The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of Pseudomonas putida becomes evident only in growing bacteria

<p>Abstract</p> <p>Background</p> <p>We have recently found that <it>Pseudomonas putida </it>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 <it>colR</it>-deficient <it>P. putida</it>.</p> <p>Results</p> <p>By using transposon mutagenesis approach we identified a set of phenol-tolerant derivatives of <it>colR</it>-deficient strain. Surprisingly, half of independent phenol tolerant clones possessed miniTn5 insertion in the <it>ttgABC </it>operon. However, though inactivation of TtgABC efflux pump significantly enhanced phenol tolerance, it did not affect phenol-enhanced autolysis of the <it>colR </it>mutant on glucose medium indicating that phenol- and glucose-caused stresses experienced by the <it>colR</it>-deficient <it>P. putida </it>are not coupled. Inactivation of TtgABC pump significantly increased the phenol tolerance of the wild-type <it>P. putida </it>as well. Comparison of phenol tolerance of growing <it>versus </it>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 <it>ttgC- </it>and <it>colRttgC-</it>deficient strains revealed that their membrane permeabilization by phenol resembles that of the wild-type and the <it>colR </it>mutant, respectively. However, cell division of <it>P. putida </it>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 <it>colR</it>-mutant strain than in the wild-type <it>P. putida</it>.</p> <p>Conclusions</p> <p>ColRS signal system and TtgABC efflux pump are involved in the phenol tolerance of <it>P. putida</it>. 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.</p

The ColRS Two-Component System Regulates Membrane Functions and Protects Pseudomonas putida against Phenol

As reported, the two-component system ColRS is involved in two completely different processes. It facilitates the root colonization ability of Pseudomonas fluorescens and is necessary for the Tn4652 transposition-dependent accumulation of phenol-utilizing mutants in Pseudomonas putida. To determine the role of the ColRS system in P. putida, we searched for target genes of response regulator ColR by use of a promoter library. Promoter screening was performed on phenol plates to mimic the conditions under which the effect of ColR on transposition was detected. The library screen revealed the porin-encoding gene oprQ and the alginate biosynthesis gene algD occurring under negative control of ColR. Binding of ColR to the promoter regions of oprQ and algD in vitro confirmed its direct involvement in regulation of these genes. Additionally, the porin-encoding gene ompA(PP0773) and the type I pilus gene csuB were also identified in the promoter screen. However, it turned out that ompA(PP0773) and csuB were actually affected by phenol and that the influence of ColR on these promoters was indirect. Namely, our results show that ColR is involved in phenol tolerance of P. putida. Phenol MIC measurement demonstrated that a colR mutant strain did not tolerate elevated phenol concentrations. Our data suggest that increased phenol susceptibility is also the reason for inhibition of transposition of Tn4652 in phenol-starving colR mutant bacteria. Thus, the current study revealed the role of the ColRS two-component system in regulation of membrane functionality, particularly in phenol tolerance of P. putida

Pseudouridines of tRNA Anticodon Stem-Loop Have Unexpected Role in Mutagenesis in Pseudomonas sp.

Pseudouridines are known to be important for optimal translation. In this study we demonstrate an unexpected link between pseudouridylation of tRNA and mutation frequency in Pseudomonas species. We observed that the lack of pseudouridylation activity of pseudouridine synthases TruA or RluA elevates the mutation frequency in Pseudomonas putida 3 to 5-fold. The absence of TruA but not RluA elevates mutation frequency also in Pseudomonas aeruginosa. Based on the results of genetic studies and analysis of proteome data, the mutagenic effect of the pseudouridylation deficiency cannot be ascribed to the involvement of error-prone DNA polymerases or malfunctioning of DNA repair pathways. In addition, although the deficiency in TruA-dependent pseudouridylation made P. putida cells more sensitive to antimicrobial compounds that may cause oxidative stress and DNA damage, cultivation of bacteria in the presence of reactive oxygen species (ROS)-scavenging compounds did not eliminate the mutator phenotype. Thus, the elevated mutation frequency in the absence of tRNA pseudouridylation could be the result of a more specific response or, alternatively, of a cumulative effect of several small effects disturbing distinct cellular functions, which remain undetected when studied independently. This work suggests that pseudouridines link the translation machinery to mutation frequency

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Practical Course in Genetics

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Mutation Frequency and Spectrum of Mutations Vary at Different Chromosomal Positions of <em>Pseudomonas putida</em>

<div><p>It is still an open question whether mutation rate can vary across the bacterial chromosome. In this study, the occurrence of mutations within the same mutational target sequences at different chromosomal locations of <em>Pseudomonas putida</em> was monitored. For that purpose we constructed two mutation detection systems, one for monitoring the occurrence of a broad spectrum of mutations and transposition of IS element IS<em>1411</em> inactivating LacI repressor, and another for detecting 1-bp deletions. Our results revealed that both the mutation frequency and the spectrum of mutations vary at different chromosomal positions. We observed higher mutation frequencies when the direction of transcription of the mutational target gene was opposite to the direction of replisome movement in the chromosome and <em>vice versa</em>, lower mutation frequency was accompanied with co-directional transcription and replication. Additionally, asymmetry of frameshift mutagenesis at homopolymeric and repetitive sequences during the leading and lagging-strand replication was found. The transposition frequency of IS<em>1411</em> was also affected by the chromosomal location of the target site, which implies that regional differences in chromosomal topology may influence transposition of this mobile element. The occurrence of mutations in the <em>P. putida</em> chromosome was investigated both in growing and in stationary-phase bacteria. We found that the appearance of certain mutational hot spots is strongly affected by the chromosomal location of the mutational target sequence especially in growing bacteria. Also, artificial increasing transcription of the mutational target gene elevated the frequency of mutations in growing bacteria.</p> </div