VceC is the outer membrane component of the major facilitator (MF) VceAB-VceC multiple-drug-resistant (MDR) efflux pump of Vibrio cholerae. TolC is the outer membrane component of the resistance-nodulationdivision AcrAB-TolC efflux pump of Escherichia coli. Although these proteins share little amino acid sequence identity, their crystal structures can be readily superimposed upon one another. In this study, we have asked if TolC and VceC are interchangeable for the functioning of the AcrAB and VceAB pumps. We have found that TolC can replace VceC to form a functional VceAB-TolC MDR pump, but VceC cannot replace TolC to form a functional AcrAB-VceC pump. However, we have been able to isolate gain-of-function (gof) VceC mutants which can functionally interface with AcrAB. These mutations map to four different amino acids located at the periplasmic tip of VceC. Chemical cross-linkage experiments indicate that both wild-type and gof mutant VceC can physically interact with the AcrAB complex, suggesting that these gof mutations are not affecting the recruitment of VceC to the AcrAB complex but rather its ability to functionally interface with the AcrAB pump.

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...

We investigated the association between biomarkers of dermal exposure, naphthyl-keratin adducts (NKA), and urine naphthalene biomarker levels in 105 workers routinely exposed to jet-fuel. A moderate correlation was observed between NKA and urine naphthalene levels (p = 0.061). The NKA, post-exposure breath naphthalene, and male gender were associated with an increase, while CYP2E1*6 DD and GSTT1-plus (++/+−) genotypes were associated with a decrease in urine naphthalene level (p < 0.0001). The NKA show great promise as biomarkers for dermal exposure to naphthalene. Further studies are warranted to characterize the relationship between NKA, other exposure biomarkers, and/or biomarkers of biological effects due to naphthalene and/or PAH exposure.

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