Expression Of acrAB Research Articles

Antibiotics do not induce expression of acrAB directly but via a RamA-dependent pathway.

The aim of this study was to determine if acrAB induction in Salmonella Typhimurium relies solely on RamA or if other transcriptional activator pathways are also involved, and to better understand the kinetics of induction of both acrAB and ramA. We evaluated the expression of acrAB in S. Typhimurium in response to a variety of compounds that are known to induce the expression of one or more of the transcriptional activators, MarA, SoxS, RamA, and Rob. We utilized green fluorescent protein (GFP) transcriptional reporter fusions to investigate the changes in the expression of acrAB, ramA, marA, and soxS following exposure to sub-inhibitory concentrations of antimicrobial compounds. Of the compounds tested, 13 induce acrAB expression in S. Typhimurium via RamA, MarA, SoxS, and Rob-dependent pathways. None of the tested antibiotics induced acrAB expression, and compounds that induced acrAB expression also induced a general stress response. The results from this study show that the majority of compounds tested induced acrAB via the RamA-dependent pathway. However, none of the antibiotic substrates of the AcrB efflux pump directly increased the expression of AcrAB either directly or indirectly via the induction of one of the transcriptional activators. Using a dual GFP/RFP reporter, we investigated the kinetics of the induction of ramA and acrAB simultaneously and found that acrAB gene expression was transient compared to ramA gene expression. ramA gene expression increased with time and would remain high or decrease slowly over the course of the experiment indicating that RamA exerts a wider global effect and is not limited to efflux regulation alone.

AcrAB efflux pump impacts on the survival of adherent-invasive Escherichia coli strain LF82 inside macrophages

The tripartite complex AcrAB-TolC is the major RND pump in Escherichia coli and other Enterobacteriaceae. It consists of the AcrB transporter, which is embedded in the inner membrane, the AcrA adapter located in the periplasm, and the channel protein TolC responsible for the transport of substrates towards the extracellular environment. Besides conferring resistance to many classes of antibiotics, AcrAB plays a role in the pathogenesis and virulence of several bacterial pathogens. Here we report that the AcrAB pump heavily affects the infection process of the LF82 strain, the prototype of Adherent-Invasive Escherichia coli (AIEC) which are highly abundant in the ileal mucosa of Chron disease patients. We found that the deletion of genes encoding AcrA and/or AcrB leads to decreased survival of LF82 within macrophages. Ectopic AcrAB expression in a acrAB defective mutant restores the wild type condition. Furthermore, we demonstrate that inhibition of AcrB and replacement of the transporter with an unfunctional AcrB also interfere with bacterial viability inside macrophages. Overall, these data suggest a pivotal role of the AcrAB efflux pump in bacteria-host cell interactions also in AIEC.

AcrAB, the major RND-type efflux pump of Photorhabdus laumondii, confers intrinsic multidrug-resistance and contributes to virulence in insects.

The resistance-nodulation-division (RND)-type efflux pumps AcrAB and MdtABC contribute to multidrug-resistance (MDR) in Gram-negative bacteria. Photorhabdus is a symbiotic bacterium of soil nematodes that also produces virulence factors killing insects by septicaemia. We previously showed that mdtA deletion in Photorhabdus laumondii TT01 resulted in no detrimental phenotypes. Here, we investigated the roles of the last two putative RND transporters in TT01 genome, AcrAB and AcrAB-like (Plu0759-Plu0758). Only ΔacrA and ΔmdtAΔacrA mutants were multidrug sensitive, even to triphenyltetrazolium chloride and bromothymol blue used for Photorhabdus isolation from nematodes on the nutrient bromothymol blue-triphenyltetrazolium chloride agar (NBTA) medium. Both mutants also displayed slightly attenuated virulence after injection into Spodoptera littoralis. Transcriptional analysis revealed intermediate levels of acrAB expression in vitro, in vivo and post-mortem, whereas its putative transcriptional repressor acrR was weakly expressed. Yet, plasmid-mediated acrR overexpression did not decrease acrAB transcript levels neither MDR in TT01 WT. While no pertinent mutations were detected in acrR of the same P. laumondii strain grown either on NBTA or nutrient agar, we suggest that AcrR-mediated repression of acrAB is not physiologically required under conditions tested. Finally, we propose that AcrAB is the primary RND-efflux pump, which is essential for MDR in Photorhabdus and may confer adaptive advantages during insect infection.

Adaptation of Salmonella enterica to bile: essential role of AcrAB-mediated efflux.

Adaptation to bile is the ability to endure the lethal effects of bile salts after growth on sublethal concentrations. Surveys of adaptation to bile in Salmonella enterica ser. Tyhimurium reveal that active efflux is essential for adaptation while other bacterial functions involved in bile resistance are not. Among S. enterica mutants lacking one or more efflux systems, only strains lacking AcrAB are unable to adapt, thus revealing an essential role for AcrAB. Transcription of the acrAB operon is upregulated in the presence of a sublethal concentration of sodium deoxycholate (DOC) while other efflux loci are either weakly upregulated or irresponsive. Upregulation of acrAB transcription is strong during exponential growth, and weak in stationary cultures. Single cell analysis of ethidium bromide accumulation indicates that DOC-induced AcrAB-mediated efflux occurs in both exponential and stationary cultures. Upregulation of acrAB expression may thus be crucial at early stages of adaptation, while sustained AcrAB activity may be sufficient to confer bile resistance in nondividing cells.

RamR is not involved in the regulation of ramA associated antibiotic resistance in Salmonella enterica serovar Typhi

RamA, a global transcriptional activator, belongs to the AraC/XylS family of regulatory proteins and can regulate multidrug resistance through activating the expression of AcrAB. In Salmonella spp., Klebsiella pneumonia, Enterobacter cloacae and Enterobacter aerogenes, RamR represses the transcription of gene ramA through binding to the upstream sequences of ramA. In this study, we found that the locus and transcription directions of ramA-ramR in S. Typhi GIFU10007 are different from that in S. Typhimurium (SL1344). To study the role of RamR involved in regulation of ramA in S. Typhi, we constructed ramA over-expression strain and ramR deletion mutant, and detected the expression level of ramA, and measured the growth curve of these strain in the presence of ampicillin. The results showed that RamR in S. Typhi neither repressed the expression of ramA, nor affected the bacterial resistance to ampicillin. In summary, RamR is not the repressor of RamA in S. Typhi, which is different from its role in other bacteria, such as S. Typhimurium and K. pneumoniae.

In vivo development of tigecycline resistance in Klebsiella pneumoniae owing to deletion of the ramR ribosomal binding site

Tigecycline resistance is emerging among Klebsiella pneumoniae, but knowledge regarding in vivo development of tigecycline resistance is limited. Here we report a new mechanism of tigecycline resistance in K. pneumoniae that evolved during tigecycline therapy. Klebsiella pneumoniae isolates were consecutively obtained from urine samples of a patient with scrotal abscess and urinary tract infection before and during tigecycline treatment. Two tigecycline-resistant K. pneumoniae strains (KP-3R and KP-4R; MIC = 8 µg/mL) were isolated after 41 days and 47 days of tigecycline therapy. These isolates had the same sequence type (ST11) and PFGE patterns as tigecycline-susceptible strains (KP-1S and KP-2S; MIC = 2 µg/mL) initially isolated from the patient. Compared with KP-1S and KP-2S, KP-3R and KP-4R exhibited higher expression of efflux pump AcrAB. Sequence comparison of the repressor gene ramR did not find any mutation within the open-reading frame that exist frequently in tigecycline-resistant K. pneumoniae. Instead, a 12-bp deletion of ramR upstream region including the ribosomal binding site (RBS) TGAGG was observed in KP-3R and KP-4R. qRT-PCR and immunoblotting analyses showed that KP-3R and KP-4R did not have impaired ramR transcription but had abolished RamR protein production. Furthermore, xylE reporter assay demonstrated that KP-3R and KP-4R had a defect in RamR translation caused by the 12-bp deletion. Complementing KP-3R and KP-4R with functional ramR suppressed expression of acrAB and consequently restored tigecycline susceptibility. This is the first report identifying deletion of the ramR RBS as a mechanism of in vivo tigecycline resistance in K. pneumoniae developing during tigecycline therapy.

Coordinated Expression of acrAB-tolC and Eight Other Functional Efflux Pumps Through Activating ramA and marA in Salmonella enterica serovar Typhimurium.

The aim of this study was to determine the expression of eight other functional transporter genes upon acrAB inactivation and also the expression of acrAB when the function of eight other transporters are impaired in Salmonella enterica. We used single- or multigene deletion mutants (i.e., ΔacrA, ΔacrB, ΔtolC, ΔacrAB, ΔacrEF, ΔacrD, ΔmdsABC, ΔmdtABC, ΔemrAB, ΔmacAB, ΔmdfA, ΔmdtK, ΔacrABramA, ΔacrABmarA, and ΔacrABsoxS) and real time (RT)-PCR to quantify the expression of different pump and regulator genes; infection ability was characterized by adhesion and invasion assays. The expression of acrAB operon was increased upon acrB inactivation. Single deletion of acrA or tolC also increased expression of acrB. The deletion of acrAB increased expression of eight other functional efflux pumps genes and vice versa, in which increased expression of ramA and marA was also detected. Mutants containing single deletions of functional pump genes were attenuated in cells. In conclusion, there is a feedback mechanism that coordinates regulation of AcrAB-TolC and eight other functional efflux pumps through the global transcriptional regulators ramA and marA in S. enterica serovar Typhimurium.

The Resistance Mechanism and Clonal Distribution of Tigecycline-Nonsusceptible Klebsiella pneumoniae Isolates in Korea.

PurposeTigecycline is one of the drugs used to treat multi-drug resistant Klebsiella pneumoniae (K. pneumoniae) infections, including complicated skin and soft tissue infections, complicated intra-abdominal infection, and community-acquired pneumonia in the Republic of Korea. However, since its commercial release, K. pneumoniae resistance against tigecycline has been reported, and there is a serious concern about the spread of tigecycline resistant bacteria.Materials and MethodsIn this study, we collected and analyzed 342 isolates from 23 hospitals in the Republic of Korea to determine the mechanisms of tigecycline susceptibility and their clonal types. The hospitals include several from each province in the Republic of Korea, except Jeju, an island province, and nonsusceptibility among the isolates was tested by the disk diffusion method. In our lab, susceptibility was checked again using the broth dilution method, and clonal types were determined using the multilocus sequence typing protocol. Real-time PCR was performed to measure the ramR mutation in the isolates nonsusceptible to tigecycline, which would suggest an increased expression of the AcrAB multidrug pump.ResultsFifty-six K. pneumoniae isolates were found to be nonsusceptible, 16% of the 342 collected. Twenty-seven and nine isolates of the tigecycline nonsusceptible isolates had mutations in the ramR and rpsJ genes, respectively; while 18 nonsusceptible isolates harbored the tetA gene. Comparison of isolates with and without ramR mutation showed a significant statistical difference (p<0.05) for expression of AcrAB. Moreover, the most common clonal types, as observed in our study, appear to be ST11 and ST789.ConclusionSeveral dominate clonal types infer tigecycline resistance to K. pneumoniae, including ST11, ST768, ST15, ST23, ST48, and ST307. There does not seem to be a transferrable medium, such as plasmid, for the resistance yet, although mutation of the ramR gene may be a common event, accounting for 48% of the nonsusceptibility in this study.

Expression of acrA and acrB Genes in Esherichia coli Mutants with or without marR or acrR Mutations.

The major antibiotic efflux pump of Esherichia coli is AcrAB-TolC. The first part of the pump, AcrAB, is encoded by acrAB operon. The expression of this operon can be kept elevated by overexpression of an activator, MarA following inactivation of MarR and AcrR repressors due to mutation in encoding genes, marR and acrR, respectively. The aims of this research were to use E. coli mutants with or without mutation in marR to search for the presence of possible mutation in acrR and to quantify the expression of acrAB. The DNA binding region of acrR gene in these mutants were amplified by PCR and sequenced. The relative expression of acrA and acrB were determined by real time PCR. RESULTS showed that W26 and C14 had the same mutation in acrR, but none of the mutants overexpressed acrA and acrB in comparison with wild type strain. The effect of marR or acrR mutation on acrAB overexpression is dependent on levels of resistance to tetracycline and ciprofloxacin.

Alternative explanation for indole-induced antibiotic tolerance in Salmonella

We were interested to read the recent publication by Vega et al. (1), which suggests that indole is an interspecies signal that causes Salmonella to become less susceptible to antibiotics due to activation of the oxidative stress response. However, we believe that there is an alternative explanation for their data: the drug tolerance phenotype is due to increased production of one or more multidrug efflux pumps. The drugs tested by Vega et al., ciprofloxacin and carbenicillin, are known substrates of the AcrB transporter, and indole has been shown to induce the production of efflux pumps in both Escherichia coli and Salmonella (2). In Salmonella, induction is mediated by increased expression of the transcriptional activator RamA, which regulates expression of acrAB (2). Therefore, the indole-induced drug tolerance seen by Vega et al. could result from induction of the multidrug resistance (MDR) AcrAB-TolC efflux system, leading to increased tolerance to these two drugs. This hypothesis is noted by the authors but dismissed after their RT-PCR experiments showed that expression of the ramA gene was reduced in the presence of indole. We are perplexed by this observation because it conflicts with all previous studies including measurements using gene reporter constructs, RT-PCR, microarray, and Western blotting (2), which reveal that ramA/RamA is induced by indole in numerous different Salmonella strains including strain LT2 used by Vega et al. in their experiments. Furthermore, the authors used these RT-PCR data as evidence that efflux is not involved in the phenotype. We believe that this is a mistaken assumption for two reasons. First, efflux was not measured. This is surprising because efflux of ciprofloxacin and other compounds can be measured easily using one of several published methods to quantify accumulation or efflux of fluorescent substrates (3, 4). Second, regulation of MDR efflux pumps in Gram-negative bacteria is complex and multifactorial. For instance, other transcription factors, including MarA, SoxS, and Rob, can also regulate expression of MDR efflux pumps, and indole induces expression of soxS in E. coli (5).

Regulation of acrAB expression by cellular metabolites in Escherichia coli

Multidrug efflux pumps mediate resistance to antibiotics and other toxic compounds. We studied the role of AcrAB-TolC, the main efflux pump in Escherichia coli, in regulating gene expression. Deletion mutants, an acrABp-lacZ fusion and reverse transcription-real-time quantitative PCR experiments were used to study the role of AcrAB-TolC and metabolism in regulating gene expression of the acrAB operon and its transcriptional regulators. Deletion of the acrB gene increased the expression of the acrAB operon. A similar induction of acrAB was found when acrA or tolC was deleted, and when the pump function was inhibited using phenylalanine-arginine-β-naphthylamide. The induction of acrAB in the ΔacrB strain was totally (AcrR or SoxS) or partially (SoxR or MarA) prevented when the genes for these acrAB regulators were also deleted. The expression of soxS and marA, but not of acrR, was increased in the ΔacrB strain, which also showed altered expression of many other genes related to different cellular processes, including motility. Deletion of the metabolic genes entA and entE (enterobactin biosysnthesis), glpX (gluconeogenesis), cysH (cysteine biosynthesis) and purA (purine biosynthesis) also prevented activation of the acrAB promoter in the ΔacrB strain. Addition of the enterobactin biosynthesis intermediate metabolite 2,3-dihydroxybenzoate induced the expression of acrAB. These results together suggest a model in which the AcrAB-TolC pump effluxes cellular metabolites that are toxic and/or have a signalling role. If the pump is inactivated or inhibited, these metabolites would accumulate, inactivating AcrR and/or up-regulating soxS and marA expression, ultimately triggering the up-regulation of acrAB expression to restore homeostasis.

Contribution of Insertion Within acrR to acrAB Expression and Ciprofloxacin Resistance in a Veterinary Escherichia coli Isolate

Contribution of Insertion Within acrR to acrAB Expression and Ciprofloxacin Resistance in a Veterinary Escherichia coli Isolate

Identification of Transport Proteins Involved in Free Fatty Acid Efflux in Escherichia coli

Escherichia coli has been used as a platform host for studying the production of free fatty acids (FFA) and other energy-dense compounds useful in biofuel applications. Most of the FFA produced by E. coli are found extracellularly. This finding suggests that a mechanism for transport across the cell envelope exists, yet knowledge of proteins that may be responsible for export remains incomplete. Production of FFA has been shown to cause cell lysis, induce stress responses, and impair basic physiological processes. These phenotypes could potentially be diminished if efflux rates were increased. Here, a total of 15 genes and operons were deleted and screened for their impact on cell viability and titer in FFA-producing E. coli. Deletions of acrAB and rob and, to a lower degree of statistical confidence, emrAB, mdtEF, and mdtABCD reduced multiple measures of viability, while deletion of tolC nearly abolished FFA production. An acrAB emrAB deletion strain exhibited greatly reduced FFA titers approaching the tolC deletion phenotype. Expression of efflux pumps on multicopy plasmids did not improve endogenous FFA production in an acrAB(+) strain, but plasmid-based expression of acrAB, mdtEF, and an mdtEF-tolC artificial operon improved the MIC of exogenously added decanoate for an acrAB mutant strain. The findings suggest that AcrAB-TolC is responsible for most of the FFA efflux in E. coli, with residual activity provided by other resistance-nodulation-cell division superfamily-type efflux pumps, including EmrAB-TolC and MdtEF-TolC. While the expression of these proteins on multicopy plasmids did not improve production over the basal level, their identification enables future engineering efforts.

In Vitro Selection of ramR and soxR Mutants Overexpressing Efflux Systems by Fluoroquinolones as Well as Cefoxitin in Klebsiella pneumoniae

The relationship between efflux system overexpression and cross-resistance to cefoxitin, quinolones, and chloramphenicol has recently been reported in Klebsiella pneumoniae. In 3 previously published clinical isolates and 17 in vitro mutants selected with cefoxitin or fluoroquinolones, mutations in the potential regulator genes of the AcrAB efflux pump (acrR, ramR, ramA, marR, marA, soxR, soxS, and rob) were searched, and their impacts on efflux-related antibiotic cross-resistance were assessed. All mutants but 1, and 2 clinical isolates, overexpressed acrB. No mutation was detected in the regulator genes studied among the clinical isolates and 8 of the mutants. For the 9 remaining mutants, a mutation was found in the ramR gene in 8 of them and in the soxR gene in the last one, resulting in overexpression of ramA and soxS, respectively. Transformation of the ramR mutants and the soxR mutant with the wild-type ramR and soxR genes, respectively, abolished overexpression of acrB and ramA in the ramR mutants and of soxS in the soxR mutant, as well as antibiotic cross-resistance. Resistance due to efflux system overexpression was demonstrated for 4 new antibiotics: cefuroxime, cefotaxime, ceftazidime, and ertapenem. This study shows that the ramR and soxR genes control the expression of efflux systems in K. pneumoniae and suggests the existence of efflux pumps other than AcrAB and of other loci involved in the regulation of AcrAB expression.

Salicylate reduces the antimicrobial activity of ciprofloxacin against extracellular Salmonella enterica serovar Typhimurium, but not against Salmonella in macrophages

Salicylate, a potent inducer of the MarA activator in Salmonella enterica, is the principal metabolite of aspirin, which is often consumed for medicinal and cosmetic uses. Our research was aimed at testing if salicylate activates the mar regulon in macrophage-associated Salmonella (intracellular bacteria), and investigating its effects on bacterial susceptibility to ciprofloxacin extracellularly and intracellularly. J774 macrophages were infected with S. enterica serovar Typhimurium (wild-type and marA null mutant), treated with ciprofloxacin with and without pre-exposure to salicylate, and the surviving bacteria were counted. Similar experiments were conducted with bacteria in broth (extracellular bacteria). Phe-Arg-beta-naphthylamide (PAbetaN) was added to investigate the role of efflux pumps in resistance. The transcriptional regulation of marRAB, acrAB and micF in extracellular and intracellular Salmonella Typhimurium with and without salicylate and ciprofloxacin was investigated using green fluorescent protein as a marker protein and quantitative real time PCR. Pre-exposure of Salmonella to salicylate increased the resistance of extracellular but not intracellular bacteria to ciprofloxacin, although salicylate stimulated the expression of mar genes in intracellular and extracellular bacteria. Using marA mutants and the inhibitor PAbetaN, we showed that the improved resistance in extracellular bacteria is derived from the induction of acrAB by salicylate, which is mediated by MarA. In intracellular bacteria, the expression of acrAB is already higher when compared with extracellular cells; therefore, salicylate does not result in significant acrAB induction intracellularly and subsequent resistance enhancement. Results show that conclusions raised from extracellular studies cannot be applied to intracellular bacteria, although the systems have similar functions.

Emergence of AcrAB-mediated tigecycline resistance in a clinical isolate of Enterobacter cloacae during ciprofloxacin treatment

Tigecycline resistance remains rare amongst Enterobacteriaceae in the UK, as elsewhere, but has been associated with upregulation of the AcrAB efflux system. Using isolates of an Enterobacter cloacae strain that developed tigecycline resistance in vivo during ciprofloxacin therapy as well as laboratory-selected mutants, we investigated the role of this pump and the global regulator RamA in tigecycline resistance. Laboratory mutants were selected from a susceptible clinical isolate in vitro by exposure to increasing concentrations of tigecycline. Expression of the acrAB operon and the ramA gene was monitored by real-time reverse-transcription polymerase chain reaction (RT-PCR). Overexpression of ramA was achieved using the pBAD expression vector, whilst insertional inactivation of acrB with a gentamicin resistance cassette was achieved with the bacteriophage λ Red recombination system. Increased tigecycline minimum inhibitory concentrations in the clinical isolate and a laboratory mutant were associated with increases in acrAB and ramA transcripts. Induction of increased ramA expression resulted in increased acrAB expression, whilst insertional inactivation of acrB restored full susceptibility to tigecycline. Treatment with ciprofloxacin, a substrate of AcrAB in E. cloacae, possibly selected for cross-resistance to tigecycline as a result of RamA-mediated AcrAB upregulation.

Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli

BackgroundEfflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems.ResultsEtBr transport across the cell envelope of E. coli K-12 and derivatives was analysed by a semi-automated fluorometric method. Accumulation and efflux of EtBr was studied under limiting energy supply (absence of glucose and low temperature) and in the presence and absence of the efflux pump inhibitor, chlorpromazine. The bulk fluorescence variations were also observed by single-cell flow cytometry analysis, revealing that once inside the cells, leakage of EtBr does not occur and that efflux is mediated by active transport. The importance of AcrAB-TolC, the main efflux system of E. coli, in the extrusion of EtBr was evidenced by comparing strains with different levels of AcrAB expression. An experimental model was developed to describe the transport kinetics in the three strains. The model integrates passive entry (influx) and active efflux of EtBr, and discriminates different degrees of efflux between the studied strains that vary in the activity of their efflux systems, as evident from the calculated efflux rates: = 0.0173 ± 0.0057 min-1; = 0.0106 ± 0.0033 min-1; and = 0.0230 ± 0.0075 min-1.ConclusionThe combined use of a semi-automated fluorometric method and an experimental model allowed quantifying EtBr transport in E. coli strains that differ in their overall efflux activity. This methodology can be used for the early detection of differences in the drug efflux capacity in bacteria accounting for antibiotic resistance, as well as for expedite screening of new drug efflux inhibitors libraries and transport studies across the bacterial cell wall.

Identification and characterization of the AcrR/AcrAB system of a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is one of the leading marine pathogens that can infect a wide range of cultured marine species. In this study, the acrR-acrAB cluster was cloned from TX1, a pathogenic E. tarda strain isolated from diseased fish. AcrR and AcrAB were found to be involved in resistance against acriflavine and methyl viologen, which positively regulate the expression of acrAB. AcrR negatively regulates its own expression and the expression of the acrAB operon, most likely by interacting with a 24-bp operator site that overlaps the putative promoter of acrA (P(acrA)). The repressive effect of AcrR on P(acrA) could be relieved by acriflavine, methyl viologen, and ethidium bromide, the presence of each of which enhanced transcription from P(acrA). Interruption of the regulated expression of acrR by introducing into TX1 a plasmid that overexpresses acrR affected growth under stress conditions, AI-2 production, and bacterial virulence. In addition, mutational analyses identified a constitutively active AcrR mutant (named N215), which exhibits full repressor activity but is impaired in its ability to interact with the inducer. Overexpression of N215 produced the same kind of but moderately stronger effect on TX1 compared to that produced by overexpression of the wild-type acrR.

AcrAB Multidrug Efflux Pump Regulation in Salmonella enterica serovar Typhimurium by RamA in Response to Environmental Signals

Salmonella enterica serovar Typhimurium has at least nine multidrug efflux pumps. Among these pumps, AcrAB is effective in generating drug resistance and has wide substrate specificity. Here we report that indole, bile, and an Escherichia coli conditioned medium induced the AcrAB pump in Salmonella through a specific regulator, RamA. The RamA-binding sites were located in the upstream regions of acrAB and tolC. RamA was required for indole induction of acrAB. Other regulators of acrAB such as MarA, SoxS, Rob, SdiA, and AcrR did not contribute to acrAB induction by indole in Salmonella. Indole activated ramA transcription, and overproduction of RamA caused increased acrAB expression. In contrast, induction of ramA was not required for induction of acrAB by bile. Cholic acid binds to RamA, and we suggest that bile acts by altering pre-existing RamA. This points to two different AcrAB regulatory modes through RamA. Our results suggest that RamA controls the Salmonella AcrAB-TolC multidrug efflux system through dual regulatory modes in response to environmental signals.

Crystal Structure of the Transcriptional Regulator AcrR from Escherichia coli

The AcrAB multidrug efflux pump, which belongs to the resistance nodulation division (RND) family, recognizes and extrudes a wide range of antibiotics and chemotherapeutic agents and causes the intrinsic antibiotic resistance in Escherichia coli. The expression of AcrAB is controlled by the transcriptional regulator AcrR, whose open reading frame is located 141 bp upstream of the acrAB operon. To understand the structural basis of AcrR regulation, we have determined the crystal structure of AcrR to 2.55-Å resolution, revealing a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. Each monomer of AcrR forms a multientrance pocket of 350 Å3 in the ligand-binding domain. The ligand-binding pocket is surrounded with mostly hydrophobic residues. In addition, a completely buried negatively charged glutamate, expected to be critical for drug binding, is located at the center of the binding pocket. The crystal structure provides novel insight into the mechanisms of ligand binding and AcrR regulation.