Regulation of the transposase of Tn4652 by the transposon-encoded protein TnpC

Abstract

Transposition is a DNA reorganization reaction potentially deleterious for the host. The frequency of transposition is limited by the amount of transposase. Therefore, strict regulation of a transposase is required to keep control over the destructive multiplication of the mobile element. We have shown previously that the expression of the transposase (tnpA) of the Pseudomonas putida PaW85 transposon Tn4652 is positively affected by integration host factor. Here, we present evidence that the amount of the transposase of Tn4652 in P. putida cells is controlled by the transposon-encoded protein (TnpC). Sequence analysis of the 120-amino-acid-long TnpC, coded just downstream of the tnpA gene, showed that it has remarkable similarity to the putative polypeptide encoded by the mercury resistance transposon Tn5041. As determined by quantitative Western blot analysis, the abundance of TnpA was reduced up to 10-fold in the intact tnpC background. In vivo experiments using transcriptional and translational fusions of the tnpA gene and the reporter gene gusA indicated that TnpC operates in the regulation of the transposase of Tn4652 at the post-transcriptional level.

FIG. 1

Sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis demonstrating overexpression and purification of His-tagged TnpA in E. coli BL21(DE3). Lane 1, crude extract from E. coli BL21(DE3)(pET19-tnpA); lane 2, as described for lane 1, but induced with 0.4 mM IPTG; lane 3, purified His-TnpA; lane 4, standard molecular weight markers.

FIG. 2

Western immunoblot analyses of P. putida PaW85 and PRS2000 cell lysates by using anti-TnpA polyclonal antibodies. Lane 1, purified TnpA protein; lane 2, crude extract from P. putida PaW85(pEST1354); lane 3, crude extract from P. putida PaW85[pKTtnpA(D/H)]; lane 4, crude extract from P. putida PRS2000(pEST1354); lane 5, crude extract from P. putida PRS2000[pKTtnpA(D/H)]; lane 6, crude extract from P. putida PRS2000[pKTtnpA(D/P)]; lanes 7 through 12, gradual dilutions of crude extracts of P. putida PRS2000[pKTtnpA(D/P)*]; lane 13, crude extract from P. putida PRS2000(pKTGC/tnpA); lane 14, crude extract from P. putida PRS2000(pKTGC/tnpAC). The amount of crude lysate was 40 μg per lane except that for lanes 8 to 12, gradual dilutions of cell lysate of P. putida PRS2000[pKTtnpA(D/P)*] were used.

FIG. 3

Genetic organization of tnpA and tnpC in the right arm of the Tn4652. Right inverted repeat of Tn4652 is marked by a black triangle. Restriction sites relevant to this study are indicated. The arrows indicate the direction of transcription of the tnpA and tnpC genes. The promoter of the tnpA gene is designated p_tnpA.

FIG. 4

Alignment of the deduced amino acid sequence of TnpC of Tn4652 with the putative 120-amino-acid-long polypeptide encoded by Tn5041 (16). Identical amino acids are indicated between the two aligned sequences in boldface. Similar amino acids are marked by plus signs.

FIG. 5

(A) GUS activities measured in P. putida PRS2000 carrying different translational fusion plasmids either together with the tnpC gene or without the tnpC gene. Na-benzoate (10 mM) was used for the induction of tnpC. Data (means ± standard deviations) of at least five independent experiments are presented. (B) Schematic presentation of the translational fusions of the 5′ end of the tnpA gene with the reporter gene gusA. For each fusion, the PDEL2-GC promoter is indicated by an open box, the 5′ region of tnpA is marked by a line, and the translation initiation codon ATG of tnpA is indicated by a black diamond.

FIG. 6

(A) GUS activities measured in P. putida PRS2000 carrying the different transcriptional fusions of the tnpAC region with the reporter gene gusA. Plasmids with disrupted tnpC are marked by asterisks in the text. Data (means ± standard deviations) of at least five independent experiments are presented. pNP, p-nitrophenol; OD590, optical density at 590 nanometers. (B) Schematic depiction of plasmids with transcriptional fusions employed in GUS activity assays. Restriction sites used for construction of deletion derivatives of pKT-ACG are indicated. The EcoRI restriction site in the 3′ end of tnpC is artificial, designed by using oligonucleotide TnpCEco (Materials and Methods). The direction of transcription from the tnpA promoter is indicated by an arrow.