Topological analysis of a haloacid permease of a Burkholderia sp. bacterium with a PhoA-LacZ reporter.
BACKGROUND
2-Haloacids can be found in the natural environment as degradative products of natural and synthetic halogenated compounds. They can also be generated by disinfection of water and have been shown to be mutagenic and to inhibit glyceraldehyde-3-phosphate dehydrogenase activity. We have recently identified a novel haloacid permease Deh4p from a bromoacetate-degrading bacterium Burkholderia sp. MBA4. Comparative analyses suggested that Deh4p is a member of the Major Facilitator Superfamily (MFS), which includes thousands of membrane transporter proteins. Members of the MFS usually possess twelve putative transmembrane segments (TMS). Deh4p was predicted to have twelve TMS. In this study we characterized the topology of Deh4p with a PhoA-LacZ dual reporters system.


RESULTS
Thirty-six Deh4p-reporter recombinants were constructed and expressed in E. coli. Both PhoA and LacZ activities were determined in these cells. Strength indices were calculated to determine the locations of the reporters. The results mainly agree with the predicted model. However, two of the TMS were not verified. This lack of confirmation of the TMS, using a reporter, has been reported previously. Further comparative analysis of Deh4p has assigned it to the Metabolite:H+ Symporter (MHS) 2.A.1.6 family with twelve TMS. Deh4p exhibits many common features of the MHS family proteins. Deh4p is apparently a member of the MFS but with some atypical features.


CONCLUSIONS
The PhoA-LacZ reporter system is convenient for analysis of the topology of membrane proteins. However, due to the limitation of the biological system, verification of some of the TMS of the protein was not successful. The present study also makes use of bioinformatic analysis to verify that the haloacid permease Deh4p of Burkholderia sp. MBA4 is a MFS protein but with atypical features.
Tse
Yuk Man
YM
Molecular Microbiology Laboratory, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong. mavericktse@gmail.com
Yu
Manda
M
Tsang
Jimmy S H
JS
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D019117:Burkholderia / Q000201:enzymology* / Q000235:genetics; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D017930:Genes, Reporter; D054879:Halogenation; D007763:Lac Operon; D026901:Membrane Transport Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli