mhpT encodes an active transporter involved in 3-(3-hydroxyphenyl)propionate catabolism by Escherichia coli K-12.
Escherichia coli K-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome, mhpT was proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated that mhpT is essential for 3HPP catabolism in E. coli K-12 W3110 at pH 8.2. Uptake assays with (14)C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpT containing recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpT containing the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital for E. coli K-12 W3110 growth on this substrate under basic conditions.
Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
Ying
Y
Xu
Bing
B
Chen
Hongjun
H
Chao
Ning-Yi
NY
Zhou
D019943:Amino Acid Substitution; D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism; D002250:Carbon Radioisotopes / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D003373:Coumaric Acids / Q000378:metabolism*; D004252:DNA Mutational Analysis; D048168:Escherichia coli K12 / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D018613:Microscopy, Confocal; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D018892:Proton-Motive Force; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
D027321:Anion Transport Proteins; D002250:Carbon Radioisotopes; D003373:Coumaric Acids; D029968:Escherichia coli Proteins; C000588660:MhpT protein, E coli; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C038547:dihydro-3-coumaric acid; D002244:Carbon