Abstract
Pseudomonas azelaica HBP1 is one of the few bacteria known to completely mineralize the biocide and toxic compound 2-hydroxybiphenyl (2-HBP), but the mechanisms of its tolerance to the toxicity are unknown. By transposon mutant analysis and screening for absence of growth on water saturating concentrations of 2-HBP (2.7 mM) we preferentially found insertions in three genes with high homology to the mexA, mexB, and oprM efflux system. Mutants could grow at 2-HBP concentrations below 100 μM but at lower growth rates than the wild-type. Exposure of the wild-type to increasing 2-HBP concentrations resulted in acute cell growth arrest and loss of membrane potential, to which the cells adapt after a few hours. By using ethidium bromide (EB) as proxy we could show that the mutants are unable to expel EB effectively. Inclusion of a 2-HBP reporter plasmid revealed that the wild-type combines efflux with metabolism at all 2-HBP concentrations, whereas the mutants cannot remove the compound and arrest metabolism at concentrations above 24 μM. The analysis thus showed the importance of the MexAB-OprM system for productive metabolism of 2-HBP.
Highlights
Bacteria degrading xenobiotic compounds often face the difficulty that the chemicals are very difficult to metabolize but are very toxic (van der Meer, 2006)
TRANSPOSON INSERTIONS IN THE mexAB-oprM GENE CLUSTER Replica plating of some 10,000 P. azelaica transposon mutants from eight independently created mutant libraries resulted in ∼1% of colonies growing on succinate but unable to grow with 2.7 mM (460 mg/L) 2-HBP as a sole carbon source
We found that 28 insertions had occurred at different positions in a gene cluster homologous to mexAB-oprM (9 insertions occurred in mexA, 12 in mexB, and 7 in oprM), specifying a multidrug resistance efflux pump (Figure 1)
Summary
Bacteria degrading xenobiotic compounds often face the difficulty that the chemicals are very difficult to metabolize but are very toxic (van der Meer, 2006). Mechanistic studies of chemical toxicity to biological membranes have underscored two general processes: baseline toxicity or narcosis, and uncoupling (van Wezel and Opperhuizen, 1995). Partitioning of hydrophobic organic compounds into cellular membranes can be described by the octanol-water coefficient (Escher et al, 2008) as well as by their dissociation constants (Escher et al, 2000, 2002). Such calculations predict accumulations for strongly lipophilic compounds of thousandfold or more in the membrane compared to the cytoplasm or extracellular environment (Sikkema et al, 1995)
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