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J Bacteriol, May 1998, p. 2682-2688, Vol. 180, No. 10
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Negative Regulation of IS2 Transposition
by the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex
Shiau-Ting
Hu,*
Hsuan-Chen
Wang,
Guang-Sheng
Lei, and
Shao-Hung
Wang
Department of Microbiology and Graduate
Institute of Microbiology and Immunology, National Yang-Ming
University, Taipei, Taiwan, Republic of China
Received 16 October 1997/Accepted 17 March 1998
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ABSTRACT |
Three sequences similar to that of the consensus binding sequence
of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex were found
in the major IS2 promoter region. Experiments were performed to determine whether the cAMP-CRP complex plays a role in the
regulation of IS2 transposition. In the gel retardation assay, the cAMP-CRP complex was found to be able to bind the major IS2 promoter. A DNA footprinting assay confirmed that the
cAMP-CRP complex binds to the sequences mentioned above. With an
IS2 promoter-luciferase gene fusion construct, the cAMP-CRP
complex was shown to inhibit transcription from the major
IS2 promoter. IS2 was found to transpose at a
frequency approximately 200-fold higher in an Escherichia coli host defective for CRP or adenyl cyclase than in a wild-type host. These results suggest that the cAMP-CRP complex is a negative regulator of IS2 transposition.
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INTRODUCTION |
The insertion sequence
IS2 is a member of the IS3 family (56, 65,
66). It is 1,331 bp in length, with a pair of 42-bp imperfect
inverted repeats (18, 26, 64). The IS2 genome contains five open reading frames (ORF1 to -5) of greater than 50 amino
acids; however, only two IS2-encoded proteins, of 14 and 46 kDa, have been detected (27, 28). The 14-kDa protein, referred to as the InsA protein (27), is encoded by ORF1.
The 46-kDa protein is designated InsAB'. It is encoded by ORF1 and ORF2
via a 
1 frameshift mechanism (28) at the frameshift signal AAAAAAG, which is located between the 3' end of ORF1 and the 5' end of
ORF2 (8, 28, 56). The mRNAs encoding both proteins are
transcribed from the promoter located within the left inverted repeat
(LIR) of IS2. This promoter has been shown to be the major promoter of IS2 (27). The production of InsAB' by
a 1 frameshift mechanism appears to be a general phenomenon in
members of the IS3 family, because it also occurs in
IS150 (56, 78), IS911 (55,
56), and IS3 (69).
The 14-kDa InsA is a DNA binding protein. It binds to the sequence
5'-TATCACTTAAATAAGTGATA-3' (27), which is located
around the 10 sequences of the major IS2 promoter (Fig.
1). Since this promoter is responsible
for the expression of both InsA and InsAB', binding of InsA to this
sequence may affect transcription. This notion is supported by the
observation of a decrease in IS2 transposition when InsA is
overexpressed (27). InsA functions as a homodimer. Dimerization of InsA takes place at the C-terminal end of the molecule,
whereas the DNA binding domain of InsA is located at its N terminus
(38).
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FIG. 1.
Locations of binding sequences for the cAMP-CRP complex
in the major IS2 promoter. The LIR of IS2 is
indicated by a large arrow above the sequence. The solid bars below the
sequence indicate the locations of the 10 and 35 sequences of the
major IS2 promoter. At the bottom, three putative cAMP-CRP
complex binding sequences, region I (IS2 nucleotide
positions 34 to 54), region II (positions 43 to 63), and region III
(positions 42 to 22), present in the promoter area are aligned with the
consensus cAMP-CRP complex binding sequence (matching bases are
underlined). The cAMP-CRP complex binding sequence (positions 37 to 53)
determined by the DNA footprinting experiment is bracketed above the
sequence, and the InsA binding site (positions 33 to 52) is indicated
below the sequence.
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The 46-kDa InsAB' protein has typical transposase motifs WxxD
(36), N3 (61), and C1 (41, 60),
collectively known as the DDE motif (20, 36, 54), located at
its C terminus (28) and a helix-turn-helix DNA binding
motif, TVSLVARQHGVAASQLFLWR, located at its N terminus
(amino acid positions 31 to 50) (38). InsAB' has the ability
to bind both terminal repeats of IS2 (28). Overexpression of InsAB' has been shown to increase IS2
transpositional recombination and formation of two transpositional
products, IS2 minicircles and figure eight molecules
(39). These observations suggest that InsAB' is a
transposase of IS2.
It is possible that host factors may affect IS2
transposition. Examination of the nucleotide sequence of the
IS2 promoter revealed three putative cyclic AMP (cAMP)-cAMP
receptor protein (CRP) complex binding sequences located in the major
IS2 promoter (Fig. 1). Since cAMP-CRP is a global
transcriptional regulator which may activate or inactivate gene
expression (34, 50, 62, 63), we investigated the possible
role of cAMP-CRP in the regulation of IS2 transposition.
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MATERIALS AND METHODS |
PCR.
PCRs were performed in a 100-µl mixture containing 10 ng of template DNA, PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100), 20 pmol of each PCR
primer, 0.2 mM (each) deoxynucleoside triphosphate, and 2 U of
TaqI DNA polymerase. Temperature cycling for PCR included 1 cycle of 95°C for 5 min; 25 cycles of 95°C for 1 min, 48°C for 1 min, and 72.5°C for 2 min; and a 10-min extension at 72.5°C. The
PCR products were electrophoresed on a 1.0% agarose gel to determine
the sizes of the amplified products.
Gel retardation assay.
Cell lysates of Escherichia
coli JM109(DE3) (73) containing pSK
CRP
(Fig. 2A), pT7-7 (74), or
pT7insA (27) were used as the sources of DNA binding
proteins in the gel retardation assay. These cell lysates were prepared
by repeated freezing-thawing and sonication of cells from
rifampin-treated cultures as described previously (27, 38).
Aliquots of clarified cell lysates containing various concentrations of
protein were used. The gel retardation reaction mixture contained the
cell lysate, a 32P-labeled DNA fragment, 50 mM Tris-HCl (pH
7.4), 70 mM KCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, 7 mM
MgCl2, 3 mM CaCl2, 10% glycerol, 25 µg of
herring sperm DNA, and 200 µg of bovine serum albumin/ml in a total
volume of 25 µl. The reaction mixture was incubated at room
temperature for 25 min. After the addition of 5 µl of a DNA
electrophoresis loading buffer (1 µg of bovine serum albumin/ml, 50%
glycerol, 0.01% xylene cyanol) to each reaction mixture, the mixtures
were electrophoresed on a 5% native polyacrylamide gel (16). Retarded protein-DNA complex bands were visualized by autoradiography of the gel.
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FIG. 2.
Plasmids used in this study. The shaded regions in panel
D are IS2 sequences which remained. Abbreviations:
crp, CRP gene; Ampr, ampicillin resistance gene;
Cmr, chloramphenicol resistance gene; Kmr,
kanamycin resistance gene; luxAB1, luciferase gene from
Vibrio harveyi (45); PinsA, the
insA gene promoter, which is also the major IS2
promoter.
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In situ DNA footprinting.
The DNA-protein reaction mixtures
described above were electrophoresed on a 5% native acrylamide gel.
After being washed with 200 ml of 50 mM Tris-HCl (pH 8.0) solution, the
whole gel was soaked at room temperature for 8 min in an in situ DNA
footprinting solution containing 1 mM 1,10-phenanthroline, 0.225 mM
CuSO4, and 29 mM 3-mercaptopropionic acid as described
previously (27, 37). The gel was washed with water and then
exposed to an X-ray film to detect protein-bound DNA bands. The gel was
then aligned with the autoradiogram, and the portions of the gel
containing the bands were isolated. The DNA present in the gel slices
was eluted by soaking the gel in 0.5 ml of a solution of 0.5 M ammonium acetate and 1 mM EDTA overnight. The eluted DNA was precipitated with
ethanol and then electrophoresed on a 6% DNA sequencing gel.
Luciferase assay.
One hundred microliters of 1% (vol/vol)
n-decyl-aldehyde (in ethanol) was added to 500 µl of a
late-log-phase (A600 = 0.8) culture of E. coli containing appropriate plasmids. The reaction mixture was
incubated at room temperature for 10 s, and then the luciferase
activity was measured with a luminometer (AutoLumat LB 593; EG & G,
BERTHOLD, Bad Wildbad, Germany). The bioluminescence generated from
each culture was shown as relative light units (RLU).
Transposition assay.
Transposition assays and the
determination of transposition frequencies were performed as described
previously (26). A kanamycin resistance (Kmr)
gene was inserted into IS2 so that transposition of
IS2 could be detected by determining kanamycin resistance.
pMIS2K (Fig. 2D), which carries this kanamycin gene-marked
IS2, was introduced into the isogenic E. coli
strains TP7811 (xyl araH1 his), TP7839 (xyl araH1 his
crp39), and TP7860 (xyl araH1 his cya)
(4) containing an F-derived plasmid, pCJ105, which served as
the target for IS2 transposition. These three isogenic
E. coli strains also harbor IS2, which provides
the IS2 transposase in the transposition assay. Since pCJ105
carries a chloramphenicol resistance gene, transposition of
IS2 onto pCJ105 will render pCJ105 able to confer on an
E. coli host both kanamycin- and chloramphenicol-resistant phenotypes. To determine the transposition frequency,
pCJ105::IS2 was mated out from TP7811, TP7839, or
TP7860 to HB101 (5) by conjugation. The transconjugants were
selected on Luria-Bertani agar containing chloramphenicol (50 µg/ml),
kanamycin (50 µg/ml), and streptomycin (50 µg/ml), since HB101 is
resistant to streptomycin. The transposition frequency was calculated
by dividing the number of HB101 cells that were resistant to kanamycin,
chloramphenicol, and streptomycin by those that were resistant to only
chloramphenicol and streptomycin.
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RESULTS |
Binding of cAMP-CRP to the major IS2 promoter.
To
determine whether the cAMP-CRP complex has the ability to bind the
major IS2 promoter, the gel retardation assay was performed. A cell lysate containing overexpressed CRP was used as the source of
CRP for this experiment. To overexpress CRP, a 0.7-kb DNA fragment containing the crp gene was amplified by PCR with genomic
DNA isolated from E. coli XL1-Blue (7) as the
template and primers CRP-N (5'-TTATCTGGCTCTGGAGAAAGCTT-3'),
which has a HindIII site at its 3' end, and CRP-C
(5'-TCGAAGTGCATAGTTGATATCGG-3'). The PCR product was
digested with HindIII and then cloned between the
HindIII and SmaI sites of pBluescript II
SK(), generating pSKCRP (Fig. 2A). The nucleotide
sequence of the cloned crp gene was verified by sequencing.
pSKCRP was then introduced into E. coli
JM109(DE3), and the lysate of JM109(DE3) cells containing
pSKCRP was used for the gel retardation assay.
A DNA fragment referred to as P
insA, which is the 89-bp
EcoRI-
SpeI fragment containing the IS
2
LIR, was isolated from pKS
+IS2 (
28), labeled at
its 3' end with [
-
32P]dATP, and then incubated with
cell lysate of JM109(DE3) containing
pSK
CRP in the
presence or absence of cAMP. The reaction products
were then
electrophoresed on a polyacrylamide gel to detect bands
that migrated
more slowly than those in control reactions which
lacked CRP or cAMP.
The same labeled fragment was reacted with
the InsA protein to serve as
a positive DNA binding control, since
InsA is known to bind the LIR.
Binding of cAMP-CRP to the
lacZ gene promoter was also
performed to serve as an additional positive
DNA binding control,
because the cAMP-CRP complex binds to the
lacZ gene
promoter. Reaction of cAMP-CRP with a 170-bp
EcoRI-
PvuII
fragment of pBluescript II SK(+),
which does not contain a cAMP-CRP
binding site, was done to serve as a
negative control.
The results of this experiment are shown in Fig.
3. A retarded band which migrated more
slowly than the naked P
insA (Fig.
3, lane 1) was seen when
P
insA was reacted with the cell lysate
(30 µg of total
protein) containing InsA (Fig.
3, lane 2), indicating
that
P
insA has the InsA binding sequence. In the presence of 20
mM cAMP, both 30 and 15 µg of total protein of the cell lysate
of
JM109(DE3) cells containing pSK
CRP generated a retarded
band when incubated with P
insA (Fig.
3, lanes 5 and 6). This
retarded band was not seen when cAMP was
omitted in the DNA binding
reaction (Fig.
3, lane 4). Similarly,
no retarded band was seen when a
cell lysate of JM109(DE3) without
pSK
CRP was used (Fig.
3, lane 3). Although the molecular mass of
CRP is approximately 1.5 times (22 versus 14 kDa) that of InsA
(
2,
27), it generated
a retarded band which migrated to the
same level as that generated by
InsA (Fig.
3, lanes 2, 5, and
6). A possible reason is that InsA may
have the ability to bend
DNA as CRP does, which would make the
migration of DNA bands disproportional
to the size of the bound
protein. The same cell lysate containing
CRP also generated retarded
bands (Fig.
3, lane 10) in the presence
of 20 mM cAMP when reacted with
P
lacZ, which is a 277-bp
EcoRI-
PvuII
fragment containing the
lacZ gene promoter, but did not
generate
any retarded bands with the 170-bp
EcoRI-
PvuII fragment of pBluescript
II SK(+)
which did not contain a CRP binding site (Fig.
3, lane
8).
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FIG. 3.
Binding of the cAMP-CRP complex to the major
IS2 promoter. PinsA is an 89-bp
EcoRI-SpeI DNA fragment containing the major
IS2 promoter, PlacZ is a 277-bp
EcoRI-PvuII fragment containing the
lac promoter, and NS is the 170-bp
EcoRI-PvuII fragment of pBluescript II SK(+)
which does not contain a CRP binding site. Three kinds of cell lysate
were used: CRP, E. coli JM109(DE3) containing
pSKCRP; C, JM109(DE3) containing pT7-7; and InsA,
JM109(DE3) containing pT7insA. The amounts of cell lysates used for
each reaction with (+) or without () cAMP are as indicated. Solid
arrows indicate bands of free DNA fragments, and open arrows indicate
those of protein-bound DNA fragments.
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The binding sequence of the cAMP-CRP complex on the major
IS2 promoter.
The DNA footprinting experiment was
performed to determine the binding sequence of the cAMP-CRP complex on
the major IS2 promoter. The same DNA fragment,
PinsA, used for the gel retardation assay was used. This
89-bp EcoRI-BamHI fragment containing the
IS2 LIR region was labeled at the 5' EcoRI end
and then incubated with the cAMP-CRP complex before footprinting. The
same fragment was also subjected to Maxam-Gilbert sequencing reactions
(44). All the reaction mixtures were electrophoresed on a
5% DNA sequencing gel. A footprint was seen on DNA reacted with the
cAMP-CRP complex when the gel was autoradiographed (Fig.
4). The binding sequence of the cAMP-CRP
complex was deduced to be 5'-CTATCACTTATTTAAGT-3' (Fig. 4)
by comparing the footprinting patterns of PinsA incubated with (Fig. 4, lane 4) or without (Fig. 4, lane 3) cAMP-CRP complex with
the banding patterns of the Maxam-Gilbert G (Fig. 4, lane 1) and G+A
(Fig. 4, lane 2) sequencing reactions. This sequence is complementary
to the sequence 5'-ACTTAAATAAGTGATAG-3' (IS2 nucleotide positions 37 to 53), which is located on the upper strand of
the sequence shown in Fig. 1.
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FIG. 4.
Determination of the cAMP-CRP complex binding sequence
in the major IS2 promoter. The 89-bp
EcoRI-SpeI DNA fragment containing the cAMP-CRP
binding site was labeled by the Klenow enzyme with
[-32P]dATP at the EcoRI end, incubated with
the cell lysate containing cAMP-CRP, and subjected to in situ DNA
footprinting. Lanes 1 and 2, Maxam-Gilbert (44) G and G+A
reactions, respectively, of the DNA fragment; lane 3, footprinting
reaction of unbound DNA; lane 4, footprinting reaction of the
cAMP-CRP-bound DNA. The binding sequence of the cAMP-CRP complex is
deduced from the footprint as shown. The uppercase letters on the right
represent sequences actually determined from the gel; the lowercase
letters were filled in on the basis of known sequences.
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Effect of cAMP-CRP on the transcription of the major
IS2 promoter.
To determine whether the cAMP-CRP
complex has any effect on transcription from the major IS2
promoter, the major IS2 promoter was fused with a
promoterless luciferase gene and assayed for transcription in the
presence or absence of cAMP-CRP. The 6.8-kb SalI-ScaI fragment containing the IS2
promoter fused with the lacZ structural gene was isolated
from pInsApLacZ (27) and then ligated with the 3.2-kb
SalI-HincII fragment containing the p15A replication origin and the chloramphenicol resistance gene of pACYC184,
resulting in pACPIS2-LacZ (Fig. 2B). To replace the lacZ
gene with the luciferase gene luxAB1 (45), the
plasmid pACPIS2-LacZ was digested with ClaI. The
ClaI ends were filled in with the Klenow enzyme, and the
fragment was further digested with HindIII to delete the
lacZ gene, producing a 3.2-kb DNA fragment. This 3.2-kb
fragment was then ligated with the 2.3-kb
HindIII-Ecl136II DNA fragment containing the
luxAB1 (45) gene from pUCD1752 (a gift from
C. I. Kado), generating pACPIS2-Lux (Fig. 2C).
The plasmid pACPIS2-Lux was introduced into the isogenic
E. coli strains TP7811, TP7839, and TP7860, and the transformed cells
were then assayed for the production of luciferase. TP7811 is
wild type
for cAMP-CRP, TP7839 is defective for CRP, and TP7860
is unable to
produce cAMP. The major IS
2 promoter was found to
be able to
drive the expression of the luciferase gene in TP7811
and produced
2.7 × 10
5 RLU of luciferin (Table
1). However, a fourfold increase
(10.8
× 10
5 RLU) in the production of luciferase was
seen when the same plasmid
was introduced into the CRP
TP7839. A more profound (17.1-fold) increase in the production
of
luciferase was observed when pACPIS2-Lux was introduced into
the
Cya
TP7860. To ensure that the difference in the
expression of the
luciferase gene was not due to a difference in the
copy number
of pACPIS2-Lux in different hosts, plasmid DNA was isolated
from
the same numbers of TP7811, TP7839, and TP7860 cells and then
quantitated. No difference in the copy number of pACPIS2-Lux in
TP7811,
TP7839, or TP7860 was observed. These results indicate
that the major
IS
2 promoter is more active in the absence of CRP
or cAMP
and suggest that the cAMP-CRP complex is a negative regulator
of the
major IS
2 promoter.
Effect of cAMP-CRP on IS2 transposition.
Since the
major IS2 promoter is responsible for the transcription of
the IS2 transposase InsAB', the effect of cAMP-CRP on IS2 transposition was examined. A plasmid containing a
mini-IS2 with a kanamycin resistance marker was constructed
as follows. The plasmid pKS+ISF (27) was
digested with AccI and HpaI to delete the
IS2 sequence from nucleotide 578 to 1173, resulting in the
plasmid pKS+ISFd1. IS2 nucleotides 103 to 440 were then removed by deleting the 337-bp
XhoI-SmaI fragment of pKS+ISFd1. The
1.3-kb HincII fragment containing a kanamycin resistance gene from pUC4K (Pharmacia Biotech, Uppsala, Sweden) was then inserted
into the blunt-ended XhoI and SmaI sites of
pKS+ISFd1, generating pMIS2K (Fig. 2D). Since a total of
937 bp of internal IS2 sequence in pMIS2K were deleted, all
functional genes of IS2 were destroyed and the mini-IS2 in
this plasmid could transpose only in hosts that harbor IS2.
To ensure that strains TP7811, TP7839, and TP7860 contained
IS
2, PCR was performed with primers IS
700-720
(5'-ATGCGCCAGAATGCGCTGTTG-3')
and IS
1294-1273
(5'-TTAACCCATTACAAGCCCGCTG-3'), which amplify
IS
2
from nucleotide 700 to 1294. An expected 595-bp fragment was
amplified
from all three hosts. This fragment was sequenced, and
the sequence was
verified to be derived from IS
2. pMIS2K was then
introduced
into TP7811, TP7839, and TP7860 containing pCJ105.
The frequency of
IS
2 transposition onto pCJ105 in each host was
then
determined by mating pCJ105 out to another host. The results
of this
experiment are summarized in Table
2. In
the wild-type
host (TP7811), IS
2 transposed at a frequency
of 10
5. A 130-fold increase in transposition frequency
was observed
in the CRP
host, TP7839. A much higher
transposition frequency (290-fold
increase) was seen in the
Cya
host, TP7860. These results indicate that
IS
2 transposes more
efficiently in hosts defective in CRP or
adenyl cyclase, suggesting
that IS
2 transposition is
negatively regulated by the cAMP-CRP
complex.
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DISCUSSION |
The consensus binding sequence for the cAMP-CRP complex is
5'-AAnTGTGAnnTnnnnCAnATT-3' (11). This sequence
is found in the major IS2 promoter at three regions:
IS2 nucleotide positions 34 to 54, 43 to 63, and 42 to 22 (Fig. 1). The sequence of region III (IS2 nucleotides 42 to
22) is located on the lower strand of the IS2 LIR, whereas
those of region I (IS2 nucleotides 34 to 54) and region II
(IS2 nucleotides 43 to 63) are on the upper strand. Seven
residues in region I, eight in region II, and six in region III conform
to this 13-residue consensus cAMP-CRP binding sequence. This finding
suggests that IS2 transposition may be subject to cAMP-CRP
regulation. This hypothesis is supported by the demonstration that the
cAMP-CRP complex binds to the major IS2 promoter (Fig. 3 and
4) and that binding of the cAMP-CRP complex to the IS2
promoter has a negative effect on transcription from this promoter
(Table 1). As a consequence, the production of transposase is decreased
and IS2 transposition frequency is reduced. This supposition
was demonstrated in this study by the observation that IS2
transposition frequency is higher in E. coli mutants defective in CRP or adenyl cyclase than in a wild-type host (Table 2).
It is also possible that the binding of the cAMP-CRP complex to the LIR
interferes with the binding of the IS2 transposase to the
same region to initiate transposition. This possibility remains to be
investigated.
The cAMP-CRP complex was determined to bind the sequence
5'-ACTTAAATAAGTGATAG-3' located at IS2
nucleotides 37 to 53 (Fig. 1). This sequence is located within region I
of the three putative cAMP-CRP binding sites mentioned above. This
cAMP-CRP binding sequence overlaps almost entirely with that of the
InsA binding sequence, which is located at IS2 nucleotides
33 to 52 (Fig. 1). This area covers the entire 10 sequence and its
flanking regions of the major IS2 promoter. We have
previously shown that the binding of InsA to this region also
suppresses transcription from this promoter and thus decreases
IS2 transposition frequency (27). In this study,
we have demonstrated that the cAMP-CRP complex binds to the same region
and has the same suppressive effect as the InsA on IS2
transposition. InsA is a native IS2 protein, whereas CRP is
a host protein. It is conceivable that the host has a mechanism to
limit IS2 transposition, since overtransposition could be
detrimental to the host, but it is quite intriguing to find that
IS2 produces a protein to suppress its own transposition. It
remains to be investigated whether InsA and the cAMP-CRP complex
compete with each other for binding to the same site. It appears that
InsA and the cAMP-CRP complex do not bind concomitantly, since a cell lysate containing both proteins did not produce a band that migrated more slowly than the one produced by either InsA or the cAMP-CRP complex alone in the gel retardation assay (data not shown). It is
unknown whether there is a mechanism to coordinate the binding of these
two negative regulators. How IS2 derepresses the suppression by InsA or the cAMP-CRP complex also remains to be studied.
The cAMP-CRP complex of E. coli is involved in the
activation or repression (34, 50, 62, 63) of many genes. For
example, the cAMP-CRP complex alone activates the transcription of
lacp1 (42),
galp1 (48, 49), malTp
(67), PC and PBAD of the AraCBAD operon (40), papp (15), and
gyrA (19). In the absence of the CytR repressor,
it also activates deop2 (46, 53, 72), tsx-p2 (17), nupG
(47), and cdd (25). The promoters
malKp and malEp (62, 67) require both
the MalT and the cAMP-CRP complexes, and that of the ansB
gene (31) requires both the FNR and the cAMP-CRP complexes
for transcriptional activation.
In this study, the binding of the cAMP-CRP complex was found to have a
negative effect on transcription from the major IS2 promoter. Negative regulation by the cAMP-CRP complex has also been
demonstrated to occur on
lacp2/p3 (13, 42,
79), galp2 (48),
proPp1 (80), and crp
(1, 30). In addition, the cAMP-CRP complex together with the
CytR repressor inhibits the transcription of
deop2 (46, 53, 58, 72),
cytRp (23, 52), tsx-p2
(17), nupG (51), and cdd
(25).
The effect of cAMP-CRP on IS2 transposition described in
this report is the first example of regulation of the transposition of
transposable elements by the cAMP-CRP complex. It is not known whether
the cAMP-CRP complex has a similar effect on other members of the
IS3 family. We have searched for the presence of the
cAMP-CRP binding sequence on the transposable elements of the
IS3 family (56, 65, 66) and found that 9 of 11 members of the IS3 family have a putative CRP binding
sequence on the promoter region (Table 3)
which has 7 or more bp matched with the 13-bp consensus sequence, suggesting that the cAMP-CRP complex also regulates their
transpositions. We also found that 11 of 13 non-IS3
transposable elements have 7 or more bp that matched with the putative
CRP binding sequence (Table 4). Whether
the cAMP-CRP complex has any effect on the transposition of these
transposable elements remains to be determined.
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ACKNOWLEDGMENTS |
We thank C.-H. Lee for critical reading and editing of the
manuscript, S.-C. Lo and S.-T. Liu for helpful comments on this work,
and S. Tarbor and H. Aiba for providing plasmids and host cells.
This work was supported by grant NSC86-2314-B-010-032 from the National
Science Council, Taiwan, R.O.C., to S.-T. Hu.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, National Yang-Ming University, Taipei, Taiwan. Phone:
011-886-2-2826-7107. Fax: 011-886-2-2821-2880. E-mail:
tingnahu{at}mailsrv.ym.edu.tw.
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Abstract
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