Insights into Pseudomonas putida KT2440 response to phenol-induced stress by quantitative proteomics

To gain insight into the global mechanism underlying phenol toxicity and tolerance in bacteria, we have generated a two-dimensional protein reference map and used it to identify variations in protein expression levels in Pseudomonas putida KT2440 following exposure to sub-lethal inhibitory concentrations of this solvent. Inspection of the two-dimensional gel electrophoresis gels revealed that 1 h following sudden cell exposure to two different concentrations of phenol, leading to the inhibition of exponential growth (600 mg/L) or to growth arrest for, at least, 4 h before inhibited growth resumption (800 mg/L), the amount of 68 proteins was increased while the amount of 13 proteins was reduced. The up-regulated proteins include proteins involved in the: (i) oxidative stress response (AhpC, SodB,Tpx and Dsb); (ii) general stress reponse (UspA, HtpG, GrpE and Tig); (iii) energetic metabolism (AcnB, AtpH, Fpr, AceA, NuoE, and MmsA-1); (iv) fatty acid biosynthesis (FabB, AccC-1 and FabBx1); (v) inhibition of cell division (MinD); (vi) cell envelope biosynthesis (LpxC, VacJ, and MurA); (vii) transcription regulation (OmpR and Fur); and (viii) transport of small molecules (TolC, BraC, AotJ, AapJ, FbpA and OprQ). Among the down-regulated proteins are those involved in nucleotide biosynthesis (PurM, PurL, PyrH and Dcd) and cell motility (FliC). The information emerging from this genome expression profiling and the detailed investigation of the biological role of candidate genes, as targets of phenol toxicity or as determinants of phenol resistance in P. putida KT2440, will allow more rationale strategies for developing bacteria with greater solvent tolerance with impact in bioremediation and whole-cell biotransformations in media with organic solvents.