Abstract
BackgroundWe have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the colR-deficient P. putida.ResultsBy using transposon mutagenesis approach we identified a set of phenol-tolerant derivatives of colR-deficient strain. Surprisingly, half of independent phenol tolerant clones possessed miniTn5 insertion in the ttgABC operon. However, though inactivation of TtgABC efflux pump significantly enhanced phenol tolerance, it did not affect phenol-enhanced autolysis of the colR mutant on glucose medium indicating that phenol- and glucose-caused stresses experienced by the colR-deficient P. putida are not coupled. Inactivation of TtgABC pump significantly increased the phenol tolerance of the wild-type P. putida as well. Comparison of phenol tolerance of growing versus starving bacteria revealed that both ColRS and TtgABC systems affect phenol tolerance only under growth conditions and not under starvation. Flow cytometry analysis showed that phenol strongly inhibited cell division and to some extent also caused cell membrane permeabilization to propidium iodide. Single cell analysis of populations of the ttgC- and colRttgC-deficient strains revealed that their membrane permeabilization by phenol resembles that of the wild-type and the colR mutant, respectively. However, cell division of P. putida with inactivated TtgABC pump seemed to be less sensitive to phenol than that of the parental strain. At the same time, cell division appeared to be more inhibited in the colR-mutant strain than in the wild-type P. putida.ConclusionsColRS signal system and TtgABC efflux pump are involved in the phenol tolerance of P. putida. However, as they affect phenol tolerance of growing bacteria only, this indicates that they participate in the regulation of processes which are active during the growth and/or cell division. Single cell analysis data indicated that the cell division step of cell cycle is particularly sensitive to the toxic effect of phenol and its inhibition can be considered as an adaptive response under conditions of phenol stress.
Highlights
We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses
Inactivation of different genes involved in membrane, central metabolism or regulatory functions can increase phenol tolerance of colR-deficient strain The growth of colR and colS mutant cells is precluded on glucose and gluconate solid medium in the presence of 8 mM phenol, while the growth of the wild-type is not [8] (Fig. 1)
Our flow cytometry analysis of phenol-exposed P. putida cultures demonstrated that phenol only slightly increased the amount of cells with propidium iodide (PI) permeable membrane indicating that cells quite well maintain their membrane homeostasis (Fig. 5)
Summary
We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Phenol and other aromatics can be highly toxic, yet their toxicity depends on the concentration of the compound as well as on tolerance level of bacteria. Encountering a hazardous aromatic compound, several adaptive responses are triggered in bacteria to neutralize the action of a toxicant. TCSs are typically composed of a membrane-located sensor with histidine kinase activity and of a cytoplasmic response protein with a signal-accepting receiver domain. Environmental signal sensed by membrane protein is transduced to a response regulator by phosphorylation
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