4-Chlorobenzoate uptake in Comamonas sp. strain DJ-12 is mediated by a tripartite ATP-independent periplasmic transporter

Abstract

The fcb gene cluster involved in the hydrolytic dehalogenation of 4-chlorobenzoate is organized in the order fcbB-fcbA-fcbT1-fcbT2-fcbT3-fcbC in Comamonas sp. strain DJ-12. The genes are operonic and inducible with 4-chloro-, 4-iodo-, and 4-bromobenzoate. The fcbT1, fcbT2, and fcbT3 genes encode a transporter in the secondary TRAP (tripartite ATP-independent periplasmic) family. An fcbT1T2T3 knockout mutant shows a much slower growth rate on 4-chlorobenzoate compared to the wild type. 4-Chlorobenzoate is transported into the wild-type strain five times faster than into the fcbT1T2T3 knockout mutant. Transport of 4-chlorobenzoate shows significant inhibition by 4-bromo-, 4-iodo-, and 4-fluorobenzoate and mild inhibition by 3-chlorobenzoate, 2-chlorobenzoate, 4-hydroxybenzoate, 3-hydroxybenzoate, and benzoate. Uptake of 4-chlorobenzoate is significantly inhibited by ionophores which collapse the proton motive force.

FIG. 1.

Transcriptional analysis indicating inducible (A) and operonic (B) expression of the fcb gene cluster in Comamonas sp. strain DJ-12. Lanes 1 and 2 (A) show RT-PCR results amplified with total RNA extracted from cells grown on 10 mM succinate or 1 mM 4CBA, respectively. Lanes 3 and 4 (B) indicate RT-PCR products obtained using RNA prepared from cells induced with 1 mM 4CBA. fcbA, -B, and -C encode 4CBA-CoA ligase, 4CBA-CoA dehalogenase, and 4HBA-CoA thioesterase, respectively. fcbT1, -T2, and -T3 represent putative transporter genes. S, 1-kb DNA ladder.

FIG. 2.

Expression of the fcb gene operon in response to benzoate derivatives. The enzyme activities are the average of triplicate experiments. Cells were grown on succinate and exposed to one of the following inducing compounds: 4CBA, 4-chlorobenzoate; 4IBA, 4-iodobenzoate; 4BrBA, 4-bromobenzoate; 4FBA, 4-fluorobenzoate; 3CBA, 3-chlorobenzoate; 2CBA, 2-chlorobenzoate; BA, benzoate; 4HBA, 4-hydroxybenzoate.

FIG. 3.

Growth of Comamonas sp. strain DJ-12 (open circle) and MUT1 (open square) in minimal medium with 4-chlorobenzoate (A) and 4-hydroxybenzoate (B) as the sole carbon source. Optical density was measured at 600 nm.

FIG. 4.

Uptake of 4-chlorobenzoate by induced cells of Comamonas sp. strain DJ-12. (A) The curve shows a nonlinear regression fit of data to the Michaelis-Menten equation. (B) The uptake assay was performed with 4CBA-induced cells of Comamonas sp. strain DJ-12 (closed circle) and MUT1 (open circle) and uninduced DJ-12 (closed triangle). Each point in panels A and B is the average value from triplicate experiments. Error bars indicate standard deviation.

FIG. 5.

Effect of energetic inhibitors on 4-chlorobenzoate uptake in Comamonas sp. strain DJ-12. Van, sodium orthovanadate.