Characterization of the Vibrio cholerae vceCAB Multiple-Drug Resistance Efflux Operon in Escherichia coli

Abstract

Herein, we identify vceC as a component of a vceCAB operon, which codes for the Vibrio cholerae VceAB multiple-drug resistance (MDR) efflux pump, and vceR, which codes for a transcriptional autoregulatory protein that negatively regulates the expression of the vceCAB operon and is modulated by some of the substrates of this MDR efflux pump.MDR efflux pumps contribute to the intrinsic and acquired antibiotic resistance in bacteria and significantly impact the emergence of drug-resistant pathogens (9,10,16,18,24,25). These efflux systems can be amplified in resistant cells (9,11,17) and can alter their substrate profiles with mutation (6, 21), making them a major threat to the successful application of antibiotic therapy.In an earlier study, we cloned a 6.6-kb DNA fragment from Vibrio cholerae (pVC2) that encoded an MDR efflux pump that functioned in an Escherichia coli tolC mutant (3). In that study, we identified two genes, vceA and vceB, that code for a membrane fusion protein (MFP) and a cytoplasmic membrane translocase (CMT), respectively. VceB belongs to the major facilitator superfamily of proton antiporters, and the VceAB pump components share homology with the E. coli EmrAB efflux MDR pump. The substrates of this pump included hydrophobic agents, such as deoxycholate (DOC), antibiotics, such as chloramphenicol and nalidixic acid, and the proton motive force uncoupler, cyanide carbonyl m-chlorophenylhydrazone (CCCP). However, we did not identify the outer membrane efflux (channel) protein (OEP) of this MDR system. In this report, we identify VceC (4) as the OEP of this pump and demonstrate that its gene resides in an operon with vceAB (i.e., vceCAB). We also identify VceR, a transcriptional autoregulatory protein that negatively regulates the expression of the vceCAB operon and whose function is modulated by some of the substrates of this MDR system.Identification of the OEP component of vceAB MDR pump. Examination of the V. cholerae El Tor N16961 genome sequence (The Institute for Genomic Research) revealed an open reading frame (ORF) designated VC1409 (accession no. Q9KS51) which lies just upstream of vceA and codes for a putative OEP. To determine if VC1409 is essential for the functioning of the VceAB pump we examined the ability of this ORF (pVC91) to complement vceAB (pVC4) for the production of a functional VceAB pump in a tolC mutant. pVC4 has previously been described (3) and pVC91 contains the V. cholerae DNA fragment of pVC9 (3) cloned into a plasmid (pACYC184) which is compatible with pBR322 (i.e., pVC4). As expected, neither pVC4 nor pVC91 transformants could provide a tolC mutant with a functional VceAB pump. However, double transformants carrying both pVC4 and pVC91 conferred resistance to CCCP and DOC but not sodium dodecyl sulfate (SDS) or novobiocin. This substrate specificity is consistent with that of the VceAB MDR efflux pump (3) and indicates that pVC91 codes for a transacting factor(s) necessary for the functioning of VceAB. It also demonstrates that the OEP of this pump cannot repla...

Isolation and Characterization of VceC Gain-of-Function Mutants That Can Function with the AcrAB Multiple-Drug-Resistant Efflux Pump of Escherichia coli

“…To determine if VceC can replace TolC in the functioning of MDR efflux pumps which remove CCCP, NOV, or DOC, we transformed a tolC mutant (LBB1175) (13) with a plasmid carrying vceC (pVC91) (43) and examined the transformant's sensitivity to these inhibitors. As can be seen from Table 3, the presence of VceC does not increase resistance to these inhibitors or to any other inhibitors we have examined, including nalidixic acid, erythromycin, acriflavin, sodium dodecyl sulfate, and bile salts, in a tolC mutant (data not shown).…”

“…IP was performed as described by Werner et al (42) with the following modifications. VceC-associated DSP cross-linked proteins were immunoprecipitated with anti-VceC polyclonal antibody (43) and affinity purified by TrueBlot anti-rabbit immunoglobulin (Ig) IP beads (eBioscience [www.ebioscience.com]). Washed IP beads were resuspended in 60 l of Laemmli sample buffer containing 0.1% mercaptoethanol and boiled for 5 min.…”

“…However, detailed interactions between these different components, their stoichiometry, and the means by which they remove substrates from the cell are just beginning to be deciphered. Recently, the crystal structure of VceC, an OEP for the Vibrio cholerae VceAB pump (7,43), has been solved (11), and its architecture has been shown to be very similar to that of TolC (19) and OprM (3). In fact, despite a very low degree of amino acid sequence identity, these three OEP structures can be readily superimposed upon one another (11).…”

The Sphingomonas Plasmid pCAR3 Is Involved in Complete Mineralization of Carbazole

“…The products of ORF69, ORF68, and ORF67 showed 40, 54, and 53% identities with the tripartite drug efflux pumps composed of VceCAB isolated from the pathogen Vibrio cholerae (11,15,69). Vce-CAB can complement the multidrug resistance phenotype in E. coli null mutants, and the complemented strain shows resistance against the uncoupler cyanide m-chlorophenylhydrazone, the detergent sodium deoxycholate, phenylmercuric acetate, and several antibiotics such as chloramphenicol (Cm), nalixidic acid (Nal), erythromycin, and Rif (11,15,69). To assess whether the products of these genes would be functional, we compared resistance of KA1 and KA1W to the antibiotics Cm, Nal, and Rif.…”

Involvement of the SmeAB Multidrug Efflux Pump in Resistance to Plant Antimicrobials and Contribution to Nodulation Competitiveness in Sinorhizobium meliloti

“…Although several InterPro signatures for MFS drug transporters (IPR001411, IPR001958, IPR004734, IPR004751, and IPR004821) have been described, the sequence signature specific for multicomponent-type MFS drug transporters is not yet known. In this study, the occurrence of MSF and MFP genes in tandem organization was adopted as a criterion for selecting multicomponent-type MFS drug transporters, because all known genes encoding multicomponent-type MFS transporters are accompanied by MFP genes (19,28,32,34,63). A BLAST search with R. etli RmrB (19) returned 22 sequences with BLAST E values of less than 1.0.…”