Involvement of sigma(S) in starvation-induced transposition of Pseudomonas putida transposon Tn4652

Abstract

Transpositional activity of mobile elements can be induced by different environmental stresses. Here, we present evidence that transposition of Tn4652 is elevated in stationary-phase Pseudomonas putida and suppressed in an isogenic sigma(S)-defective strain. We demonstrate that transcription from the Tn4652 transposase promoter is controlled by the stationary-phase-specific sigma factor sigma(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.

FIG. 1

(A) Schematic presentation of transposition target region in plasmid pEST1332. Catechol 1,2-dioxygenase (pheB) and phenol monooxygenase (pheA) genes are indicated by grey boxes. Vector DNA of pAYC32 is depicted with a line. Different insertion sites of Tn4652 are indicated with arrows. (B) Accumulation of Phe⁺ mutants on phenol minimal plates is indicated for P. putida strain PaW85 (wt) and isogenic rpoS-defective strain PKS54 (rpoS) containing plasmid pEST1332. Each point represents the mean of five independent determinations, and error bars represent standard deviations. Dashed lines indicate the theoretical appearance of Tn4652-linked Phe⁺ mutants deduced from the results of PCR analysis of Phe⁺ colonies. Up to 30 Phe⁺ mutants were subjected to analysis on each day. (C) Viability of P. putida PaW85 (wt) and PKS54 (rpoS) carrying plasmid pAYC32 on phenol minimal plates. Each point represents the mean of five independent measurements, and error bars represent standard deviations. 1,0E + 08, e.g., marks 10⁸ viable cells.

FIG. 2

Western immunoblot analyses of Tn4652 TnpA in P. putida strain PaW85 (wt) and rpoS-defective strain PKS54 (rpoS) containing TnpA-expressing plasmid pKTtnpA(D/H). About 40 μg of crude cell lysate was loaded per lane.

FIG. 3

(A) Sequence of right end of Tn4652 containing promoter region of tnpA. The 46-bp inverted repeat is in boldface italics. The −10 hexamer of the tnpA promoter is boxed, and the transcription start of tnpA (11) is indicated by an asterisk. The potential IHF binding site is underlined. (B) Growth-dependent expression of tnpA promoter. P. putida wild-type strain PaW85 (wt) and its rpoS mutant PKS54 (rpoS) carrying either pKTlacZS/C or pKTlacZD/C were grown on Luria-Bertani medium. Plasmid pKTlacZD/C lacks the 57 nucleotides (up to the DraI restriction site; for details, see the description for panel A) of the Tn4652 right end sequence. Data (mean ± standard deviation) of at least four independent experiments are presented. OD580, optical density at 580 nm.