Two evolutionarily closely related ABC transporters mediate the uptake of choline for synthesis of the osmoprotectant glycine betaine in Bacillus subtilis

Biosynthesis of the compatible solute glycine betaine in Bacillus subtilis confers a considerable degree of osmotic tolerance and proceeds via a two-s tep oxidation process of choline, with glycine betaine aldehyde as the inte rmediate. We have exploited the sensitivity of B. subtilis strains defectiv e in glycine betaine production against glycine betaine aldehyde to select for mutants resistant to this toxic intermediate. These strains were also d efective in choline uptake, and genetic analysis proved that two mutations affecting different genetic loci (opuB and opuC) were required for these ph enotypes. Molecular analysis allowed us to demonstrate that the opuB and op uC operons each encode a binding protein-dependent ABC transport system tha t consists of four components. The presumed binding proteins of both ABC tr ansporters were shown to be lipoproteins. Kinetic analysis of [C-14]-cholin e uptake via OpuB (K-m= 1 mu M; V-max = 21 nmol min(-1) mg(-1) protein) and OpuC (K-m = 38 mu M; V-max = 75 nmol min(-1) mg(-1) protein) revealed that each of these ABC transporters exhibits high affinity and substantial tran sport capacity. Western blotting experiments with a polyclonal antiserum cr oss-reacting with the presumed substrate-binding proteins from both the Opu B and OpuC transporter suggested that the expression of the opuB and opuC o perons is regulated in response to increasing osmolality of the growth medi um. Primer extension analysis confirmed the osmotic control of opuB and all owed the identification of the promoter of this operon. The opuB and opuC o perons are located close to each other on the B. subtilis chromosome, and t heir high sequence Identity strongly suggests that these systems have evolv ed from a duplication event of a primordial gene cluster. Despite the close relatedness of OpuB and OpuC, these systems exhibit a striking difference in substrate specificity for osmoprotectants that would not have been predi cted readily for such closely related ABC transporters.