AM181176, locus-tag PFLU_0035) and clpA (locus-tag PFLU_3805) genes of P. fluorescens encoding tryptophan synthase alpha-subunit and ATP dependent Clp protease, respectively. CSM3 had transposon insertion site identical to that of CSM2. The difference in the copper tolerance between the wild-type strain and the mutants (CSM1 and CSM2) was investigated by growth inhibition experiments in LB broth with increasing concentrations of copper (Fig. 2). The BMN 673 purchase growth of the mutants was comparable to the wild-type strain grown in the presence of 2 mM copper and no copper, suggesting that the mutations did not limit the bacterial fitness in 2 mM copper. The growth of the two mutants was significantly inhibited in

4 mM copper compared with the wild-type control (P < 0.05). CSM2 and CSM1 did not grow in 4.5 and 5 mM copper, respectively. Quantitative RT-PCR analysis showed that the relative expression of clpA and trpA genes in wild type under copper stress (4 mM) was 13- and 3.2-fold, respectively, compared

with wild type grown without copper (Fig. S1). No clpA and trpA expression was detected in the mutants. Proteomic analysis of the wild type and the copper-sensitive mutant CSM2 grown without copper identified 21 protein spots with a greater than twofold change, of which the relative click here intensity of 13 protein spots decreased by 2- to 4.3-fold and eight spots increased two to eightfold. Five protein spots were selected for mass spectrometry analysis based on more than threefold changes ASK1 in protein expression and the possibility of clean excision. Expression of proteins involved in carbohydrate metabolism, energy production and tRNA processing was down-regulated in CSM2 compared with the wild type (Table 1). However, the expression

of DnaJ-class molecular chaperone and HpcH/HpaI aldolase was up-regulated compared with the wild-type strain. Interestingly, the protein expression of all the five identified spots was up-regulated in wild-type strain grown in 4 mM copper compared with the wild-type strain and CSM2 grown without copper. DnaJ-class molecular chaperone tRNA (guanine-N(7)-)- methyltransferase Energy production and conversion Ubiquinone biosynthesis protein ABC transporter-like protein Regulator of citrate/ malate metabolism Transcriptional regulatory protein MalR Amino acid metabolism Tryptophan synthase β subunit Amino acid metabolism/ TCA cycle Our next step was to investigate proteins whose expression was altered in wild type exposed to copper and which, at the same time, showed no change in CSM2 compared with the wild type. This experiment identified eight proteins that have a role in efflux of macromolecules, small molecules and ions, and act as transporters of amino acids (Table 1). Proteins related to amino acid metabolism and histidine kinase, which is part of the bacterial two-component sensor system involved in environmental sensing (Swartz et al.