MhbT Is a Specific Transporter for 3-Hydroxybenzoate Uptake by Gram-Negative Bacteria

Abstract

ABSTRACTKlebsiella pneumoniaeM5a1 is capable of utilizing 3-hydroxybenzoate via gentisate, and the 6.3-kb gene clustermhbRTDHIMconferred the ability to grow on 3-hydroxybenzoate toEscherichia coliandPseudomonas putidaPaW340. 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. putidaPaW340 containingmhbRΔTDHIMcould not grow on 3-hydroxybenzoate; however, supplyingmhbTintransallowed the bacterium to grow on the substrate.K. pneumoniaeM5a1 andP. putidaPaW340 containing recombinant MhbT transported14C-labeled 3-hydroxybenzoate but not14C-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 byP. putidaPaW340 carrying the mutant proteins. Hence, we demonstrated that MhbT is a specific 3-hydroxybenzoate transporter.