Transcription from fusion promoters generated during transposition of transposon Tn4652 is positively affected by integration host factor in Pseudomonas putida

Abstract

We have previously shown that both ends of the Tn3 family transposon Tn4652 contain integration host factor (IHF) binding sites and that IHF positively regulates expression of the Tn4652 transposase gene tnpA in Pseudomonas putida (R. Hõrak, and M. Kivisaar, J. Bacteriol. 180:2822-2829, 1998). Tn4652 can activate silent genes by creating fusion promoters during the transposition. The promoters are created as fusions between the -35 hexamer provided by the terminal inverted repeats of Tn4652 and the -10 hexamers in the target DNA. Two fusion promoters, PRA1 and PLA1, that contain sequences of the right and left termini of Tn4652, respectively, were chosen for the study of mechanisms of transcription activation. Gel mobility shift analysis using crude extracts from P. putida cells allowed us to detect specific binding of P. putida IHF to the ends of the transposon Tn4652. We found that the rate of transcription from the fusion promoter PRA1 is enhanced by IHF. Notably, the positive effect of IHF on transcription from the promoter PRA1 appeared only when cells of P. putida reached the stationary growth phase. We speculate that the intracellular concentration of IHF might be critical for the in vivo effect of IHF on transcription from the fusion promoters in P. putida. In the case of PLA1, the mechanism of transcription modulation by IHF is different than that observed for PRA1. Our results demonstrate that transcription of neighboring genes from outwardly directed promoters at the ends of a mobile DNA element could be influenced by the same factors that control transposition of the element.

FIG. 1

Nucleotide sequence of the fusion promoters PRA1 (A) and PLA1 (B). Fusions between −35 hexamer provided by the inverted repeats of Tn4652 and −10 hexamers found in the target DNA upstream of the pheBA genes created these promoters (36). The sequences of Tn4652 are shaded. Locations of −10 and −35 hexamers of the fusion promoters are indicated above the sequence. The upstream region of the promoter PRA1 overlaps with the oppositely directed promoter region of the Tn4652 transposase gene tnpA. Location of −10 hexamer of the tnpA promoter is shown below the sequence of the right end of Tn4652. Transcription start sites for the promoters, determined by primer extension experiments, are indicated by arrows. The potential IHF binding sites at the ends of Tn4652 resembling the E. coli IHF binding consensus sequence WATCAANNNNTTR are indicated above the sequences of the promoters PRA1 and PLA1 by black bars. Restriction sites relevant to the experiments presented in this paper are shown.

FIG. 2

Schematic representation of PRA1 and PLA1 promoter constructs used in this study. Plasmids pRA1, pRA1-7, and pRA1-12 (A) carry the sequences of the right end of Tn4652 extending to the DraI, HaeIII, and Eco47III sites, respectively. Plasmids pLA1, pLA1-4, pLA1-5, and pLA1-12 (B) contain the sequences of the left end of Tn4652 sequences upstream of the fusion promoter PLA1 extending to the DraI, BstUI, EcoRV, and HaeIII sites, respectively. All the plasmids are promoter-pheBA fusions in pEST1332 (29). Plasmids pRA1 and PLA1 were constructed as in our previous study (36). The promoters were initially cloned into pBluescript SK, and multicloning sites SacI and ClaI were used in order to reclone them upstream of the pheBA genes in plasmid pEST1332. The large spotted arrow represents the tnpA gene of Tn4652, and the checked arrows represent the pheB gene. Open boxes designate noncoding sequences of Tn4652, and thick lines show noncoding sequences of plasmid pEST1332 locating between the reporter gene pheB and promoters PRA1 or PLA1. Grey regions indicate the locations of −10 and −35 elements of the promoters. The small arrows denote transcription start sites of the pheB gene and the tnpA gene. The IHF binding consensus-resembling sequences at the ends of Tn4652 are indicated by brackets.

FIG. 3

Study of the effects of upstream sequences and IHF on transcription from the fusion promoters PRA1 (A) and PLA1 (B and C) by comparison of the levels of expression of C12O activity in the wild-type strain P. putida KT2442 and in its ihfA knockout derivative A8759. C12O activity was measured at different time points either in exponentially growing or stationary-phase cells of P. putida KT2442. Data (means ± standard deviations) of at least four independent experiments are presented. The growth curve of P. putida KT2442 in LB is shown (D). The growth rate of the strain A8759 is similar to that of KT2442 (not shown).

FIG. 4

Gel shift assay of in vitro binding of IHF from cell lysates of P. putida and E. coli to the ends of Tn4652. Cell lysates used were from P. putida wild-type strain KT2442 (lanes 7 and 17), P. putida A8759 defective in the ihfA gene (lanes 6 and 16), P. putida RT31 carrying the ihfA and ihfB genes under the control of Ptac promoter and lacI^q repressor (lanes 1 to 5 and 11 to 15), E. coli wild-type strain WM2015 (lanes 9 and 19), and E. coli WM2017 defective in ihfA and ihfB genes (lanes 8 and 18). No cell lysate was added to the reaction mixture in the case of lanes 10 and 20. The protein-DNA complex visible on lane 8 is of unknown origin. All lysates were prepared from stationary-phase cells.

FIG. 5

(A) Gel shift assay demonstrating suppression of the formation of the P. putida IHF complex with the right end of Tn4652 by nonlabeled DNA fragment containing Pu promoter region. Cell lysates used were from P. putida KT2442. (B) Gel shift assay of in vitro binding of P. putida and E. coli IHF to the ends of Tn4652 lacking A-T-rich regions upstream of the IHF binding core sequence (DNA probes from plasmids pRA1 and pLA1) and in the presence of A-T-rich regions (DNA probes from plasmids pRA1-7 and pLA1-4). Lysates used were from E. coli WM2015 (lanes 8, 12, 16, and 20) and P. putida KT2442 (lanes 6, 10, 14, and 18) and RT31 grown in the presence of 1 mM IPTG (lanes 7, 11, 15, and 19). No cell lysate was added to the reaction mixture in lanes 9, 13, 17, or 21. All lysates were prepared from stationary-phase cells.

FIG. 6

Study of the effect of different intracellular concentrations of IHF on transcription from the promoter PRA1 in plasmids pRA1 and pRA1-12 (A, exponentially growing cells; B, stationary-phase cells) and from the promoter PLA1 in plasmids pLA1 and pLA1-12 (C, exponentially growing cells; D, stationary-phase cells) containing different lengths of upstream sequences. P. putida RT31 carrying the ihfA and ihfB genes under the control of Ptac promoter and lacI^q repressor was grown in LB in the presence or absence of inducer IPTG. Exponentially growing cells were sampled at 6 h, and stationary-phase cells were sampled at 16 h. Data (means ± standard deviations) of at least four independent experiments are presented.

FIG. 7

Gel shift assay of IHF binding to the right end of Tn4652 in cell extracts prepared from different growth phases of cells of P. putida KT2442. No cell lysate was added to the reaction mixture in lane 5. Another complex moving faster than the IHF-specific complex is designated as X. The growth curve of this bacterium is shown in Fig. 3.