mhpT Encodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)propionate Catabolism by Escherich

• 作 者: Ying Xu Bing Chen Hongjun Chao Ning-Yi Zhou
• 机 构: Key Laboratory of Agricultural and Environmental Microbiology,Wuhan Institute of Virology,Chinese Ac
• 出 处: 第十六次全国环境微生物学学术研讨会
• 发表日期: 2013年10期

　　Escherichia coli K-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source.Among 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 located at the periphery of cells under confocal microscopy, consistent with localization to the cytoplasmic membrane.Gene knock-out and complementation studies clearly indicated that mhpT was essential for 3HPP catabolism in E.coli K-12 W3110 at pH 8.2.Uptake assays with [14C]-labeled substrates demonstrated that strain W3110 and strain W3110 mhpT 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 uptake assay was also detected at pH 6.2 and 7.2, although mhpT-deleted strain W3110 mhpThad the ability to utilize 3HPP at pH 6.2 and 7.2, it showed significantly lower transport activities than those of the wild type and complemented strains, because of the absence of MhpT-mediated transport.Especially at pH 8.2, strain W3110 mhpT almost lost the ability to accumulate 3HPP, and it indicated that the environmental pH values distinctly affected 3HPP transport.The change of Ala-272 of MhpT to a histidine surprisingly resulted in an enhanced transport activity and strain W3110 mhpT containing MhpT mutant A272H had a slightly higher growth rate than 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 to grow on this substrate under the basic condition.