Hidetada Hirakawa

Indole induces the expression of multidrug exporter genes in Escherichia coli

Our comprehensive expression cloning studies previously revealed that 20 intrinsic xenobiotic exporter systems are encoded in the Escherichia coli chromosome, but most of them are not expressed under normal conditions. In this study, we investigated the compounds that induce the expression of these xenobiotic exporter genes, and found that indole induces a variety of xenobiotic exporter genes including acrD, acrE, cusB, emrK, mdtA, mdtE and yceL. Indole treatment of E. coli cells confers rhodamine 6G and SDS resistance through the induction of mdtEF and acrD gene expression respectively. The induction of mdtE by indole is independent of the EvgSA two‐component signal transduction system that regulates the mdtE gene, but mediated by GadX. On the other hand, the induction of acrD and mdtA was mediated by BaeSR and CpxAR, two‐component systems. Interestingly, CpxAR system‐mediated induction required intrinsic baeSR genes, whereas BaeSR‐mediated induction was observed in the cpxAR gene‐deletion mutant. BaeR and CpxR directly bound to different sequences of the acrD and mdtA promoter regions. These observations indicate that BaeR is a primary regulator, and CpxR enhances the effect of BaeR.

Comprehensive Studies of Drug Resistance Mediated by Overexpression of Response Regulators of Two-Component Signal Transduction Systems in Escherichia coli

In Escherichia coli, there are 32 open reading frames (ORFs) that are assumed to be response regulator genes of two-component signal transduction systems on the basis of sequence similarities. We cloned all of these 32 ORFs into a multicopy expression vector and investigated whether or not they confer drug resistance via control of drug resistance determinants. Fifteen of these ORFs, i.e., baeR, citB, cpxR, evgA, fimZ, kdpE, narL, narP, ompR, rcsB, rstA, torR, yedW, yehT, and dcuR, conferred increased single- or multidrug resistance. Two-thirds of them conferred deoxycholate resistance. Five of them, i.e., evgA, baeR, ompR, cpxR, and rcsB, modulated the expression of several drug exporter genes. The drug resistance mediated by evgA, baeR, and cpxR could be assigned to drug exporters by using drug exporter gene knockout strains.

Roles of TolC-Dependent Multidrug Transporters of Escherichia coli in Resistance to β-Lactams

ABSTRACT AcrAB exports some β-lactam antibiotics in the periplasm out of cells via an outer-membrane channel, TolC. It has been reported that eight drug transporters in Escherichia coli cooperate with TolC. In this study, the roles of the drug exporters of E. coli in β-lactam resistance were examined. We found that five of five resistance-nodulation-cell division-type drug exporters confer β-lactam antibiotic resistance, while no other drug exporters confer any β-lactam resistance even when they cooperate with TolC.

Growth Phase-Dependent Expression of Drug Exporters in Escherichia coli and Its Contribution to Drug Tolerance

Drug exporters contribute to the intrinsic drug resistance in many organisms. Although there are at least 20 exporter genes in Escherichia coli, most of them apparently do not confer drug resistance in complex laboratory media except for the AcrAB, EmrE, and MdfA efflux systems. In this study, we comprehensively investigated the growth phase-dependent expression of drug exporter genes. The expression of acrAB, emrAB, emrD, emrE, emrKY, mdfA, and ydgFE is stable at moderate levels during any growth phase, whereas mdtEF promoter activity greatly increased with cell growth and reached the maximum level at the late stationary phase. The growth phase-dependent increase in mdtEF expression was also observed on quantitative reverse transcription-PCR analysis. As expected from the transporter expression, the stationary-phase cells actually showed MdtEF-dependent tolerance to drugs and toxic dyes. Growth phase-dependent elevation of mdtEF expression was found to be mediated by the stationary-phase sigma factor rpoS and the RpoS-dependent signaling pathway, Hfq, GadY, and GadX. The induction level was decreased by tnaAB deletion, suggesting that indole sensing stimulates this process.

Secreted indole serves as a signal for expression of type III secretion system translocators in enterohaemorrhagic Escherichia coli O157:H7.

Indole is produced by tryptophanase during growth of enteric bacteria and accumulates in the culture medium. The physiological role of indole production is poorly understood. We discovered that enterohaemorrhagic Escherichia coli (EHEC) O157:H7 with a tnaA deletion has decreased secretion of EspA and EspB via the type III secretion system and as a result there is reduced formation of attaching and effacing (A/E) lesions in HeLa cells. Addition of indole restored and enhanced secretion of EspA and EspB and formation of A/E lesions by the tnaA deletion mutant EHEC. Indole addition moderately increased the promoter activity of LEE4 genes, including espA and espB, in the locus of enterocyte effacement. Thus in EHEC indole can serve to signal EspA and EspB expression and secretion and stimulate the ability of EHEC to form A/E lesions on human cells.

Interference of bacterial cell-to-cell communication: a new concept of antimicrobial chemotherapy breaks antibiotic resistance

Bacteria use a cell-to-cell communication activity termed “quorum sensing” to coordinate group behaviors in a cell density dependent manner. Quorum sensing influences the expression profile of diverse genes, including antibiotic tolerance and virulence determinants, via specific chemical compounds called “autoinducers”. During quorum sensing, Gram-negative bacteria typically use an acylated homoserine lactone (AHL) called autoinducer 1. Since the first discovery of quorum sensing in a marine bacterium, it has been recognized that more than 100 species possess this mechanism of cell-to-cell communication. In addition to being of interest from a biological standpoint, quorum sensing is a potential target for antimicrobial chemotherapy. This unique concept of antimicrobial control relies on reducing the burden of virulence rather than killing the bacteria. It is believed that this approach will not only suppress the development of antibiotic resistance, but will also improve the treatment of refractory infections triggered by multi-drug resistant pathogens. In this paper, we review and track recent progress in studies on AHL inhibitors/modulators from a biological standpoint. It has been discovered that both natural and synthetic compounds can disrupt quorum sensing by a variety of means, such as jamming signal transduction, inhibition of signal production and break-down and trapping of signal compounds. We also focus on the regulatory elements that attenuate quorum sensing activities and discuss their unique properties. Understanding the biological roles of regulatory elements might be useful in developing inhibitor applications and understanding how quorum sensing is controlled.

AcrS/EnvR Represses Expression of the acrAB Multidrug Efflux Genes in Escherichia coli

The acrS regulatory gene is located upstream of the acrEF multidrug efflux system genes. However, the roles of AcrS in regulation of drug efflux pumps have not been clearly understood. Here we show that AcrS represses other multidrug efflux genes, acrAB, which encode a major efflux system in Escherichia coli.

Impact of the RNA chaperone Hfq on multidrug resistance in Escherichia coli

OBJECTIVES Hfq is a bacterial RNA chaperone involved in the post-transcriptional regulation of many stress-inducible genes via small non-coding RNAs. Although Hfq is related to important phenotypes including virulence in many bacterial pathogens, its role in drug resistance is unknown. The aim of this study was to investigate the role of Hfq in bacterial multidrug resistance. METHODS The hfq gene was inactivated in Escherichia coli by use of pKO3, which is a gene replacement vector. The drug susceptibility and drug accumulation of the hfq mutant were determined. The level of production of the AcrB multidrug efflux pump in this mutant was also measured. RESULTS The hfq mutant was susceptible to acriflavine, benzalkonium, cefamandole, chloramphenicol, Crystal Violet, nalidixic acid, novobiocin, oxacillin and rhodamine 6G. E. coli cells were strongly stained with rhodamine 6G compared with the wild-type on deletion of hfq, indicating that Hfq affects the accumulation of the drug in bacterial cells. The deletion of the drug efflux gene acrB impairs the effect of hfq deletion on E. coli susceptibility. Furthermore, the level of AcrB protein production was reduced in the hfq mutant, whereas hfq deletion did not affect the promoter activity of the acrAB operon. CONCLUSIONS These results indicate that Hfq regulates the drug efflux system at the post-transcriptional level and reveals the previously uncharacterized role of Hfq in bacterial multidrug resistance.

Activity of the Rhodopseudomonas palustris p-Coumaroyl-Homoserine Lactone-Responsive Transcription Factor RpaR

The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorum-sensing signal p-coumaroyl-homoserine lactone (pC-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by pC-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His(6)-RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although pC-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is pC-HSL-RpaR-activated antisense transcription of rpaR. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon.

Anaerobic p-Coumarate Degradation by Rhodopseudomonas palustris and Identification of CouR, a MarR Repressor Protein That Binds p-Coumaroyl Coenzyme A

The phenylpropanoid p-coumarate and structurally related aromatic compounds are produced in large amounts by green plants and are excellent carbon sources for many soil bacteria. Aerobic bacteria remove the acyl side chain from phenylpropanoids to leave an aromatic aldehyde, which then enters one of several possible central pathways of benzene ring degradation. We investigated the pathway for the anaerobic degradation of p-coumarate by the phototrophic bacterium Rhodopseudomonas palustris and found that it also follows this metabolic logic. We characterized enzymes for the conversion of p-coumarate to p-hydroxybenzaldehyde and acetyl coenzyme A (acetyl-CoA) encoded by the couAB operon. We also identified a MarR family transcriptional regulator that we named CouR. A couR mutant had elevated couAB expression. In addition, His-tagged CouR bound with high affinity to a DNA fragment encompassing the couAB promoter region, and binding was abrogated by the addition of nanomolar quantities of p-coumaroyl-CoA but not by p-coumarate. Footprinting demonstrated binding of CouR to an inverted repeat sequence that overlaps the -10 region of the couAB promoter. Our results provide evidence for binding of a CoA-modified aromatic compound by a MarR family member. Although the MarR family is widely distributed in bacteria and archaea and includes over 12,000 members, ligands have been identified for relatively few family members. Here we provide biochemical evidence for a new category of MarR ligand.