MhbT is a specific transporter for 3-hydroxybenzoate uptake by Gram-negative bacteria.
Klebsiella pneumoniae M5a1 is capable of utilizing 3-hydroxybenzoate via gentisate, and the 6.3-kb gene cluster mhbRTDHIM conferred the ability to grow on 3-hydroxybenzoate to Escherichia coli and Pseudomonas putida PaW340. Four of the six genes (mhbDHIM) encode enzymes converting 3-hydroxybenzoate to pyruvate and fumarate via gentisate. MhbR is a gene activator, and MhbT is a hypothetical protein belonging to the transporter of the aromatic acid/H(+) symporter family. Since a transporter for 3-hydrxybenzoate uptake has not been characterized to date, we investigated whether MhbT is responsible for the uptake of 3-hydroxybenzoate, its metabolic intermediate gentisate, or both. The MhbT-green fluorescent protein (GFP) fusion protein was located on the cytoplasmic membrane. P. putida PaW340 containing mhbRΔTDHIM could not grow on 3-hydroxybenzoate; however, supplying mhbT in trans allowed the bacterium to grow on the substrate. K. pneumoniae M5a1 and P. putida PaW340 containing recombinant MhbT transported (14)C-labeled 3-hydroxybenzoate but not (14)C-labeled gentisate and benzoate into the cells. Site-directed mutagenesis of two conserved amino acid residues (Asp-82 and Asp-314) and a less-conserved residue (Val-311) among the members of the symporter family in the hydrophilic cytoplasmic loops resulted in the loss of 3-hydroxybenzoate uptake by P. putida PaW340 carrying the mutant proteins. Hence, we demonstrated that MhbT is a specific 3-hydroxybenzoate transporter.
Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
Ying
Y
Xu
Xiaoli
X
Gao
Song-He
SH
Wang
Hong
H
Liu
Peter A
PA
Williams
Ning-Yi
NY
Zhou
D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D005841:Gentisates / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D062385:Hydroxybenzoates / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D016958:Pseudomonas putida / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D017422:Sequence Analysis, DNA
D029968:Escherichia coli Proteins; D005841:Gentisates; D062385:Hydroxybenzoates; D026901:Membrane Transport Proteins; C576688:MhbT protein, E coli; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C032948:3-hydroxybenzoic acid; C010925:2,5-dihydroxybenzoic acid