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.

Fig. 1

Choline (A) and betaine (B) uptake by P. syringae pv. tomato DC3000 and mutants lacking one or more loci encoding the CbcXWV, OpuC and BetT transporters. The wild-type DC3000 and the strains GB1–GB4 and DCΔcbc (Table 1) were grown in the absence of choline or betaine, designated no inducer, or in the presence of betaine or choline (final concentration 10 mM), allowing these compounds to serve as inducers. As a catabolic product of choline, betaine may also serve as an inducer in the choline-amended cells. Uptake was measured using 10 μM [¹⁴C]choline (A) or [¹⁴C]betaine (B). Values are mean uptake rates + standard error of the mean (SE) (n = 3).

Fig. 2

Genomic arrangement of the cbcXWV loci and related loci in (A) P. aeruginosa PA14 and PAO1 and (B) P. syringae strain B728a. The following genes are shown: gbdR [transcriptional regulator influencing betaine catabolism (Wargo et al., 2008)], sdaB (L-serine dehydratase), cbcXWV (ABC transporter), cdhR [transcriptional regulator influencing carnitine catabolism (Wargo and Hogan, 2009)], caiX (carnitine-specific binding protein), cdhCAB [carnitine catabolic proteins (Wargo and Hogan, 2009)], betX (betaine-specific binding protein) and actP (acetate permease).

Fig. 3

Complementation of P. aeruginosa and P. syringae transporter-deficient mutants by plasmids expressing cbcXWV from various strains. A. Growth of PA14, PA14 containing insertions in individual cbc genes, and the PA14 mutants containing pME6041 or pME6041 expressing cbcXWV from PAO1. B. Uptake of radiolabelled betaine, choline or carnitine (1 mM) by DC3000, B728a, B728a with insertions in the putative SBP loci Psyr_2916 (B2916Ko) and Psyr_0028 (B0028Ko), and B728a lacking opuC, betT and cbc (BT). C. Uptake of radiolabelled choline and carnitine (1 mM) by strain BT containing the vector pME6041 or pME6041 expressing cbcXWV from DC3000, B728a or PAO1. Cells were grown with 20 mM choline (A) or with 10 mM choline and 10 mM betaine (B and C) as sole C sources and optical density at 600 nm (OD₆₀₀) was recorded after 24 h (with an initial OD₆₀₀ of 0.05). Values represent the mean + SE (n = 3).

Fig. 4

Complementation of uptake activity in P. syringae and P. aeruginosa strain derivatives by plasmids expressing Psyr_2916 (caiX) from P. syringae B728a. Uptake of radiolabelled betaine, choline and carnitine (1 mM) by derivatives of strain (A) B2916Ko, in which Psyr_2916 was insertionally inactivated, (B) PA14ΔcaiX, in which PA5388 was deleted, and (C) DC3000. Strain derivatives included those expressing only pME6041 or pME6041 containing caiX_B728a (pME2916) or caiX_B728a and the adjacent putative transcriptional regulator cdhR (pME2915-16) (Wargo and Hogan, 2009). Values are mean + SE (n = 3).

Fig. 5

Complementation of betaine, choline and carnitine uptake in the QAC transporter-deficient P. syringae strain BT by vectors expressing cbcWV and cbcXWV. Cells were grown in King’s B medium, washed with water, and re-suspended in ½-21C medium for the transport assay using radiolabelled substrates at a concentration of 10 μM. Values are mean + SE (n = 3).

Fig. 6

Model of the interacting components comprising the Cbc transporter of P. aeruginosa and P. syringae. (A) Diagrammatic model and (B) list of Cbc transporter components. This study has provided functional evidence for all of the components shown.