Hence, our data show that ColRS system and TtgABC pump are involved in phenol tolerance of P. putida only under growth conditions
indicating that BAY 80-6946 ic50 especially growth-related processes of phenol tolerance are affected https://www.selleckchem.com/products/elacridar-gf120918.html by both these systems. Presence of phenol in growth medium enhances proportion of cells with higher DNA content Flow cytometry is a technique which allows to analyse microbial population at single cell level and to detect distinct subpopulations with different functional and structural parameters. We have previously shown that population of solid medium-grown P. putida is heterogeneous by its DNA content and membrane permeability to propidium iodide (PI) when analysed with flow cytometry . In order to assess how the wild-type P. putida and its colR- and ttgC-deficient derivatives change their population structure as well as membrane permeability when growing on different media supplemented with phenol, the microbial populations were analysed at single cell level. Flow cytometry analysis of solid medium-grown bacteria stained with the mixture of SYTO9 and PI demonstrated highly heterogeneous population structure with seven clearly distinguishable subpopulations (Fig. 4). Cells in the first three subpopulations, named as C1, BIBF1120 C2 and C3+, are considered completely
healthy as they do not stain with PI. These three populations differ from each other by their SYTO9 fluorescence intensity which most probably reflects their different DNA content. Next three populations, C1_perm, C2_perm and C3+_perm,
are considered together as cells with membrane permeable to PI but they can be also distinguished by different DNA content analogous to populations C1, C2 and C3+. This was supported by comparative analysis of SYTO9-only and SYTO9+PI-stained populations which revealed that subpopulations C1, C2 and C3+ observed with SYTO9 alone were equal to the sums of their respective healthy and PI-permeable subpopulations in case of SYTO9 and PI double tetracosactide staining (Additional File 2). Seventh subpopulation, marked as Dead, is clearly present only in glucose-grown colR-deficient cells (Fig. 4 and Additional File 3) and correlates with cell lysis. Therefore, this subpopulation most probably represents dead cells with strongly damaged membranes and even lowered DNA content. Latter is supported by observation that glucose-grown colR-deficient cells had subpopulation with remarkably lower green fluorescence when stained with SYTO9 only (Additional File 3). In addition, Dead subpopulation has lower side scatter (SSC) indicating that these cells have less complex intracellular structure compared to other cells (Additional File 3). Figure 4 Visualization of subpopulations by flow cytometry analysis. P.