Table of Contents:
In its natural habitat the soil bacterium Bacillus subtilis is exposed to various stressfactors. To protect itself against osmotic stress it accumulates compatible solutes in the cell. Glycine betaine is a powerful osmostress protectant for B. subtilis. It can either be imported or synthesized from the precursor choline. The OpuB and OpuC ABC-transporters both serve for choline uptake, while OpuC is promiscuous and imports also glycine betaine and a wide range of other compatible solutes. The oxidation of choline takes place via the two dehydrogenases GbsB and GbsA. The encoding genes are organized in the genome of B. subtilis as an operon. Next to the operon the gene gbsR is located. Expression of the opuB operon, along with the glycine betaine synthesis genes (gbsAB) is controlled by GbsR, a MarR-type transcriptional repressor, with choline serving as the inducer.
The two closely related ABC-type osmoprotectant uptake systems OpuB and OpuC are indispensable for acquiring a variety of compatible solutes under osmotic stress conditions in B. subtilis but possess strikingly different substrate specificities. Whereas the substrate-binding protein OpuCC recognizes a broad spectrum of compatible solutes, its 70% sequence-identical paralogue OpuBC only binds choline. In this study the OpuBC substrate binding protein of the OpuB system was substituted with OpuCC in order to analyze the functionality and substrate specificity of the hybrid ABC-transporter. The substrate profile of the narrow substrate-specific transporter OpuB can be synthetically broadened to that of the OpuC transporter by recombinantly implanting the gene for the OpuCC substrate binding protein into the opuB operon.
Molecular analysis of the difference in substrate profile has been conducted by ectopically expressing the gene for the OpuCC substrate-binding protein in a strain possessing only an intact OpuB transporter. This genetic manipulation led to the imposition of the substrate profile of the OpuC system onto OpuB. Guided by the crystal structures of the OpuBC and OpuCC substrate-binding proteins, the focus was set on residue 74 (an Asp residue in OpuBC, and a Thr residue in OpuCC) as it provides stabilizing interactions with the corresponding ligands and is critically for the stable closure of the two lobes of the binding protein upon substrate entrapment. In this study it is shown that an Asp to Thr substitution in OpuBC is sufficient to endow OpuB with the promiscuous substrate profile of OpuC. Crystal structures of the mutant OpuBC protein in complex with choline, glycine betaine, carnitine, or dimethylsulfoniopropionate (DMSP) provided detailed insight into the architecture of the ligand-binding site.
The hybrid ABC transporter OpuB::OpuCC possessed a high affinity for their substrates but the transport capacity was moderate. Suppressor mutants leading to single amino acid substitutions in the GbsR repressor controlling opuB expression greatly improved OpuB::OpuCC-mediated osmolyte transport through upregulation of opuB transcription. Each of the studied 54 mutants harbored alterations in gbsR. In most strains, gbsR was inactivated by frame-shift mutations or stop-codons. However, nine gbsR variants contained interesting mutations leading to single amino-acid substitutions. These occurred either in (i) the presumed choline binding pocket, (ii) the DNA-reading head, or (iii) in a flexible linker region connecting the winged HTH-motif with the dimerization domain of GbsR. DNA-binding studies with representatives of these three classes revealed that the corresponding GbsR variants were all defective in DNA-binding.
In addition, a new member of the Opu family of transporters within the genus Bacillus was found and characterized, OpuF. It is a representative of a sub-group of ABC transporters in which the substrate-binding protein is fused to the trans-membrane domain. A common property of the examined OpuF systems is their substrate profile; OpuF allows the import of glycine betaine, proline betaine, homobetaine and dimethylsulfoniopropionate (DMSP). An in silico model of the substrate-binding protein domain was established. It revealed the presence of an aromatic cage, a structural feature characteristic for ligand-binding sites present in components of compatible solute importers.