ISOLATION AND CHARACTERIZATION OF A PUTATIVE MULTIDRUG-RESISTANCE PUMP FROM VIBRIO-CHOLERAE

Multidrug-resistant strains of Vibrio cholerae (the causative agent of the diarrhoeal disease cholera) have recently been described. In an a ttempt to identify a homologue of the Escherichia coli TolC in V. chol erae, we isolated a DNA fragment (pVC) that enabled an E. coli tolC mu tant to grow in the presence of 0.05% deoxycholate (DOG). However, oth er TolC defects were not complemented. Nucleotide sequence analysis of this fragment revealed the presence of two open reading frames (ORF1 and ORF2) separated by 9 bp and encoding 42.4 and 55.8 kDa proteins re spectively. The translational products of these two ORFs correlated cl osely with the molecular weights of the predicted proteins. The deduce d amino acid sequences of ORF1 and ORF2 showed a high degree of simila rity with conserved regions of the E. coli efflux pump proteins, EmrA and EmrB. The presence of pVC2 within the E. coli efflux pump mutants defective in either the emrAB or the acrAB genes provided the mutants with resistance against several antibiotics. A V. cholerae isogenic mu tant defective in ORF2 was constructed by gene replacement. Characteri zation of this mutant has shown it to be more sensitive to CCCP, PMA, PCP, nalidixic acid and DOC than the parent strain. These results sugg est that ORF1 and ORF2 constitute an operon encoding two components of a putative multidrug resistance pump in V. cholerae. In addition, the presence of both structural and functional similarities between VceAB and EmrAB suggests that VceAB is a homologue of EmrAB.