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J Bacteriol, June 1998, p. 2822-2829, Vol. 180, No. 11
Estonian Biocentre and Institute of Molecular
and Cell Biology, Tartu University, EE2400 Tartu, Estonia
Received 17 December 1997/Accepted 29 March 1998
Tn4652 is a derivative of the toluene degradation
transposon Tn4651 that belongs to the Tn3
family of transposons (M. Tsuda and T. Iino, Mol. Gen. Genet.
210:270-276, 1987). We have sequenced the transposase gene
tnpA of transposon Tn4652 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 Tn5041. Homology with other Tn3
family transposases was only moderate (about 20 to 24% identity),
suggesting that Tn4652 and Tn5041 are distantly
related members of the Tn3 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
Tn4652 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
Tn4652 on the promoter activity. Also, an in vitro binding assay demonstrated that both ends of Tn4652 bind IHF from a
cell lysate of E. coli.
Transposons are discrete DNA
segments that can move from one genetic location to another. This
process does not involve homologous recombination systems of the host
but requires a gene product encoded by the moving element
itself Transposition of a mobile element is precisely controlled and depends
on the availability of the active transposase. Moreover, in several
cases the transposition reaction itself is controlled and modulated by
some other transposon-encoded protein(s) and/or host factors
(29). One of the host factors participating in the
transposition is integration host factor (IHF) (17, 32, 41,
42).
IHF is a sequence-specific sharply DNA bending heterodimeric
protein which is involved in a variety of cellular processes including
Many mobile DNA elements carry IHF-binding sites at one or both termini
(14, 18, 25, 32, 46). For According to Kleckner (28), transposable elements from
bacteria can be divided into three classes. Class II contains
evolutionarily related elements mostly belonging to the Tn3
family of transposons. Tn3 family transposons translocate
replicatively and generate 5-bp direct duplications of the target DNA
(40). Members of the Tn3 family exhibit similar
inverted repeats 35 to 48 bp in length and similar transposases.
Comparison of the Tn3 family transposases showed their
clustering into three subgroups (26). Tn3 and
Tn21 subgroups associate transposons from gram-negative bacteria, while transposons from gram-positive bacteria belong to the
third subgroup. Transposases of IS1071 and recently
characterized mercury resistance transposon Tn5041 are more
diverse and cannot be included to any of these three subgroups
(26).
Pseudomonas putida PaW85 carries in its chromosome
transposon Tn4652, a 17-kb derivative of the 56-kb toluene
degradation transposon Tn4651 coding for xyl
genes (43). Tsuda and Iino (43) have shown that
Tn4652 belongs to the Tn3 family of transposons, as determined from its transposition properties. Genetic analysis on
Tn4652 localized the putative transposase gene to a 3.0-kb segment at the end of the right arm of the element (43).
However, regulation of the Tn4652 transposase gene as well
as the mechanism of transposition reactions of Tn4652 have
remained unexplored.
This study aims to elucidate the regulation of the Tn4652
transposase gene. We sequenced the Tn4652 transposase gene
tnpA and localized the promoter of the gene to the right end
of the element. Analysis of the deduced amino acid sequence of the
tnpA gene revealed highest homology (96.2% identity) with
the transposase of Tn5041. Study of the regulation of the
tnpA promoter from Tn4652 demonstrated that (i)
the promoter was active in P. putida but silent in
Escherichia coli and (ii) the IHF-binding site at positions Bacterial strains, plasmids, and media.
The bacterial
strains and plasmids used in this study are described in Table
1. Construction of the new
broad-host-range promoter-probe vector pKTlacZ is depicted in Fig.
1. Bacteria were grown on LB medium
(33). Antibiotics were added at the indicated final
concentrations: for E. coli, ampicillin at 100 µg/ml and
tetracycline at 15 µg/ml; for P. putida, carbenicillin at
1,500 µg/ml and streptomycin at 500 µg/ml. P. putida was
incubated at 30°C, and E. coli was incubated at 37°C.
Early-stationary-phase cultures were used for enzyme assays. E. coli was transformed with plasmid DNA as described by Hanahan
(23). P. putida was electrotransformed by using
the protocol of Sharma and Schimke (39).
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Expression of the Transposase Gene tnpA
of Tn4652 Is Positively Affected by Integration Host
Factor
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
transposase. Transposase interacts site specifically with the
ends of the transposon, cleaves the DNA at both termini of the element,
and carries out the strand transfer reaction (reviewed in references
22 and 34).
site-specific recombination, transposition, replication, and
positive and negative control of gene expression (15). IHF has been found to regulate gene expression in a number of gram-negative bacteria (21). IHF genes from diverse bacterial species are well conserved (8, 12). In most cases, the role of IHF is architectural: it facilitates the formation of nucleoprotein complexes through strong bending of DNA. However, activation of transcription from pL1 and Mu phage Pe promoters involves direct interaction of
IHF with RNA polymerase (20, 44).
(Tn1000), it was
shown that binding of IHF to the ends of the transposon facilitates binding of transposase (46). Mostly, IHF affects
transposition positively (10, 35, 42). For example, in the
well-studied Mu phage transposition, IHF acts positively both by
enhancing transcription from the early promoter Pe and favoring the
stabile synaptic complex formation that is required in the initial step of transposition (2, 44). However, there are also reports about the negative role of IHF on transposition (17, 41).
73 to 85 relative to the transcription start point affected transcription from the tnpA promoter in P. putida
positively. Gel mobility shift experiments with cell lysates of
E. coli and P. putida were carried out to examine
binding of IHF to the ends of Tn4652 in vitro.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used
View larger version (15K):
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FIG. 1.
Map of the broad-host-range promoter probe vector
pKTlacZ. An about 5-kb HindIII-PstI fragment
carrying the lacZ gene originates from plasmid pKRZ-1
(37). After this fragment was cloned into pBluescriptSK(+)
it was recut with XhoI and SmaI and subcloned
into pKT240 opened with XhoI and Ecl136II.
Suitable cloning sites are BamHI, HindIII
(two sites), SmaI, XhoI, and SalI.
DNA manipulations and mRNA mapping. DNA sequencing was performed with a Sequenase version 2.0 DNA sequencing kit (Amersham). Subclones of the tnpA promoter region (Table 1) were obtained by cloning PCR products. The following oligonucleotides, containing suitable restriction sites (SacI and ClaI; boldfaced) and complementary to nucleotides (nt) 1 to 21, 40 to 63, and 101 to 122 relative to the right end of the Tn4652, were used in cloning: Osac (5'-CGTGAGCTCGGGGTTATGCCGAGATAAGGC-3'), Oihf (5'-CGTGAGCTCTGTAAATATATGATTTAAAAGG-3'), and Ocla (5'-CGTATCGATCAGCATAGACGGCTAGCCAG-3'). Locations of these oligonucleotides are shown in Fig. 2A.
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120 to 136
relative to the start codon of the reporter gene pheB, was
used in the primer extension analysis.
Enzyme assays.
The catechol 1,2-dioxygenase (C12O) assay was
carried out as described by Hegeman (24). The
-Galactosidase (-Gal) assay was performed as specified by Miller
(33). Protein concentration in cell lysates was measured by
the Bradford method (7).
Gel mobility shift assay. Cell lysates used in gel shift assays were prepared from 30-ml early-stationary-phase cultures. The cells were pelleted and sonicated in 1× binding buffer (25 mM Tris-HCl [pH 7.5], 0.05 mM EDTA, 5 mM dithiothreitol, 25 mM NaCl, 50 mM KCl, 5% glycerol). Protein concentration in cleared lysates was 15 to 20 mg/ml; 1 to 3 µl of undiluted lysate or lysate diluted in 1× binding buffer was used in gel shift assays.
The following DNA fragments were used in gel shift binding assays: (i) a 108-bp DNA restriction fragment containing the right end of transposon Tn4652 up to the NheI restriction site (Fig. 2A); (ii) a 140-bp DNA restriction fragment containing the left end of the transposon up to the Bpu1102I restriction site (Fig. 2B); and (iii) a 140-bp DNA restriction fragment containing a 129-bp DpnI segment of the Pu promoter region cloned into pUC18 (Table 1). These DNA fragments were end labeled with [
-32P]dCTP, using the Klenow fragment of DNA
polymerase I, and subsequently purified through an polyacrylamide gel.
The binding reaction was carried out in a volume of 20 µl. About 1 ng
(1,000 cpm) of DNA probe was incubated at 20°C for 20 min with
different cell lysates in 1× binding buffer containing 1 µg of
bovine serum albumin and 5 µg of salmon sperm DNA. The following
specific nonlabeled competitor DNAs containing IHF-binding sites were
generated by PCR: (i) a 122-bp fragment of the right end of
Tn4652, amplified by using primers Osac and Ocla (Fig. 2A);
(ii) a 132-bp fragment of the left end of Tn4652, amplified
by using primers Osac and Ohind (5'-CGTAAGCTTCCTCAATGGATGGCTGAAG-3' [Fig. 2B]);
and (iii) a 250-bp DNA fragment including a 129-bp DpnI
segment of the Pu promoter region cloned into pUC18 (Table 1),
amplified by using pUC18 reverse and forward primers. When the specific
competitor DNA was used, the cell lysate was added last to the binding
reaction. After incubation, the reaction mixture was loaded on a
1-h-prerun 5% nondenaturing polyacrylamide gel. Electrophoresis was
carried out at room temperature in 0.5× Tris-borate-EDTA buffer at 10 V/cm for 2 h. The gels were dried and autoradiographed or exposed to a phosphorimager screen.
Nucleotide sequences accession numbers. The 3,348-bp sequence of the right arm of Tn4652 has been assigned accession no. X83686 in the EMBL database. The accession number of the 604-bp-long sequence of the left end of Tn4652 is X83687.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Sequence of the Tn4652 transposase shows highest homology with the putative transposase of Tn5041. Genetic analysis has localized the transposase gene of Tn4652 to the right arm of the transposon (43). A 3.2-kb DraI-HindIII fragment from Tn4652 DNA, known to contain the transposase gene tnpA, was subcloned into the pBluescript KS(+) vector. Sequencing of the DNA fragment revealed a single 3,012-bp open reading frame (ORF) directed inward from the right end of the transposon. The ORF has two potential ATG start codons, separated by 6 bp (Fig. 2A). Since the potential ribosome-binding site overlaps the first ATG, initiation of translation of tnpA from the second ATG is more likely. The predicted protein, starting from the second ATG, is 1,001 amino acids long, with a calculated molecular mass of 114 kDa. Sequence comparison with the translated sequences of genes in the EMBL database by using the FASTA and BLAST programs revealed a high degree of homology of the Tn4652 tnpA with the putative transposase of the mercury resistance transposon Tn5041 (96.2% identity). Homology with other transposases of Tn3 family transposons (Tn501, Tn1721, Tn1546, Tn21, Tn4430, Tn3926, Tn2501, Tn3, Tn4556, Tn1000, and IS1071) was much lower (about 20 to 24% identity and 30 to 36% similarity). In most of the Tn3 family transposons, the 3' ends of the transposase genes terminate within one of the terminal repeats of the element (40). Contrary to that, the direction of the tnpA gene of Tn4652 is opposite, starting from the right end of the transposon. Multiple alignment of Tn3 family transposase sequences homologous to Tn4652 transposase was performed via the CBRG server (http://cbrg.inf.ethz.ch/) by using the Darwin program. Alignment revealed stronger conservation in C termini of these proteins (data not shown). The phylogenetic tree of the entire protein sequences demonstrated that the Tn4652 transposase is quite distantly related to other members of the Tn3 family and might constitute a new Tn3 family subgroup together with Tn5041 (Fig. 3).
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Mapping of the tnpA promoter. The ORF of tnpA gene starts at 152 bp from the right end of transposon Tn4652. To map the tnpA promoter, we constructed plasmid p1332S/C by cloning the 122-bp DNA segment covering the right end of the transposon upstream of the promoterless pheBA operon in plasmid pEST1332 (Table 1 and Fig. 2A). In addition, plasmids p1332D/C and p1332S/N, containing Tn4652 right-end DNA from nt 58 to 122 and from nt 1 to 104, respectively, were constructed (Table 1 and Fig. 2A). E. coli HB101 and P. putida PaW85 and PRS2000 were transformed with these plasmids. As many transposase promoters are downregulated by transposon-encoded repressor proteins (29), the transposon Tn4652-free P. putida strain PRS2000 was used as a reference strain to distinguish potential effects of chromosomally encoded transposon protein(s) on the promoter activity in strain PaW85.
The C12O assay was carried out to study expression of the reporter gene pheB in the plasmids constructed. P. putida PaW85 and PRS2000 harboring plasmids p1332S/C and p1332D/C revealed promoter activity (Fig. 4A), but no C12O activity was detected in bacteria carrying plasmid p1332S/N, indicating that the tnpA promoter was disrupted in this construct (data not shown). Data in Fig. 4A show that C12O activities measured in P. putida PRS2000 were more than twofold higher than those measured in P. putida PaW85. Additionally, bacteria harboring p1332S/C revealed about twofold-higher enzyme activities than bacteria containing p1332D/C. None of the promoter constructs studied revealed activity in E. coli (Fig. 4A).
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70-recognized promoter consensus
TATAAT, was found 10 bp upstream of the transcription start
point. However, the 35 region of the promoter was not homologous with
70-recognized consensus hexamer TTGACA.
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The IHF-binding site affects positively transcription from the
tnpA promoter.
Results presented in Fig. 4A suggest
that the region from bp 1 to 56 bp of the right end of
Tn4652 has a positive effect on the transcription from the
tnpA promoter (compare p1332S/C and p1332D/C). Sequence
analysis of the transposon right end revealed a potential IHF-binding
site flanking the DraI site in p1332S/C (Fig. 2A). To test
the effect of the presence of an IHF-binding site upstream of the
tnpA promoter on expression of the reporter gene, the enzyme
assay using the widely used -Gal reporter system was performed.
For that purpose, we constructed plasmids pKTlacZS/C, pKTlacZIHF/C, and pKTlacZD/C by cloning different DNA
fragments from the tnpA promoter region (bp 1 to 122, 39 to
122, and 58 to 122, respectively) upstream of the -Gal gene
lacZ in the broad-host-range vector pKTlacZ (Table 1, Fig.
1, and Fig. 2). Plasmid pKTlacZIHF/C, which contains an IHF site
adjacent to the DraI site, lacks the last 39 bp from the
transposon end. Results of the -Gal assay presented in Fig. 4B
confirmed previous data obtained with the C12O reporter system (Fig.
4A): the presence of the Tn4652 terminal sequences (bp 1 to
57) upstream of the tnpA promoter enhances transcription
from the promoter. Moreover, while in the C12O reporter system the
positive effect was nearly twofold, the -Gal system exhibited
five- to sixfold enhancement. In Tn4652-free
P. putida PRS2000, the presence of an IHF-binding site
upstream of the DraI site was sufficient to complement the
positive effect of the transposon right end to the tnpA
promoter activity (Fig. 4B; compare pKTlacZIHF/C with pKTlacZD/C and
pKTlacZS/C). However, in Tn4652-containing P. putida PaW85, the positive effect of an IHF-binding site in plasmid p1332IHF/C was lower than in p1332S/C (Fig. 4B).
-Gal reporter was used in E. coli (data not
shown).
tnpA promoter activity in ihfA-deficient
P. putida A8759.
To elucidate the role of IHF in
the tnpA promoter activity, a C12O assay using P. putida KT2442 and in its ihfA-deficient derivative
P. putida A8759 was carried out. Usage of the -Gal reporter system was excluded since both of these strains carry a copy
of the lacZ gene under the control of the Pu promoter in the
chromosome (Table 1). In addition to plasmids p1332S/C and p1332D/C
characterized before, plasmid p1332IHF/C was constructed analogously to
pKTlacZIHF/C (Table 1, Fig. 1, and Fig. 2). Figure 4C shows that
enzyme activities in ihfA-deficient P. putida A8759 harboring either p1332S/C or p1332IHF/C were about
twofold lower than in bacteria carrying plasmid p1332D/C. Thus, the DNA
region containing the IHF-binding site had no enhancing effect on the tnpA promoter activity in the ihfA-deficient
P. putida strain. In contrast, an obvious negative
effect of terminal sequences of Tn4652 on transcription from
the tnpA promoter could be seen in the
ihfA-deficient P. putida strain A8759.
E. coli IHF specifically binds to both ends of
Tn4652.
Sequence analysis of the left terminus of
Tn4652 revealed two potential IHF-binding sites from bp 44 to 56 and from bp 59 to 71 (Fig. 2B). To test the possibility that IHF
can bind to both ends of the transposon, a gel mobility shift assay was
carried out. For binding reactions, crude lysates prepared from both
E. coli and P. putida PaW85 cells were
used. Figure 6 demonstrates that IHF from
E. coli specifically retards DNA fragments containing either the left end (Fig. 6A) or right end (Fig. 6B) of the transposon. No probe retardation was detected when cell extract from E. coli WM2017 defective for IHF was used (Fig. 6, lanes 3, 8, and
9). Complementation of this IHF-negative strain with plasmid pHN carrying ihfA and ihfB restored the shift (Fig.
6, lanes 4, 10, and 11). However, we could not detect any specific
shift with cell lysate from P. putida PaW85 either with
the right end or with the left end of the transposon (data not shown).
Additionally, a gel shift assay with the DNA fragment of the Pu
promoter region known to contain an IHF-binding site (1, 13)
was carried out as a control to test whether this site binds IHF from
cell lysate of P. putida. However, although the DNA
segment of the Pu promoter region specifically bound IHF from
E. coli (Fig. 6C, lanes 13 and 14), no probe
retardation was detected in the cell lysate from P. putida (Fig. 6C, lanes 17 and 18).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Many transposons require bacterial host proteins for transposition. IHF is known to participate in transposition of several transposons (32, 41, 42, 47), and it also modulates transposase expression in some cases (44). The experiments presented in this report show that transcription from the Tn4652 transposase promoter is positively affected by IHF.
We found that both ends of Tn4652 contain sequences similar
to the IHF-binding consensus sequence (Fig. 2). The putative
IHF-binding site at the right end of the transposon is located at
positions from 73 to 85 relative to the transcription start point
of the tnpA gene. Transposase promoter constructs carrying
sequences of the right end of Tn4652 including an
IHF-binding site revealed enhanced activity of the reporter gene
pheB or lacZ in P. putida in
comparison with the constructs lacking the IHF site upstream of the
tnpA promoter (Fig. 4A and B). Enzyme assay using the
P. putida ihfA-defective strain A8759 confirmed that
IHF was involved in stimulation of transcription from the
tnpA promoter. No positive effect of the IHF-binding site on
promoter activity was detected in this strain (Fig. 4C). In contrast,
the right end of the transposon had a negative effect on
tnpA promoter activity when IHF was absent: both constructs
p1332S/C and p1332IHF/C containing the IHF site exhibited even lower
enzyme activity than p1332D/C in the ihfA-defective strain
A8759 (Fig. 4C). This finding indicates that the IHF site, if not
occupied by IHF protein, can suppress the tnpA promoter activity. It is known that IHF is involved in activation of the Pe
promoter of bacteriophage Mu by a dual mechanism. IHF stimulates transcription from the Pe promoter directly and also indirectly via alleviation of the H-NS-mediated repression (44).
Analogously, we suggest that binding of IHF to the right end of
Tn4652 enhances transcription from the tnpA
promoter not only directly but also indirectly by competing with some
unknown negatively acting factor for the binding site.
Enzyme assay demonstrated that transcription from the tnpA promoter was higher in the Tn4652-free P. putida strain PRS2000 than that in strains PaW85 and KT2442, which contain a copy of Tn4652 in the chromosome (Fig. 4A and C). We propose that the chromosomally located copy of Tn4652 may code for functions affecting the tnpA promoter activity in P. putida PaW85 and KT2442. Since terminal sequences of transposons are presumed to bind transposase, it is possible that transcription from the tnpA promoter is modulated by the transposase of Tn4652, too.
Enzyme assay revealed that the Tn4652 tnpA gene promoter is
silent in E. coli (Fig. 4A). Comparison of promoter
specificities of RNA polymerases from E. coli and
Pseudomonas spp. revealed that they transcribe similarly
well different promoters of both species (16, 19).
Considering these experiments, we do not believe that the difference
between the E. coli and P. putida polymerases causes the silence of the tnpA promoter in
E. coli. The possibility that activation of the
transcription from the promoter needs some Tn4652-encoded
factor could be also eliminated because the promoter is functional in
Tn4652-free P. putida strain PRS2000. Thus,
the presence of some host factor specific to P. putida
is required for the promoter function. We propose two alternative explanations for the silence of the tnpA promoter in
E. coli. First, transcription initiation from the
tnpA promoter needs an activator protein that is missing in
E. coli. Many 70-dependent promoters
lacking a well-conserved 35 region are known to be subjected to
activation by the regulatory proteins (9). Correspondingly,
the 35 region of the tnpA promoter revealed no homology
with the 70-recognized 35 consensus hexamer although
the 10 region TATGCT of the tnpA promoter was
considerably homologous with the 70-recognized 10
hexamer consensus sequence TATAAT. On the other hand, the
tnpA promoter might not be necessarily recognized by 70. Therefore, an alternative sigma factor, absent in
E. coli, might be required for promoter activation.
This possibility is illustrated by the fact that alternative sigma
factors of pseudomonads, not complemented in E. coli,
are essential for the expression of several iron-regulated promoters of
Pseudomonas strains (11, 38).
Up to now, there had been no reports about in vitro binding experiments with P. putida IHF. Using the gel mobility shift assay, we demonstrated that both ends of Tn4652 can bind IHF from cell lysate of E. coli (Fig. 6A and B). However, we could not detect an IHF-caused shift under the same conditions when the cell extract of P. putida PaW85 was used. We have also carried out gel shift experiments with lysate of an E. coli IHF-defective mutant complemented with plasmids carrying cloned IHF genes of P. putida. However, we did not detect IHF-caused retardation of a DNA fragment containing the right end of the transposon or the Pu promoter region of the TOL plasmid as a control (data not shown). This indicates that the properties of P. putida and E. coli IHF are different, and the experimental conditions used in in vitro binding assay were not optimal for the binding of P. putida IHF. However, in vivo experiments with hybrid IHF protein containing P. putida and E. coli subunits have showed that the hybrid protein efficiently functioned as a regulator of the pL promoter in E. coli (8), which suggests that in vivo binding properties of the hybrid IHF protein may be similar to those of E. coli IHF. Nevertheless, it would be interesting to compare the properties of IHF purified from P. putida with that from E. coli.
Gel mobility shift experiments with the transposon right-end DNA probe and crude lysate of P. putida PaW85 confirmed formation of two specific complexes (C1 and C2). Probably neither of them corresponded to IHF bound to the probe (Fig. 7), because these complexes were also detected by using cell lysate from P. putida ihfA-defective strain A8759 (Fig. 7A). Also, neither the Tn4652 left-end nor the Pu promoter-region DNA containing an IHF site suppressed formation of these complexes. Therefore, we consider that complexes detected by using the right end of the transposon represent some other protein(s) bound to the probe. Since C1 and C2 were formed with cell lysate from Tn4652-free P. putida PRS2000 as well (data not shown), we suggest that some P. putida host protein(s) participates in these complexes. Although the identity of the protein(s) is not established, it is tempting to speculate that complexes C1 and C2 contain the repressor protein which acts negatively on transcription from the tnpA promoter in a P. putida ihfA-deficient strain (Fig. 4C). Still, we cannot exclude the possibility that an activator, essential for the activity of the tnpA promoter in P. putida, was bound to the right end of Tn4652 in the gel shift assay. However, further experiments are needed to test these possibilities.
Our results demonstrate that IHF from E. coli binds
specifically to both ends of Tn4652, just adjacent to the
terminal inverted repeats that are presumed to bind the transposase.
Other mobile elements are also known to contain IHF-binding sites at
one or both ends (18, 25, 32, 46). It is known that
transposase of (Tn1000) transposon and IHF bind
cooperatively to both ends of the element (46).
Additionally, IHF is required in in vitro reactions of IS10
transposition (35). Therefore, we suggest that besides
activation of the tnpA promoter, IHF may participate in
Tn4652 transposition also either by modulating the binding of transposase to the ends of the transposon or by influencing formation of nucleoprotein complexes needed in subsequent transposition reactions.
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ACKNOWLEDGMENTS |
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We thank V. de Lorenzo for kindly providing P. putida KT2442 and A8759, W. Messer for E. coli
WM2015 and WM2017, J. F. Gardner for plasmid pHN, and
A. M. Chakrabarty for plasmid pKRZ-1. We also thank T. Alamäe, V. Kõiv, and A. Tamm for critically reading the manuscript and for helpful discussions. We are grateful to A. Abroi for discussions and for obliging help in the computer analysis.
This work was supported by grant 2323 from the Estonian Science Foundation, grant LCO000 from the International Science Foundation, and grant LKH100 from the Joint Program of the Government of Estonia and the International Science Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Estonian Biocentre and Institute of Molecular and Cell Biology, Tartu University, 23 Riia Str., EE2400 Tartu, Estonia. Phone: 372-7-465015. Fax: 372-7-420286. E-mail: rhorak{at}ebc.ee.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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