title: Existence of a robust haloacid transport system in a Burkholderia species bacterium.
abstract: Bacterium Burkholderia sp. MBA4 can utilize haloacids as the sole carbon and energy source for growth. We have previously reported that a haloacid operon, encoding for a dehalogenase (Deh4a) and an associated permease (Deh4p), was responsible for the transformation and uptake of haloacids in MBA4. A disruption of deh4p in MBA4 caused a decrease in monochloroacetate (MCA) uptake, confirming its role as a haloacid transporter. However, this disruptant retained 68% of its MCA-uptake activity indicating the possibility of an alternative system. In this study, we report the identification of a second MCA-inducible haloacid transporter (Dehp2) in MBA4. Its function was confirmed by gene disruption and heterologous expression in Escherichia coli. A dehp2(-) mutant has 30% less, and an E. coli expressing Dehp2 has 40% more, of wildtype MCA-uptake activity. Quantitative RT-PCR illustrated that the minor loss of MCA-uptake activity in single disruptants of deh4p and dehp2 was partly due to a compensatory expression of the alternative gene. Competition assay and kinetics study revealed that Deh4p has a higher affinity for MCA while Dehp2 prefers chloropropionate. A deh4p(-)dehp2(-) double mutant retained 36% of MCA-uptake activity, indicating a robustness of the haloacid uptake systems. The MCA uptake activities mediated by Deh4p, Dehp2 and the uncharacterized system were completely abolished by protonophore carbonyl cyanide 3-chlorophenylhydrazone, suggesting that transmembrane electrochemical gradient is the driving force for MCA uptake.
authors.affiliation: Molecular Microbiology Laboratory, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China. h0892012@hku.hk
authors.forename: Xianbin
authors.initials: X
authors.lastname: Su
authors.forename: Jimmy S H
authors.initials: JS
authors.lastname: Tsang
mesh_terms: D001667:Binding, Competitive; D001692:Biological Transport; D019117:Burkholderia / Q000378:metabolism*; D002244:Carbon / Q000737:chemistry; D062845:Chloroacetates / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004563:Electrochemistry / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006867:Hydrolases / Q000737:chemistry*; D007700:Kinetics; D008954:Models, Biological; D008957:Models, Genetic; D009154:Mutation; D009876:Operon; D013997:Time Factors
chemical_list: D062845:Chloroacetates; D002244:Carbon; D006867:Hydrolases; C034507:2-haloacid dehalogenase