Dataset Information

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

ABSTRACT: We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

SUBMITTER: Chen C

PROVIDER: S-EPMC5503199 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

Chen Chiliang C Malek Adel A AA Wargo Matthew J MJ Hogan Deborah A DA Beattie Gwyn A GA

Molecular microbiology 20091117 1

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated b ...[more]

PMID: 19919675

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Project description:Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport.IMPORTANCEVibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.

Project description:Multiple transporters mediate osmoregulatory solute accumulation in Escherichia coli K-12. The larger genomes of naturally occurring strains such as pyelonephritis isolates CFT073 and HU734 may encode additional osmoregulatory systems. CFT073 is more osmotolerant than HU734 in the absence of organic osmoprotectants, yet both strains grew in high osmolality medium at low K(+) (micromolar concentrations) and retained locus trkH, which encodes an osmoregulatory K(+) transporter. Both lacked the trkH homologue trkG. Transporters ProP and ProU account for all glycine-betaine uptake activity in E. coli K-12 and CFT073, but not in HU734, yet elimination of ProP and ProU impairs the growth of HU734, but not CFT073, in high osmolality human urine. No known osmoprotectant stimulated the growth of CFT073 in high osmolality minimal medium, but putative transporters YhjE, YiaMNO, and YehWXYZ may mediate uptake of additional osmoprotectants. Gene betU was isolated from HU734 by functional complementation and shown to encode a betaine uptake system that belongs to the betaine-choline-carnitine transporter family. The incidence of trkG and betU within the ECOR collection, representatives of the E. coli pathotypes (PATH), and additional strains associated with urinary tract infection (UTI) were determined. Gene trkG was present in 66% of the ECOR collection but only in 16% of the PATH and UTI collections. Gene betU was more frequently detected in ECOR groups B2 and D (50% of isolates) than in groups A, B1, and E (20%), but it was similar in overall incidence in the ECOR collection and in the combined UTI and PATH collections (32 and 34%, respectively). Genes trkG and betU may have been acquired by lateral gene transfer, since trkG is part of the rac prophage and betU is flanked by putative insertion sequences. Thus, BetU and TrkG contribute, with other systems, to the osmoregulatory capacity of the species E. coli, but they are not characteristic of a particular phylogenetic group or pathotype.

Dataset Information

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

Publications

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

Similar Datasets

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