The goals of this project are to characterize the multiple transport activities of the TetA(L) transporter from Bacillus subtilis, and to determine the regulatory mechanisms that increase tetA(L) expression in response to tetracycline (Tc), elevated concentrations of Na+ or K+, and elevated pH. Recently, we discovered that the B. subtilis tetA(L) gene, a homologue of Gram-positive plasmid tet genes, encodes the dominant monovalent cation/H+ antiporter of B. subtilis, which functions in Na+-resistance and pH homeostasis. TetA(L) is also a metal-Tc/H+ antiporter, and deletion of the tetA(L) gene increases sensitivity of the organism to Tc. TetA(L)'s function as a Na+(K+)/H+ antiporter that regulates cytoplasmic pH is apparently necessary for viability at pH 7.0 and above unless there is a second-site mutation that up-regulates expression of a compensatory antiporter. Transport studies indicate that there are distinct substrate domains for metal-Tc versus Na+. Initial studies of gene regulation provided evidence for distinct mechanisms of tetA(L) regulation that correlate with the physiology of the system. There are four specific aims. First, rigorous demonstration of the multifunctional nature of TetA(L) will be sought through purification and reconstitution of the protein whereupon its transport activities will be evaluated and characterized in the absence of other proteins Second, topological studies of TetA(L) will be conducted using gene fusions and immunological approaches These topological studies will serve as a prelude to the third specific aim, which is to employ several mutagenesis approaches to define substrate domains and possible regions of interaction in the protein. Fourth, specific mutations and gene fusions will be used to study the multiple modes of tetA(L) regulation, at least one of which apparently involves a global type regulator of both tetA(L) and at least one other antiporter-encoding gene. The proposed studies offer an unusual opportunity for developing new insights into fundamental problems of multidrug membrane transporters, the origins of antibiotic resistance determinants, and the regulatory mechanisms for coordinating challenges of pH, salt, and antibiotics in Gram-positive bacteria.
