of S. enterica by introducing the cloned mutant soxR gene from the clinical strain. The results show that constitutive expression of soxRS can contribute to antibiotic resistance in clinically relevant S. enterica.200111120941
888750.9989RpoE is a Putative Antibiotic Resistance Regulator of Salmonella enteric Serovar Typhi. Bacterial antimicrobial resistance has been associated with the up regulation of genes encoding efflux pumps and the down regulation of genes encoding outer membrane proteins (OMPs). Gene expression in bacteria is primarily initiated by sigma factors (σ factors) such as RpoE, which plays an important role in responding to many environmental stresses. Here, we report the first observation that RpoE serves as an antibiotic resistance regulator in Salmonella enteric serovar Typhi (S. Typhi). In this study, we found that the rpoE mutant (ΔrpoE) of S. Typhi GIFU10007 has elevated resistance to several antimicrobial agents, including β-lactams, quinolones, and aminoglycosides. Genomic DNA microarray analysis was used to investigate the differential gene expression profiles between a wild type and rpoE mutant in response to ampicillin. The results showed that a total of 57 genes displayed differential expression (two-fold increase or decrease) in ΔrpoE versus the wild-type strain. The expressions of two outer membrane protein genes, ompF and ompC, were significantly down-regulated in ΔrpoE (six and seven-fold lower in comparison to wild-type strain) and RamA, a member of the efflux pump AraC/XylS family, was up-regulated about four-fold in the ΔrpoE. Our results suggest RpoE is a potential antimicrobial regulator in S. Typhi, controlling both the down regulation of the OMP genes and up-regulating the efflux system.201626742769
618660.9989A triclosan-ciprofloxacin cross-resistant mutant strain of Staphylococcus aureus displays an alteration in the expression of several cell membrane structural and functional genes. Triclosan is an antimicrobial agent found in many consumer products. Triclosan inhibits the bacterial fatty acid biosynthetic enzyme, enoyl-ACP reductase (FabI). Decreased susceptibility to triclosan correlates with ciprofloxacin resistance in several bacteria. In these bacteria, resistance to both drugs maps to genes encoding multi-drug efflux pumps. The focus of this study was to determine whether triclosan resistance contributes to ciprofloxacin resistance in Staphylococcus aureus. In S. aureus, triclosan resistance maps to a fabI homolog and ciprofloxacin resistance maps to genes encoding DNA gyrase, topoisomerase IV and to the multi-drug efflux pump, NorA. Using a norA overexpressing mutant, we demonstrated that upregulation of NorA does not lead to triclosan resistance. To further investigate triclosan/ciprofloxacin resistance in S. aureus, we isolated triclosan/ciprofloxacin-resistant mutants. The mutants were screened for mutations in the genes encoding the targets of triclosan and ciprofloxacin. One mutant, JJ5, was wild-type for all sequences analyzed. We next monitored the efflux of triclosan from JJ5 and determined that triclosan resistance in the mutant was not due to active efflux of the drug. Finally, gene expression profiling demonstrated that an alteration in cell membrane structural and functional gene expression is likely responsible for triclosan and ciprofloxacin resistance in JJ5.200717997080
632570.9989Repressed multidrug resistance genes in Streptomyces lividans. Multidrug resistance (MDR) systems are ubiquitously present in prokaryotes and eukaryotes and defend both types of organisms against toxic compounds in the environment. Four families of MDR systems have been described, each family removing a broad spectrum of compounds by a specific membrane-bound active efflux pump. In the present study, at least four MDR systems were identified genetically in the soil bacterium Streptomyces lividans. The resistance genes of three of these systems were cloned and sequenced. Two of them are accompanied by a repressor gene. These MDR gene sequences are found in most other Streptomyces species investigated. Unlike the constitutively expressed MDR genes in Escherichia coli and other gram-negative bacteria, all of the Streptomyces genes were repressed under laboratory conditions, and resistance arose by mutations in the repressor genes.200312937892
64380.9989Effect of overexpression of small non-coding DsrA RNA on multidrug efflux in Escherichia coli. OBJECTIVES: Several putative and proven drug efflux pumps are present in Escherichia coli. Because many such efflux pumps have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbour such large sets of multidrug efflux genes. To understand how bacteria utilize these multiple efflux pumps, it is important to elucidate the process of pump expression regulation. The aim of this study was to determine a regulator of the multidrug efflux pump in this organism. METHODS: We screened a genomic library of E. coli for genes that decreased drug susceptibility in this organism. The library was developed from the chromosomal DNA of the MG1655 strain, and then the recombinant plasmids were transformed into an acrB-deleted strain. Transformants were screened for resistance to various antibiotics including oxacillin. RESULTS: We found that the multidrug susceptibilities of the acrB-deleted strain were decreased by the overexpression of small non-coding DsrA RNA as well as by the overexpression of known regulators of multidrug efflux pumps. Plasmids carrying the dsrA gene conferred resistance to oxacillin, cloxacillin, erythromycin, rhodamine 6G and novobiocin. DsrA decreased the accumulation of ethidium bromide in E. coli cells. Furthermore, expression of mdtE was significantly increased by dsrA overexpression, and the decreased multidrug susceptibilities modulated by DsrA were dependent on the MdtEF efflux pump. CONCLUSIONS: These results indicate that DsrA modulates multidrug efflux through activation of genes encoding the MdtEF pump in E. coli.201121088020
845590.9989RT-PCR: characterization of long multi-gene operons and multiple transcript gene clusters in bacteria. Reverse transcription (RT)-PCR is a valuable tool widely used for analysis of gene expression. In bacteria, RT-PCR is helpful beyond standard protocols of northern blot RNA/DNA hybridization (to identify transcripts) and primer extension (to locate their start points), as these methods have been difficult with transcripts that are low in abundance or unstable, similar to long multi-gene operons. In this report, RT-PCR is adapted to analyze transcripts that form long multi-gene operons--where they start and where they stop. The transcripts can also be semiquantitated to follow the expression of genes under different growth conditions. Examples using RT-PCR are presented with two different multi-gene systems for metal cation resistance to silver and mercury ions. The silver resistance system [9 open reading frames (ORFs); 12.5 kb] is shown by RT-PCR to synthesize three nonoverlapping messenger RNAs that are transcribed divergently. In the mercury resistance system (8 ORFs; 6.3 kb), all the genes are transcribed in the same orientation, and two promoter sites produce overlapping transcripts. For RT-PCR, reverse transcriptase enzyme is used to synthesize first-strand cDNA that is used as a template for PCR amplification of single-gene products, from the beginning, middle or end of long multi-gene, multi-transcript gene clusters.199910572645
644100.9989The MarR repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli: prototypic member of a family of bacterial regulatory proteins involved in sensing phenolic compounds. BACKGROUND: The marR gene of Escherichia coli encodes a repressor of the marRAB operon, a regulatory locus controlling multiple antibiotic resistance in this organism. Inactivation of marR results in increased expression of marA, which acts at several target genes in the cell leading to reduced antibiotic accumulation. Exposure of E. coli to sodium salicylate (SAL) induces marRAB operon transcription and antibiotic resistance. The mechanism by which SAL antagonizes MarR repressor activity is unclear. MATERIALS AND METHODS: Recombinant plasmid libraries were introduced into a reporter strain designed to identify cloned genes encoding MarR repressor activity. Computer analysis of sequence databases was also used to search for proteins related to MarR. RESULTS: A second E. coli gene, MprA, that exhibits MarR repressor activity was identified. Subsequent database searching revealed a family of 10 proteins from a variety of bacteria that share significant amino acid sequence similarity to MarR and MprA. At least four of these proteins are transcriptional repressors whose activity is antagonized by SAL or by phenolic agents structurally related to SAL. CONCLUSIONS: The MarR family is identified as a group of regulatory factors whose activity is modulated in response to environmental signals in the form of phenolic compounds. Many of these agents are plant derived. Some of the MarR homologs appear more likely to control systems expressed in animal hosts, suggesting that phenolic sensing by bacteria is important in a variety of environments and in the regulation of numerous processes.19958521301
771110.9989The multiple antibiotic resistance operon of enteric bacteria controls DNA repair and outer membrane integrity. The multiple antibiotic resistance (mar) operon of Escherichia coli is a paradigm for chromosomally encoded antibiotic resistance in enteric bacteria. The locus is recognised for its ability to modulate efflux pump and porin expression via two encoded transcription factors, MarR and MarA. Here we map binding of these regulators across the E. coli genome and identify an extensive mar regulon. Most notably, MarA activates expression of genes required for DNA repair and lipid trafficking. Consequently, the mar locus reduces quinolone-induced DNA damage and the ability of tetracyclines to traverse the outer membrane. These previously unrecognised mar pathways reside within a core regulon, shared by most enteric bacteria. Hence, we provide a framework for understanding multidrug resistance, mediated by analogous systems, across the Enterobacteriaceae. Transcription factors MarR and MarA confer multidrug resistance in enteric bacteria by modulating efflux pump and porin expression. Here, Sharma et al. show that MarA also upregulates genes required for lipid trafficking and DNA repair, thus reducing antibiotic entry and quinolone-induced DNA damage.201729133912
6198120.9989BC4707 is a major facilitator superfamily multidrug resistance transport protein from Bacillus cereus implicated in fluoroquinolone tolerance. Transcriptional profiling highlighted a subset of genes encoding putative multidrug transporters in the pathogen Bacillus cereus that were up-regulated during stress produced by bile salts. One of these multidrug transporters (BC4707) was selected for investigation. Functional characterization of the BC4707 protein in Escherichia coli revealed a role in the energized efflux of xenobiotics. Phenotypic analyses after inactivation of the gene bc4707 in Bacillus cereus ATCC14579 suggested a more specific, but modest role in the efflux of norfloxacin. In addition to this, transcriptional analyses showed that BC4707 is also expressed during growth of B. cereus under non-stressful conditions where it may have a role in the normal physiology of the bacteria. Altogether, the results indicate that bc4707, which is part of the core genome of the B. cereus group of bacteria, encodes a multidrug resistance efflux protein that is likely involved in maintaining intracellular homeostasis during growth of the bacteria.201222615800
6217130.9988Identification of the sigmaB regulon of Bacillus cereus and conservation of sigmaB-regulated genes in low-GC-content gram-positive bacteria. The alternative sigma factor sigma(B) has an important role in the acquisition of stress resistance in many gram-positive bacteria, including the food-borne pathogen Bacillus cereus. Here, we describe the identification of the set of sigma(B)-regulated genes in B. cereus by DNA microarray analysis of the transcriptome upon a mild heat shock. Twenty-four genes could be identified as being sigma(B) dependent as witnessed by (i) significantly lower expression levels of these genes in mutants with a deletion of sigB and rsbY (which encode the alternative sigma factor sigma(B) and a crucial positive regulator of sigma(B) activity, respectively) than in the parental strain B. cereus ATCC 14579 and (ii) increased expression of these genes upon a heat shock. Newly identified sigma(B)-dependent genes in B. cereus include a histidine kinase and two genes that have predicted functions in spore germination. This study shows that the sigma(B) regulon of B. cereus is considerably smaller than that of other gram-positive bacteria. This appears to be in line with phylogenetic analyses where sigma(B) of the B. cereus group was placed close to the ancestral form of sigma(B) in gram-positive bacteria. The data described in this study and previous studies in which the complete sigma(B) regulon of the gram-positive bacteria Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus were determined enabled a comparison of the sets of sigma(B)-regulated genes in the different gram-positive bacteria. This showed that only three genes (rsbV, rsbW, and sigB) are conserved in their sigma(B) dependency in all four bacteria, suggesting that the sigma(B) regulon of the different gram-positive bacteria has evolved to perform niche-specific functions.200717416654
268140.9988Amplification of bacitracin transporter genes in the bacitracin producing Bacillus licheniformis. We have amplified the previously cloned and sequenced genes of the bacitracin exporter (bcr), a member of the ATP-binding transport protein family, within the chromosome of the bacitracin producing Bacillus licheniformis. Amplification of the transporter genes was followed by greatly increased bacitracin resistance. Antibiotic production was enhanced at a low level of bcr genes amplification. An enlarged increase in the copy number of the bcr genes negatively affects the overall growth of bacteria.19979418256
292150.9988Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Tetracycline-resistance determinants encoding active efflux of the drug are widely distributed in gram-negative bacteria and unique with respect to genetic organization and regulation of expression. Each determinant consists of two genes called tetA and tetR, which are oriented with divergent polarity, and between them is a central regulatory region with overlapping promoters and operators. The amino acid sequences of the encoded proteins are 43-78% identical. The resistance protein TetA is a tetracycline/metal-proton antiporter located in the cytoplasmic membrane, while the regulatory protein TetR is a tetracycline inducible repressor. TetR binds via a helix-turn-helix motif to the two tet operators, resulting in repression of both genes. A detailed model of the repressor-operator complex has been proposed on the basis of biochemical and genetic data. The tet genes are differentially regulated so that repressor synthesis can occur before the resistance protein is expressed. This has been demonstrated for the Tn10-encoded tet genes and may be a common property of all tet determinants, as suggested by the similar locations of operators with respect to promoters. Induction is mediated by a tetracycline-metal complex and requires only nanomolar concentrations of the drug. This is the most sensitive effector-inducible system of transcriptional regulation known to date. The crystal structure of the TetR-tetracycline/metal complex shows the Tet repressor in the induced, non-DNA binding conformation. The structural interpretation of many noninducible TetR mutants has offered insight into the conformational changes associated with the switch between inducing and repressing structures of TetR. Tc is buried in the core of TetR, where it is held in place by multiple contacts to the protein.19947826010
267160.9988Molecular characterization of resistance-nodulation-division transporters from solvent- and drug-resistant bacteria in petroleum-contaminated soil. PCR assays for analyzing resistance-nodulation-division transporters from solvent- and drug-resistant bacteria in soil were developed. Sequence analysis of amplicons showed that the PCR successfully retrieved transporter gene fragments from soil. Most of the genes retrieved from petroleum-contaminated soils formed a cluster (cluster PCS) that was distantly related to known transporter genes. Competitive PCR showed that the abundance of PCS genes is increased in petroleum-contaminated soil.200515640241
445170.9988Selection of Shigella flexneri candidate virulence genes specifically induced in bacteria resident in host cell cytoplasm. We describe an in vivo expression technology (IVET)-like approach, which uses antibiotic resistance for selection, to identify Shigella flexneri genes specifically activated in bacteria resident in host cell cytoplasm. This procedure required construction of a promoter-trap vector containing a synthetic operon between the promoterless chloramphenicol acetyl transferase (cat) and lacZ genes and construction of a library of plasmids carrying transcriptional fusions between S. flexneri genomic fragments and the cat-lacZ operon. Clones exhibiting low levels (<10 micro g ml-1) of chloramphenicol (Cm) resistance on laboratory media were analysed for their ability to induce a cytophatic effect--plaque--on a cell monolayer, in the presence of Cm. These clones were assumed to carry a plasmid in which the cloned fragment acted as a promoter/gene which is poorly expressed under laboratory conditions. Therefore, only strains harbouring fusion-plasmids in which the cloned promoter was specifically activated within host cytoplasm could survive within the cell monolayer in the presence of Cm and give a positive result in the plaque assay. Pai (plaque assay induced) clones, selected following this procedure, were analysed for intracellular (i) beta-galactosidase activity, (ii) proliferation in the presence of Cm, and (iii) Cm resistance. Sequence analysis of Pai plasmids revealed genes encoding proteins of three functional classes: external layer recycling, adaptation to microaerophilic environment and gene regulation. Sequences encoding unknown functions were also trapped and selected by this new IVET-based protocol.200212390353
6188180.9988Quinolone mode of action. Physical studies have further defined interactions of quinolones with their principal target, DNA gyrase. The binding of quinolones to the DNA gyrase-DNA complex suggests 2 possible binding sites of differing affinities. Mutations in either the gyrase A gene (gyrA) or the gyrase B gene (gyrB) that affect quinolone susceptibility also affect drug binding, with resistance mutations causing decreased binding and hypersusceptibility mutations causing increased binding. Combinations of mutations in both GyrA and GyrB have further demonstrated the contribution of both subunits to the quinolone sensitivity of intact bacteria and purified DNA gyrase. A working model postulates initial binding of quinolones to proximate sites on GyrA and GyrB. This initial binding then produces conformational changes that expose additional binding sites, possibly involving DNA. Quinolones also inhibit the activities of Escherichia coli topoisomerase IV (encoded by the parC and parE genes), but at concentrations higher than those inhibiting DNA gyrase. The patterns of resistance mutations in gryA and parC suggest that topoisomerase IV may be a secondary drug target in E. coli and Neisseria gonorrhoeae. In contrast, in Staphylococcus aureus these patterns suggest that topoisomerase IV may be a primary target of quinolone action. Regulation of expression of membrane efflux transporters may contribute to quinolone susceptibility in both Gram-positive and Gram-negative bacteria. The substrate profile of the NorA efflux transporter of S. aureus correlates with the extent to which the activity of quinolone substrates is affected by overexpression of NorA. In addition, the Emr transporter of E. coli affects susceptibility to nalidixic acid, and the MexAB OprK transport system of Pseudomonas aeruginosa affects susceptibility to ciprofloxacin.(ABSTRACT TRUNCATED AT 250 WORDS)19958549276
6326190.9988Identification of novel metronidazole-inducible genes in Mycobacterium smegmatis using a customized amplification library. The incidence of antibiotic resistance in pathogenic bacteria is rising. Bacterial resistance may be a natural defense of organisms, or it may result from spontaneous mutations or the acquisition of exogenous resistance genes. We grew spontaneous metronidazole-resistant Mycobacterium smegmatis mutants on solid medium cultures and employed differential expression using a customized amplification library to analyze the global gene profiles of metronidazole-resistant mutants under hypoxic conditions. In total, 66 genes involved in metronidazole resistance were identified and functionally characterized using the gene role category of M. smegmatis. Overall, genes associated with cell wall synthesis, such as methyltransferase and glycosyltransferase, and genes encoding drug transporters were highly expressed. The genes may be involved in the natural drug resistance of mycobacteria by increasing mycobacterial cell wall permeability and the efflux pumps of active drugs. In addition, the genes may play a role in dormancy. The genes identified in this study may lead to a better understanding of the mechanisms of metronidazole resistance during dormancy.200818373646