Chimeric analysis of AcrA function reveals the importance of its C-terminal domain in its interaction with the AcrB multidrug efflux pump

Abstract

AcrAB-TolC is the major, constitutively expressed efflux protein complex that provides resistance to a variety of antimicrobial agents in Escherichia coli. Previous studies showed that AcrA, a periplasmic protein of the membrane fusion protein family, could function with at least two other resistance-nodulation-division family pumps, AcrD and AcrF, in addition to its cognate partner, AcrB. We found that, among other E. coli resistance-nodulation-division pumps, YhiV, but not MdtB or MdtC, could also function with AcrA. When AcrB was assessed for the capacity to function with AcrA homologs, only AcrE, but not YhiU or MdtA, could complement an AcrA deficiency. Since AcrA could, but YhiU could not, function with AcrB, we engineered a series of chimeric mutants of these proteins in order to determine the domain(s) of AcrA that is required for its support of AcrB function. The 290-residue N-terminal segment of the 398-residue protein AcrA could be replaced with a sequence coding for the corresponding region of YhiU, but replacement of the region between residues 290 and 357 produced a protein incapable of functioning with AcrB. In contrast, the replacement of residues 357 through 397 of AcrA still produced a functional protein. We conclude that a small region of AcrA close to, but not at, its C terminus is involved in the interaction with its cognate pump protein, AcrB.

FIG. 1.

Overexpression of E. coli MFPs in HNCE3 cells induced with 0.1 mM IPTG. Crude membrane extracts were separated in an SDS-10% polyacrylamide gel stained with Coomassie blue.

FIG. 2.

Expression of RND proteins from the genome of E. coli in HNCE1a (lanes a) and HNCE1b (lanes b) cells induced with 0.1 mM IPTG. Crude membrane extracts were prepared as described in Materials and Methods and separated in an SDS-7.5% polyacrylamide gel stained with Coomassie blue.

FIG. 3.

Construction of acrA-yhiU chimeras. Precise locations of the fusion junctions between the two MFP genes are indicated in residue numbers and as conserved sequence identities at each junction site. Each construct was assayed for expression (see Fig. 4) and function (see Table 5) in HNCE3 cells.

FIG. 4.

Expression of acrA-yhiU chimeric protein products in HNCE3 cells induced with 0.1 mM IPTG. Proteins in crude membrane fractions were separated and visualized as described in the legend to Fig. 1.