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We found that certain Str-, Gen- or Rif- mutants derived from Pseudomonas putida KH146-2, which are resistant to streptomycin, gentamicin or rifampicin, respectively, are tolerant to the aromatic compound 4-hydroxybenzoate (4HBA). The minimum inhibitory concentration (MIC) of 4HBA as the sole carbon source for the wild-type strain was 1%, whereas the MIC for the mutants was 1.7%. Frequency of 4HBA-tolerant mutants among spontaneous Str-, Gen- and Rif- mutants was 5-15%, 3-5%, and 3% respectively. These 4HBA-tolerant mutants also tolerated to a variety of organic chemicals such as 3-hydroxybenzoate, aliphatic and heterocyclic compounds, chlorobenzoates, as well as organic solvents toluene and m-xylene. The Str mutants had a point mutation in the rpsL gene, which produces the ribosomal protein S12. The Rif mutants were found to have a point mutation in the rpoB gene, which encodes the RNA polymerase beta-subunit. Mutation points in Gen mutants still remain unknown. Str-, Gen- and Rif-phenotypes occurred in spontaneous 4HBA-tolerant mutants which had been selected by successively increasing concentrations (from 0.8% to 5%) of 4HBA. Complementation experiments with one of the Str mutants demonstrated a causal relationship between a rpsL mutation (str-1) and 4HBA tolerance. Uptake experiments using [14C]-4HBA revealed that apparent ability of 4HBA to be taken up by the membrane transport system was reduced two to threefold in the mutants compared to the wild-type strain, accounting at least partly for the enhanced tolerance to 4HBA. Our approaches thus could be effective in improvement of tolerance to aromatic compounds of bacteria applicable for bioremediation.

We found that certain Str-, Gen- or Rif- mutants derived from Pseudomonas putida KH146-2, which are resistant to streptomycin, gentamicin or rifampicin, respectively, are tolerant to the aromatic compound 4-hydroxybenzoate (4HBA). The minimum inhibitory concentration (MIC) of 4HBA as the sole carbon source for the wild-type strain was 1%, whereas the MIC for the mutants was 1.7%. Frequency of 4HBA-tolerant mutants among spontaneous Str-, Gen- and Rif- mutants was 5-15%, 3-5%, and 3% respectively. These 4HBA-tolerant mutants also tolerated to a variety of organic chemicals such as 3-hydroxybenzoate, aliphatic and heterocyclic compounds, chlorobenzoates, as well as organic solvents toluene and m-xylene. The Str mutants had a point mutation in the rpsL gene, which produces the ribosomal protein S12. The Rif mutants were found to have a point mutation in the rpoB gene, which encodes the RNA polymerase beta-subunit. Mutation points in Gen mutants still remain unknown. Str-, Gen- and Rif-phenotypes occurred in spontaneous 4HBA-tolerant mutants which had been selected by successively increasing concentrations (from 0.8% to 5%) of 4HBA. Complementation experiments with one of the Str mutants demonstrated a causal relationship between a rpsL mutation (str-1) and 4HBA tolerance. Uptake experiments using [14C]-4HBA revealed that apparent ability of 4HBA to be taken up by the membrane transport system was reduced two to threefold in the mutants compared to the wild-type strain, accounting at least partly for the enhanced tolerance to 4HBA. Our approaches thus could be effective in improvement of tolerance to aromatic compounds of bacteria applicable for bioremediation.