MhbT is a specific transporter for 3-hydroxybenzoate uptake by Gram-negative bacteria

Appl Environ Microbiol. 2012 Sep;78(17):6113-20. doi: 10.1128/AEM.01511-12. Epub 2012 Jun 22.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Cell Membrane / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / growth & development
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Gene Deletion
  • Genes, Reporter
  • Genetic Complementation Test
  • Gentisates / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Hydroxybenzoates / metabolism*
  • Klebsiella pneumoniae / genetics
  • Klebsiella pneumoniae / growth & development
  • Klebsiella pneumoniae / metabolism*
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Pseudomonas putida / genetics
  • Pseudomonas putida / growth & development
  • Pseudomonas putida / metabolism*
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / genetics
  • Sequence Analysis, DNA

Substances

  • Escherichia coli Proteins
  • Gentisates
  • Hydroxybenzoates
  • Membrane Transport Proteins
  • MhbT protein, E coli
  • Mutant Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • 3-hydroxybenzoic acid
  • 2,5-dihydroxybenzoic acid

Associated data

  • GENBANK/AY648560