Topological analysis of a haloacid permease of a Burkholderi a sp. bacterium with a PhoA-LacZ reporterReport as inadecuate




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BMC Microbiology

, 9:233

First Online: 31 October 2009Received: 25 June 2009Accepted: 31 October 2009

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.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2180-9-233 contains supplementary material, which is available to authorized users.

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Author: Yuk Man Tse - Manda Yu - Jimmy SH Tsang

Source: https://link.springer.com/article/10.1186/1471-2180-9-233







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