In addition to phenol stress, the colR-deficient bacteria experience serious glucose-related stress resulting in lysis of a subpopulation of cells [10]. Importantly, cell lysis does not occur on medium with gluconate which is degraded like glucose through Entner-Doudoroff pathway. To test whether inactivation of the TtgABC efflux pump would affect phenol stress only on glucose or it would have a more general role in phenol tolerance,
the growth of newly constructed ttgB- and ttgC-deficient strains were examined both on glucose and gluconate minimal media supplemented with different concentrations of phenol (Fig. 1). In accordance with the learn more transposon mutagenesis screen, the disruption of the ttgABC operon made P. putida colR-deficient cells more selleckchem resistant to phenol, and this behaviour was observed on both, glucose and gluconate medium. However, LDN-193189 since the ttgB- and ttgC-deficiency enhanced phenol tolerance also in the wild-type background (Fig. 1), we consider that the TtgABC efflux pump is related to a general
tolerance of bacteria to phenol rather than to a particular phenotype of the colR mutant. Increased phenol tolerance per se does not alleviate the phenol-enhanced autolysis of glucose-grown colR-deficient cells neither does it restore transposition of Tn4652 in the colR mutant In our previous study we showed that phenotypes of the colR-deficient bacteria such as membrane leakiness and cell lysis, which are related with growth on glucose, became more prominent if phenol was added to the medium [10]. For instance, glucose-induced release of cytoplasmic β-galactosidase into the growth medium due to the autolysis of the colR mutant was
significantly enhanced if phenol was supplied [10]. In order to find out whether the increased phenol 4��8C tolerance can alleviate glucose-induced and phenol-enhanced autolysis of the colR-deficient strain, the ttgC-knockout derivatives were subjected to β-galactosidase assay. To calculate the percentage of unmasked β-galactosidase activity which was used as an indicator of membrane leakiness and cell lysis, the enzyme activity was measured both in suspension of cells permeabilized with SDS and chloroform (total activity), and in that of intact, non-permeabilized cells. In accordance with our previous results only 4% of total β-galactosidase activity was measurable using non-permeabilized wild-type cells regardless of the presence of phenol in the growth medium [10] (Fig. 2). At the same time, about 15% of total β-galactosidase activity was detectable in case of the colR-deficient cells grown on glucose minimal plates, and up to 30% when cells were grown on glucose medium supplemented with 1 mM phenol [10] (Fig. 2). The phenol tolerant ttgC single mutant behaved in this test like the wild-type strain (Fig. 2).