Characterization of a MexAB-OprM efflux system necessary for productive metabolism of Pseudomonas azelaica HBP1 on 2-hydroxybiphenyl

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.

The TetR-Type MfsR Protein of the Integrative and Conjugative Element (ICE) ICE clc Controls both a Putative Efflux System and Initiation of ICE Transfer

“…S3). However, independent experimental repetitions measuring growth rates in liquid cultures, growth yields, and ethidium-bromide partitioning in individual cells using flow cytometry (33) were not able to confirm any increase of chromium(III) chloride sensitivity for P. putida UWC1 carrying ICEclc-⌬mfsABC seen with Biolog compared to that of P. putida UWC1 carrying wild-type ICEclc. Finally, none of the aromatic substrates for which catabolism is encoded on ICEclc (7), such as 3-CBA, 3-chlorocatechol, or 2-aminophenol, led to a derepression of the P mfsA -mcherry fusion in P. putida UWC1 carrying ICEclc when added to the culture media (data not shown).…”

Insights into Mobile Genetic Elements of the Biocide-Degrading Bacterium Pseudomonas nitroreducens HBP-1

“…Selection of P. putida UWCGC transconjugants from matings with P. nitroreducens HBP-1 on minimal medium agar with 5 mM 2-HBP and Gm yielded no colonies, possibly because of the toxicity of 2-HBP [51]. In contrast, selection on 2.5 mM salicylate and Gm yielded eight colonies among 8.6 × 10 8 donor cells, which would be equivalent to a transfer rate of 10 −8 per donor CFU.…”

Genome Sequence of Pseudomonas azelaica Strain Aramco J

“…The hbp genes involved in the conversion of 2-HBP (and 2,2′-dihydroxybiphenyl) to benzoate (salicylate) and 2-hydroxy-pentadienoate ( 4 ) are located within an integrative-conjugative element (ICEhbp) similar to that found in P. azelaica HBP1 ( 6 ). Next to the hbp genes are located the sal and dmp genes encoding a salicylate monooxygenase and a catechol meta -cleavage pathway for the metabolism of salicylate and 2-hydroxy-pentadienoate, respectively.…”

“…Moreover, 2-HBP is a bulk chemical with biocidal properties that is primarily used as an agricultural fungicide and therefore can be found in persistent low quantities in sewage effluents ( 2 ). However, bacterial degradation of 2-HBP is an uncommon trait, and only a few bacteria are known to completely metabolize 2-HBP ( 3 , 4 ). Here, we report the genome sequence of a bacterium, the Pseudomonas azelaica Aramco J strain, isolated from an oil-contaminated soil sample from Abu Ali Island, Saudi Arabia, that uses 2-HBP, as well as other hydroxybiphenyls such as 2,2′-dihydroxybiphenyl and 3-hydroxybiphenyl, as the only carbon source.…”