Transpositional activity of mobile elements can be induced by different environmental stresses. Here, we present evidence that transposition of Tn 4652 is elevated in stationary-phase Pseudomonas putida and suppressed in an isogenic ς ^S -defective strain. We demonstrate that transcription from the Tn 4652 transposase promoter is controlled by the stationary-phase-specific sigma factor ς ^S . To our knowledge, this is the first example of direct stationary-phase-specific regulation of a mobile element transposase. Data presented in this report support the idea that activation of transposition under stressful conditions could be an inducible process.

A novel experimental system to study mutation in starving bacteria was designed, relying on the activation of a promoterless phenol degradation operon of Pseudomonas putida . The Phe ⁺ (phenol-utilizing) mutants accumulated in the starving culture of P. putida in the presence of phenol but not in the absence of it. We ruled out the possibility that the absence of phenol eliminates Phe ⁺ mutants from the starving population. Sequence analysis of the Phe ⁺ mutants revealed that base substitutions, deletions, and insertion of Tn 4652 can result in creation of a sequence similar to the σ ⁷⁰ -specific promoter consensus. One particular C → A transversion was predominant in the Phe ⁺ mutants that arose in the starving population under selection for phenol use. In contrast, various deletions were the most frequent Phe ⁺ mutants occurring in a culture growing without selection. The accumulation rate of the Phe ⁺ mutants on selective plates was found to be higher for bacteria plated from stationary-phase culture than that from exponentially growing cells. This suggests that some specific processes, occurring predominantly in stationary-phase cells, facilitate generation and/or fixation of such mutations.

In Pseudomonas putida PaW85, the ortho-cleavage pathway is used for catechol degradation. The 11.4-kb XhoI fragment cloned from phenol degradation plasmid pEST1226 into pKT240 (recombinant plasmid pAT1140) contains the inducible pheBA operon that encodes catechol 1,2-dioxygenase (gene pheB) and phenol monooxygenase (gene pheA), the first two enzymes for the phenol degradation pathway. The promoter of the pheBA operon is mapped 1.5 kb upstream of the pheB gene. The plasmid pAT1140, when introduced into P. putida PaW85, enables the bacteria to use the hybrid plasmid-chromosome-encoded pathway for phenol degradation. The synthesis of the plasmid-encoded phenol monooxygenase and catechol 1,2-dioxygenase is induced by cis,cis-muconate. The expression studies of the deletion subclones derived from pAT1140 revealed that the transcription of the pheBA operon is positively controlled by a regulatory protein that is chromosomally encoded in P. putida. cis,cis-Muconate in cooperation with positive transcription factor CatR activates the transcription of the chromosomal ortho-pathway genes catA and catBC in P. putida (R. K. Rothmel, T. L. Aldrich, J. E. Houghton, W. M. Coco, L. N. Ornston, and A. M. Chakrabarty, J. Bacteriol. 172:922-931, 1990). The inability to express the pheBA operon in a P. putida CatR- background and activation of transcription of the pheBA operon in Escherichia coli in the presence of the catR-expressing plasmid demonstrated that the transcription of the pheBA operon in P. putida PaW85 carrying pEST1226 is controlled by the chromosomally encoded CatR.

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 Tn 4652 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 Tn 4652 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 .

Chromosomal toxin-antitoxin (TA) systems are widespread among free-living bacteria and are supposedly involved in stress tolerance. Here, we report the first TA system identified in the soil bacterium Pseudomonas putida. The system, encoded by the loci PP1586-PP1585, is conserved in pseudomonads and belongs to the HigBA family. The new TA pair was named GraTA for the growth rate-affecting ability of GraT and the antidote activity of GraA. The GraTA system shares many features common to previously described type II TA systems. The overexpression of GraT is toxic to the antitoxin deletion mutants, since the toxin's neutralization is achieved by binding of the antitoxin. Also, the graTA operon structure and autoregulation by antitoxin resemble those of other TA loci. However, we were able to delete the antitoxin gene from the chromosome, which shows the unusually mild toxicity of innate GraT compared to previously described toxins. Furthermore, GraT is a temperature-dependent toxin, as its growth-regulating effect becomes more evident at lower temperatures. Besides affecting the growth rate, GraT also increases membrane permeability, resulting in higher sensitivity to some chemicals, e.g., NaCl and paraquat. Nevertheless, the active toxin helps the bacteria survive under different stressful conditions and increases their tolerance to several antibiotics, including streptomycin, kanamycin, and ciprofloxacin. Therefore, our data suggest that GraT may represent a new class of mild chromosomal regulatory toxins that have evolved to be less harmful to their host bacterium. Their moderate toxicity might allow finer growth and metabolism regulation than is possible with strong growth-arresting or bactericidal toxins.

Tn 4652 is a derivative of the toluene degradation transposon Tn 4651 that belongs to the Tn 3 family of transposons (M. Tsuda and T. Iino, Mol. Gen. Genet. 210:270–276, 1987). We have sequenced the transposase gene tnpA of transposon Tn 4652 and mapped its promoter to the right end of the element. The deduced amino acid sequence of tnpA revealed 96.2% identity with the putative transposase of Tn 5041 . Homology with other Tn 3 family transposases was only moderate (about 20 to 24% identity), suggesting that Tn 4652 and Tn 5041 are distantly related members of the Tn 3 family. Functional analysis of the tnpA promoter revealed that it is active in Pseudomonas putida but silent in Escherichia coli , indicating that some P. putida -specific factor is required for the transcription from this promoter. Additionally, tnpA promoter activity was shown to be modulated by integration host factor (IHF). The presence of an IHF-binding site upstream of the tnpA promoter enhanced the promoter activity. The positive role of IHF was also confirmed by the finding that the enhancing effect of IHF was not detected in the P. putida ihfA -deficient strain A8759. Moreover, the Tn 4652 terminal sequences had a negative effect on transcription from the tnpA promoter in the ihfA -defective strain. This finding suggests that IHF not only enhances transcription from the tnpA promoter but also alleviates the negative effect of terminal sequences of Tn 4652 on the promoter activity. Also, an in vitro binding assay demonstrated that both ends of Tn 4652 bind IHF from a cell lysate of E. coli.