Mutagenic analysis of the conserved residues of a haloacid permease

Haloacetic acids are common environmental pollutants and have been shown to be mutagenic. Burkholderia caribensis MBA4, which utilizes monobromoacetate as a growth substrate, was isolated from soil and has been shown to produce an inducible dehalogenase. By means of chromosome walking we have identified a haloacid transporter protein gene (deh4p) downstream of the dehalogenase gene. Comparative analysis showed that Deh4p is a major facilitator superfamily protein, which usually possesses twelve transmembrane segments (TMS). Recombinant DNA technique based on a PhoA-LacZ dual reporters system was used to analyze the topology of Deh4p and the results showed that the N- and the C-termini of the protein were located in the cytoplasm. However, only eight TMS were detected. The first two and the last two putative TMS were not found. ClustalW alignment of Deh4p with some closely related transporter classification TC2.A.1.6 proteins has identified the presence of many conserved residues. An assay system that examined the functional property of the haloacid permease was constructed and used for analysis of mutations on some of these conserved residues. An MBA4 mutant defective in the haloacid operon was used as the host and plasmids with a haloacid operon were transformed into the cell. Charged or potentially phosphorylated residues were selected for modification. A total of sixteen deh4p mutants were generated and tested. The results showed that six of these mutants impeded the growth of the bacterium in utilizing chloroacetate as a growth substrate. Most of these critical mutations were located in the putative cytoplasmic loops of the protein.link_to_subscribed_fulltex