Abstract
Background2-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.ResultsThirty-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.ConclusionThe 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.
Highlights
Haloacids are metabolic products of naturally occurring compounds [1,2,3] and are disinfection by-products of sewage and water [4,5]
A soil-borne Burkholderia sp., MBA4, has been isolated for its ability to grow on monobromoacetate (MBA) [8]
Hydropathy analysis of Deh4p Computational analysis of Deh4p has categorized it as a Major Facilitator Superfamily (MFS) protein
Summary
Haloacids are metabolic products of naturally occurring compounds [1,2,3] and are disinfection by-products of sewage and water [4,5]. It has been shown that some haloacids are toxic and mutagenic [6,7]. Microorganisms capable of degrading these haloacids can be found in the natural environment. A soil-borne Burkholderia sp., MBA4, has been isolated for its ability to grow on monobromoacetate (MBA) [8]. This bacterium produces a haloacid dehalogenase that allows the cell to grow on MBA. Since MBA is a more potent mutagen than ethylmethane sulfonate [9] one would not expect an uptake (page number not for citation purposes)
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