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

Abstract

SummaryBiosynthesis of the compatible solute glycine betaine in Bacillus subtilis confers a considerable degree of osmotic tolerance and proceeds via a two-step oxidation process of choline, with glycine betaine aldehyde as the intermediate. We have exploited the sensitivity of B. subtilis strains defective in glycine betaine production against glycine betaine aldehyde to select for mutants resistant to this toxic intermediate. These strains were also defective in choline uptake, and genetic analysis proved that two mutations affecting different genetic loci (opuB and opuC ) were required for these phenotypes. Molecular analysis allowed us to demonstrate that the opuB and opuC operons each encode a binding protein-dependent ABC transport system that consists of four components. The presumed binding proteins of both ABC transporters were shown to be lipoproteins. Kinetic analysis of [ 14 C]-choline uptake via OpuB (K m 1 mM; V max 21 nmol min À1 mg À1 protein) and OpuC (K m 38 mM;V max 75 nmol min À1 mg À1 protein) revealed that each of these ABC transporters exhibits high af®nity and substantial transport capacity. Western blotting experiments with a polyclonal antiserum cross-reacting with the presumed substrate-binding proteins from both the OpuB and OpuC transporter suggested that the expression of the opuB and opuC operons is regulated in response to increasing osmolality of the growth medium. Primer extension analysis con®rmed the osmotic control of opuB and allowed the identi®cation of the promoter of this operon. The opuB and opuC operons are located close to each other on the B. subtilis chromosome, and their high sequence identity strongly suggests that these systems have evolved 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 speci®city for osmoprotectants that would not have been predicted readily for such closely related ABC transporters.

Variation in the Group BStreptococcusCsrRS Regulon and Effects on Pathogenicity

“…Of these, possible virulence genes include SAG_1135 and SAG_ 1137 that encode homologs of Gls24, a stress response protein shown to contribute to virulence in experimental enterococcal infection (18,28). A similar pattern of regulation was noted for SAG_1796 and SAG_1797, which are predicted to encode a glycine/betaine osmoregulation system implicated in adaptation to osmotic stress in other species (13,14,19,31). Differential CsrS-dependent regulation of these loci in the 2603 strain background may account for the unexpectedly high virulence of strain 2603⌬csrS.…”

“…Gls24 has been implicated in stress response and virulence in Enterococcus faecalis (18,28). The GBS CsrRS also regulates expression of two separate operons predicted to encode components of a glycine/betaine osmoregulation system (SAG_1796 to -1797 and SAG_0241 to -0244), a system that mediates adaptation to osmotic stress in Bacillus subtilis and Lactococcus lactis (13,14,19,31). Although their role in GBS is undefined, expression of two predicted transcriptional regulators is also controlled by CsrRS: SAG_0712 encoding a putative regulator of the OmpR family and SAG_0938 encoding a predicted GntR family transcriptional regulator (11,27).…”

T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis

“…A 2.75 kb SalI-EcoRI DNA fragment (9ykkE-proB-proA9) from pBKB26 was cloned into the SalI-and EcoRI-restricted pBluescriptSK 2 vector (Stratagene), thereby yielding plasmid pJS3. For the construction of the proBA gene disruption mutation, a HindIII and BclI fragment (224 bp) was deleted from plasmid pJS3 and replaced with a chloramphenicol-resistance cassette derived from plasmid pRMK59 (Kappes et al, 1999), resulting in plasmid pJS8. A proBA-treA reporter gene fusion was constructed as follows.…”

Crystal Structure of the Substrate-Binding Domain from Listeria monocytogenes Bile-Resistance Determinant BilE

“…The predicted functional transporter is a homodimer ( Figure 1B). Although the architecture of BilE is similar to Lactococcus lactis OpuA, the individual domains share more sequence similarity with the equivalent subunits from Listeria monocytogenes OpuC [30], Bacillus subtilis OpuB and OpuC [31], and Archaeoglobus fulgidus ProX [32,33] (Figure 1C). These are all Type I ABC importers that contribute to osmo-or thermotolerance through the import of quaternary ammonium compounds (QACs) which act as compatible solutes either directly (e.g., glycine betaine), or as metabolic precursors (e.g., choline).…”

“…The sequence of BilEB is most similar to AfProX (33% identity, 51% similarity), although one binding site tyrosine is substituted for a phenylalanine. Figure 2 shows a multiple sequence alignment of BilEB, AfProX, and other selected homologs in which these four tyrosine residues are conserved: OpuBC and OpuCC from Bacillus subtilis [31]; OpuCC from Listeria monocytogenes [30], Staphylococcus aureus [41], Pseudomonas aeruginosa and Pseudomonas syringae [42]; OsmX (OpuCC) from Salmonella typhimurium [43,44]; and YehZ from Escherichia coli [45]. The known ligands of these proteins include glycine betaine, proline betaine, choline, acetylcholine, choline-O-sulfate, carnitine, and ectoine ( Figure S3).…”