After 10 minutes about 70% of the cells were alive independent of their genetic background. By 20 minutes more than 99% of P. putida wild-type as well as of colR-, ttgC- and colRttgC-deficient cells were dead (not able to form colonies on selective media) and after 30 minutes of treatment with 50 mM phenol the count of viable cells of all strains had dropped by four orders of magnitude. This data suggests that the cell membrane of the colR-deficient strain is not more permeable to phenol than

the membrane of the wild-type cells. ColRS system and TtgABC efflux pump affect phenol tolerance only in growing bacteria To further investigate variation in phenol sensitivity between the wild-type, colR, ttgC and colRttgC mutant strains

we next monitored the 24-hour-viability CX-4945 of bacteria treated with different concentrations of phenol. To evaluate the effect of different physiological conditions, liquid M9 minimal medium contained either 10 mM glucose, 10 mM gluconate or no carbon source at all. As expected, significant differences between the wild-type and colR-deficient strains became evident when phenol tolerance was tested on glucose minimal medium. However, differently from solid glucose medium where colR mutant is able to grow at phenol click here concentration as high as 6 mM (Fig. 1), growth of the colR mutant in liquid glucose medium was restricted already at 2-6 mM phenol concentration. Moreover, whilst the presence of 4-6 mM phenol allowed growth of the wild-type, then the colR mutant started to die at these phenol concentrations and only less than 10% of inoculated cells could survive during the incubation for 24 hours (Fig. 3A). Another interesting phenomenon detected by us was a specific vulnerability of the glucose-grown colR-deficient strain to intermediate phenol concentrations (4-8 mM), IKBKE which is in contrast with its wild-type-like tolerance to high phenol concentrations (10-16 mM) (Fig. 3A). This data correlates well with

our finding that the colR mutant possesses wild-type-like survival in phenol killing assay (see above) and indicates that in totally stressed cells the phenol tolerance is not influenced by ColRS system any more. Analysis of the ttgC mutants revealed that the effect of the ttgC disruption on phenol tolerance in the liquid glucose medium was negligible compared to its effect on the solid medium (compare Fig. 1 and 3A). Compared to the wild-type strain, the ttgC mutant tolerated higher phenol concentrations on solid glucose medium (Fig. 1) while in liquid medium there were no differences in phenol tolerance between these two strains (Fig. 3A). Also in the colR-deficient background the effect of ttgC disruption was stronger on solid than in liquid glucose medium (compare Fig. 1 and 3A).