It is notable that glucose-dependent cell lysis of colR-mutant was significantly enhanced if phenol was present in the growth medium [10]. Identification of ColRS-regulated genes has selleck pointed to cell membrane as a potential target of this

particular TCS. Namely, the operon locating just downstream of colRS genes that codes for a probable lipopolysaccharide kinase and a methyltransferase is positively controlled by ColR both in P. putida and P. fluorescens [11, 12]. In addition, P. putida ColRS system negatively regulates transcription of oprQ and algD genes that code ACP-196 order for outer membrane porin and alginate biosynthesis enzyme, respectively [8]. Genome-wide search for ColR regulon in P. putida has revealed several other ColR-regulated membrane proteins such as lipid A 3-O-deacylase PagL and diacylglycerol kinase DgkA involved in metabolism of lipopolysaccharides and phospholipides, respectively [12]. Importantly, the presence of phenol in growth medium significantly enhances the effect of ColR on its target promoters [8, 12] pointing once more to increased phenol sensitivity selleck compound of the colR mutant P. putida. Many ColR-regulated genes have been tested with respect to their

potential participation in the phenol tolerance of P. putida. However, despite several efforts we could not identify so far any particular ColR target gene responsible for reduced phenol tolerance of the colR-deficient P. putida (our unpublished data). Here, to further unravel the role of ColRS system in phenol tolerance, we report on a transposon mutagenesis performed in a colR-deficient about strain to search for suppressors of phenol sensitivity. This screen disclosed several genes, disruption of which enhanced phenol tolerance of the colR mutant. Additionally, we show that phenol sensitivity of the colR-deficient bacteria becomes evident only under growth-permitting conditions

and not if bacteria are starving for a carbon source. Population analysis at single cell level indicated that particularly cell division is inhibited under condition of phenol stress. Methods Bacterial strains and media All strains used in this study are derivatives of P. putida PaW85 [13], which is isogenic to fully sequenced KT2440 [14]. To study the role of ColRS system, previously constructed colR- and colS-knockout derivatives of P. putida PaW85, PaWcolR and PaWcolS [9] were exploited. Escherichia coli strains DH5α [15] and CC118 λpir [16] were used for DNA cloning procedures, and HB101 [17] as a host for helper plasmid pRK2013 [18]. E. coli was grown at 37°C and P. putida at 30°C. Bacteria were grown in Luria-Bertani (LB) medium [19] or in M9 minimal medium [20] containing either 10 mM glucose or 10 mM gluconate. Phenol concentrations in minimal media are specified in the text, as they varied between the experiments.