From substrate specificity to promiscuity: hybrid ABC transporters for osmoprotectants

Abstract

The ABC-transporters OpuB and OpuC from Bacillus subtilis function as osmoprotectant import systems. Their structural genes have most likely evolved through a duplication event but the two transporters are remarkably different in their substrate profile. OpuB possesses narrow substrate specificity, while OpuC is promiscuous. We assessed the functionality of hybrids between these two ABC-transporters by reciprocally exchanging the coding regions for the OpuBC and OpuCC substrate-binding proteins between the corresponding opuB and opuC operons. Substantiating the critical role of the binding protein in setting the substrate specificity of ABC transporters, OpuB::OpuCC turned into a promiscuous system, while OpuC::OpuBC now exhibited narrow substrate specificity. Both hybrid transporters possessed a high affinity for their substrates but the transport capacity of the OpuB::OpuCC system was moderate due to the synthesis of only low amounts of the xenogenetic OpuCC protein. Suppressor mutations causing single amino acid substitutions in the GbsR repressor controlling the choline to glycine betaine biosynthesis pathway greatly improved OpuB::OpuCC-mediated compatible solute import through transcriptional up-regulation of the hybrid opuB::opuCC operon. Collectively, we demonstrate for the first time that one can synthetically switch the substrate specificity of a given ABC transporter by combining its core components with a xenogenetic ligand-binding protein.

Arsenobetaine: an ecophysiologically important organoarsenical confers cytoprotection against osmotic stress and growth temperature extremes

“…These compounds were extracted with 80% ethanol from the cells and their intracellular pools were measured by LC‐ESI‐MS. Strain TMB118 was used as a control for the true import of glycin betaine, arsenobetaine, choline or arsenocholine as it lacks the OpuA, OpuB, OpuC and OpuD transporters (Teichmann et al ., ). The TMB118 control strain had apparent pool sizes of 18 mM glycine betaine, and 3 mM arsenobetaine respectively.…”

OpuF, a New Bacillus Compatible Solute ABC Transporter with a Substrate-Binding Protein Fused to the Transmembrane Domain

“…As a consequence, the amino acid sequences of their components are closely related, yet their substrate profiles are strikingly different. OpuB is a rather specific transporter (mainly for choline), while OpuC is highly promiscuous (46,53,82). We used the amino acid sequence of OpuCC, the SBP of the OpuC system (26,46,76,82), as a query to analyze the phylogenomic distribution of ABC transport systems in members of the genus Bacillus.…”

“…OpuB is a rather specific transporter (mainly for choline), while OpuC is highly promiscuous (46,53,82). We used the amino acid sequence of OpuCC, the SBP of the OpuC system (26,46,76,82), as a query to analyze the phylogenomic distribution of ABC transport systems in members of the genus Bacillus. For this database search, we relied on the Integrated Microbial Genomes & Microbiomes (IMG/M) comparative data analysis system of the U.S. Department of Energy (83).…”

“…This chassis strain was constructed by transformation of strain TMB118 (strain JH642 with ΔopuA::tet, ΔopuC::spc, ΔopuD::kan, and ΔopuB::ery mutations) (82) with chromosomal DNA of the B. subtilis strain CAB5 (strain 168 with a ΔgbsR::zeo mutation), thereby yielding strain LTB52, a derivative of TMB118 carrying the Δ(gbsR::zeo) gene disruption mutation ( Table 1). To obtain appropriate control strains for our experiments, we inserted the empty pX vector (99) and plasmid pChen3 carrying the B. subtilis opuC wild-type operon (82) into the genome of the LTB52 chassis strain, yielding strains HKB12 (amyE::pChen3) and HKB13 (amyE::pX), respectively (Table 1).…”

“…The glycine betaine concentration used in the experiments varied between 3 M and 120 M and was spiked with a 1 M concentration of the radiolabeled substrate. The details of this type of transport assay haven been reported (24,37,82).…”