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Journal of Bacteriology, May 2000, p. 2953-2959, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Interaction of the FimB Integrase with the
fimS Invertible DNA Element in Escherichia coli
In Vivo and In Vitro
Lesley S.
Burns,
Stephen
G. J.
Smith, and
Charles J.
Dorman*
Department of Microbiology, Moyne Institute
of Preventive Medicine, University of Dublin, Trinity College,
Dublin 2, Republic of Ireland
Received 23 December 1999/Accepted 21 February 2000
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ABSTRACT |
The FimB protein is a site-specific recombinase that inverts the
fimS genetic switch in Escherichia coli. Based
on amino acid sequence analysis alone, FimB has been assigned to the
integrase family of tyrosine recombinases. We show that amino acid
substitutions at positions R47, H141, R144, and Y176, corresponding to
highly conserved members of the catalytic motif of integrase proteins, render FimB incapable of inverting the fimS element in
vivo. The arginine substitutions reduced the ability of FimB to bind to fimS in vivo or in vitro, while the substitution R144Q
resulted in a protein unable to bind independently to the half sites
located at the left end of fimS in phase-on bacteria. These
data confirm that FimB is an integrase and suggest that residue R144
has a role in binding to a specific component of the fim switch.
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TEXT |
FimB protein is one of two
site-specific recombinases that invert the fim switch
(fimS) in Escherichia coli (20, 24). Inversion of the 314-bp switch is the basis of phase-variable expression of type 1 fimbriae (1, 21). FimB can invert
it in either direction with approximately equal facility, whereas FimE
(the other recombinase) inverts the switch predominantly in the
on-to-off direction (17, 26, 38). When expressed in the same
strain, FimE is completely dominant to FimB under standard growth
conditions (16).
The FimB and FimE proteins are closely related in amino acid sequence
(48% identical) and are encoded by tandemly arranged genes on the
chromosome located adjacent to the switch (Fig.
1) (25). The fimS
element harbors the fimAp promoter for transcription of the
fimA gene encoding the subunit protein of type 1 fimbriae. When fimAp is directed toward fimA, the bacteria
are fimbriate (phase on); when it is directed away, the bacteria are
afimbriate (phase off) (Fig. 1). Both FimB and FimE require the
accessory proteins integration host factor (IHF) and the
leucine-responsive regulatory protein (Lrp) for efficient inversion of
the switch (7, 11, 14, 35). The nucleoid-associated protein
H-NS plays a poorly understood inhibitory role, at least in
FimB-promoted switching (10, 23, 24, 31, 37).
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FIG. 1.
The fimS invertible element and its
surroundings. The relevant section of the fim locus is shown
in wild-type, CJD808 (fimS locked on), and CJD957
(fimS locked off) strains. The fimA promoter is
represented by the angled arrow labeled PfimA. In phase on,
this arrow is directed towards fimA; in phase off, it is
directed the other way. The regulatory genes fimB and
fimE are insertionally inactivated in strains CJD808 and
CJD957, locking the switch in those genetic backgrounds. The converging
arrowheads that bound the fimS element represent the IRL and
IRR. Below, the DNA sequences of the IRL and IRR are shown, together
with those of the half sites to which the Fim recombinases bind. The
9-bp inverted repeats are in boldface type, and the half sites
(identified by Gally et al. [17]) are underlined. For
clarity, spaces have been introduced 5' and 3' to the invariant CA
dinucleotide motif within each half site. The asterisks indicate the 4 bp (5'-AATT-3') deleted in the IRL of strain CJD1353. The transcription
start site of fimA lies immediately to the left of the IRR
and is represented by "+1" at the start of a horizontal arrow.
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Inversion of fimS involves site-specific recombination
between two 9-bp inverted repeats that border the switch (Fig.
1). Each repeat is flanked by two Fim recombinase binding sites
(known as half sites), each of which contains the essential core
dinucleotide 5'-CA (17). Previous work has shown that there
are four binding sites for FimB (and FimE) at fimS
(17). These half sites flank the 9-bp inverted repeats and
can be designated IRL (left-hand inverted repeat)-outside, IRL-inside,
IRR (right-hand inverted repeat)-outside, and IRR-inside. The
IRL-outside and IRR-outside sites are constant in phase-on and
phase-off cells, while the inside sites vary as fimS inverts
(Fig. 1). This variation is important for the directionality of the
FimB- and FimE-promoted DNA inversion event (26).
Integrases are usually associated with integration and excision events
or the resolution of dimeric structures through recombination between
directly repeated copies of specific sequences. The Fim integrases are
unusual in promoting an inversion event, something that is more often
associated with members of the invertase family of site-specific
recombinases. The fim system is unusual in having two
integrases. In other cases where two are found (such as the Xer-dif system), the two proteins cooperate to promote
recombination (6). In the case of fim, FimB and
FimE operate independently of one another (25). Finally, at
approximately 200 amino acids, the Fim proteins are the smallest
members of the Int family (over 100 recombinases), making them an
attractive subject for structure-function studies.
Based on their amino acid sequences, FimB and FimE have been assigned
to the integrase family of tyrosine site-specific recombinases (11, 14). Although the family members display a great deal of sequence heterogeneity, each integrase possesses a catalytic motif
of amino acids that are almost absolutely conserved throughout the
family. Originally, it was believed that there were just four of these:
two arginines, a histidine, and a tyrosine that occurred in the order
RHRY to form a catalytic tetrad for DNA cleavage, strand exchange, and
religation during the recombination event (2, 3, 15, 18, 30,
39). Recently, a fifth catalytic residue (a lysine) has been
identified (8). In this investigation, we sought to
identify the four amino acids in FimB that correspond to the RHRY
members of the catalytic domain by mutation and to study the
interaction of wild-type and mutant FimB proteins with the
fimS element in vivo and in vitro.
Identification of four members of the FimB catalytic motif.
Amino acid sequence alignment with the integrase family was used to
identify candidates for the RHRY members of the catalytic motif of FimB
(11). These residues were R47, H141, R144, and Y176.
The substitutions R47A, H141A, R144Q, and Y176F were made by
site-directed mutagenesis using either the QuikChange kit
(Stratagene) (R47A and H141A) or the unique-site elimination
method (Pharmacia Amersham Biotech) (R144Q and Y176F). For unique-site
elimination mutagenesis, a unique HindIII site was
eliminated using primer H3Nsi (Table 1).
For mutagenesis of the fimB gene, plasmids pSLD203 (FimB)
and pSGS223 (maltose binding protein [MBP]-FimB fusion) (10 ng) (Table 2) were used as
substrates. The oligonucleotides (100 pmol each) used for mutagenesis
were obtained from MWG Biotech and are listed in Table 1. Standard
recombinant DNA techniques were employed throughout this work
(36).
Each mutant FimB protein was compared with the wild type in an in vivo
recombination assay in which inversion of the
fimS element
was measured on the chromosome by PCR as previously described
(
38). In this assay, cells that were phase on gave rise to
two
DNA bands of 293 and 433 bp; phase-off cells gave bands of 539
and
187 bp. Mixed populations of phase-on and phase-off cells
displayed
bands of all four sizes. The in vivo assay for FimB
function involved
complementation of a
fimB gene knockout mutation.
Plasmids
harboring wild-type or mutant
fimB genes were introduced
to
E. coli tester strains (
4) in which
fimS was locked in either
the on or the off orientation
(CJD808 and CJD957, respectively)
(Fig.
1). The bacterial strains were
grown as described previously
(
12,
38). CJD808 (on) and
CJD957 (off) each had knockout insertion
mutations in both the
fimB and
fimE genes on their chromosomes.
In the
locked-on strain, the
fimA promoter was directed towards
the
fimA-lacZ reporter gene fusion, and this strain produced red
colonies on MacConkey lactose indicator plates. In the locked-off
strain, the
fimA promoter was directed away from the
fimA-lacZ reporter gene fusion, and this strain
produced white colonies
on the indicator plates. When incoming
plasmids expressed functional
FimB protein, they complemented the
fimB knockout mutation and
restored phase-variable
fimA-lacZ expression, giving rise to sectored
colonies on
the MacConkey lactose plates. The data obtained showed
that while the
plasmid with the wild-type
fimB gene could reactivate
inversion in both the phase-on and the phase-off
fimB fimE
tester
strains, the plasmids with the mutant
fimB gene
copies could not
(Fig.
2). These data
confirmed that the four residues that had
been substituted were indeed
critical for the DNA inversion activity
of the FimB recombinase. Since
it was possible that the amino
acid substitutions had disabled the DNA
binding activity of the
mutants, it was necessary to examine them in
more detail in order
to distinguish between defects in DNA binding and
catalytic defects
which, while permitting binding, prevented
site-specific recombination
from proceeding.
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FIG. 2.
Inversion of the fimS element by FimB and its
mutant derivatives. Inversion of fimS was detected by a PCR
assay. The structures of the phase-on and phase-off substrates are
summarized at the top, together with details of the oligonucleotide primers used to amplify the inversion
products. Gels with the PCR products obtained with phase-on and
phase-off substrates are shown below. The positions to which PCR
products diagnostic of phase-on or phase-off switches migrate are
indicated to the right of each gel. Inversion of fimS is
seen only in the case of the wild-type FimB protein (third lane from
the left). DNA size markers are shown in the first lane; pUC18 is the
vector-only control.
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Binding of FimB proteins to the IRR in vivo.
In previous
work it was found that when FimB was expressed from a multicopy
plasmid it could repress transcription from the fimAp
promoter by a promoter occlusion mechanism, and this binding could be
demonstrated by in vivo footprinting (13). In other words,
in phase-on bacteria, the binding of FimB to the IRR on the chromosome
resulted in repression of the fimA promoter. This offered a
method for assessing the binding of FimB to the IRR in vivo, since
binding of the protein to this sequence simultaneously placed the
protein directly on the transcription start site for the
fimA gene in a phase-on strain (Fig. 1) and the effect on beta-galactosidase expression from the fimA-lacZ reporter
gene fusion could be assayed readily. Furthermore, recent work with the
FimE recombinase showed that a similar effect could be achieved with
that protein in vivo, a result that has been validated by in vitro
binding studies (38). Beta-galactosidase activity was assayed by the method of Miller (29).
The wild-type FimB protein repressed expression of the
fimA-lacZ fusion by 90% in the phase-on tester strain
CJD808 (Fig.
3). Transcription repression
was also seen with the mutant proteins
with the H141A or Y176F
substitution; little repression was seen
with the proteins with
the R47A or the R144Q substitution (Fig.
3). Assays were repeated at
least three times. These data suggested
that two of the
catalytic-motif substitutions (R47A and R144Q)
had significantly
altered the ability of FimB to bind to the IRR
in vivo. Therefore, it
was decided to test the interactions of
the wild-type and mutant
proteins with both the left (IRL) and
the right (IRR) repeats in vitro.
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FIG. 3.
Indirect measurement by a promoter occlusion test of
FimB binding to the IRR. The data shown are beta-galactosidase
activities for cultures harboring plasmids expressing no recombinase
(pUC18), wild-type FimB, or mutant derivatives of FimB. These
experiments were repeated on three separate occasions; typical data are
shown. The error bars indicate standard deviations.
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Interaction of wild-type and mutant FimB proteins with IRL and IRR
in both orientations in vitro.
Sufficient quantities of FimB to
perform electrophoretic mobility shift assays were not obtainable from
lysates of strains expressing multicopy FimB. Instead, FimB was found
to be expressed better as an MBP fusion. Therefore, the in vitro
binding assay was carried out with MBP fusions to the wild-type and
mutant FimB proteins. First, the fimB open reading
frame was amplified with primers FimBR and FimBL (Table 1) and then
inserted between the XmnI and BamHI sites of
pMalC2, resulting in plasmid pSGS223. In this construct, the
fimB open reading frame was inserted downstream and in frame
with the malE gene to create an MBP-FimB fusion protein. This protein could be overexpressed and was soluble (see below). In
vivo, the MBP-FimB hybrid had full wild-type FimB activity (data not
shown). The expression of wild-type and mutant FimB proteins was
confirmed by Western blotting using an anti-FimB antibody from New
Zealand White rabbits immunized with a multiple-antigen peptide
(Research Genetics) (Fig. 4). This
peptide (PLLNKEVQALKNWLS) corresponded to amino acids 80 to 94 of
FimB, and the procedure used for raising antibodies and performing
Western immunoblot analyses was essentially identical to that described
previously for FimE (38).
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FIG. 4.
Detection of hybrid MBP-FimB protein by Western
blotting. Polypeptides expressed from multicopy recombinant
plasmids were detected by antibody against FimB (A) or MBP (B). The
MBP-FimE fusion protein is included as a negative control in panel A
(third lane from the left). The positions of native MBP (44 kDa) and
MBP fusions (66 kDa) are indicated.
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The strain CJD931, which is deficient in the
fimS-binding
proteins FimB, FimE, IHF, and Lrp (Table
2), was transformed with
plasmids expressing wild-type or mutant MBP-FimB protein fusions.
Lysates were prepared and used in electrophoretic mobility shift
assays
(
38) with radiolabeled DNA fragments consisting of the
IRL
or IRR sequence amplified from
fimS in the on or off
orientation.
The wild-type FimB protein shifted the mobilities of all
four
DNA fragments, producing two shifted species called SS and DS
(Fig.
5). The MBP protein alone did not
affect the mobilities
of the
fimS DNA fragments. The upper
band (DS) was consistent
with occupation of both half sites at each
inverted repeat (Fig.
1), while the lower band (SS) represented
occupation of just one
half site. These results were consistent with
previous work showing
that two shifted complexes arise when the Fim
recombinases interact
with
fimS in vitro (
17,
38). In the IRL-on experiment, the
SS species ran as a doublet,
perhaps reflecting the previously
described ability of integrases to
bend their DNA substrates (
20).
In agreement with the in
vivo data from the
fimAp promoter occlusion
experiments (see
above), the amino acid substitutions R47A and
R144Q each altered the
ability of FimB to bind to IRR-on in vitro
(compare Fig.
3 and
5).
However, while R47A could still bind to
IRR-off, R144Q could not.
Therefore, R144Q could not bind to IRR
in either the on or the
off orientation.
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FIG. 5.
Electrophoretic mobility shift analysis of
FimB-containing lysate interactions with IRR or IRL sequences from
fimS. Data are shown for the IRR and IRL amplified from
phase-on or phase-off fimS elements. Lanes 1, no added
protein; lanes 2, MBP alone; lanes 3, MBP-FimB; lanes 4, MBP-FimB R47A;
lanes 5, MBP-FimB H141A; lanes 6, MBP-FimB R144Q; lanes 7, MBP-FimB
Y176F. Species corresponding to complexes with two bound protein
protomers (DS) and a single bound protomer (SS) are shown. Also
indicated are an unknown complex (UC) that migrated near the top of all
lanes and the unbound radiolabeled probe (P). The IRL DNA fragment was
111 bp, while the IRR DNA fragment was 205 bp.
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At IRL, only R144Q was affected in its ability to bind. At IRL-off,
R144Q produced little electrophoretic mobility shift (Fig.
5), while at
IRL-on it created the shifted species that was consistent
with
occupation of both half sites (DS) but not the species indicative
of
single-half-site occupancy (SS) (Fig.
5). This interaction
with IRL-on
was investigated in greater detail by comparing the
abilities of
wild-type FimB and R144Q to bind as their concentrations
were increased
(Fig.
6). While the wild-type protein
produced
two electrophoretically shifted species indicative of
single-half-site
and dual-half-site occupancy at IRL-on, the R144Q
mutant protein
could only produce the species indicative of binding to
the two
IRL-on half sites, and much more of the mutant protein was
needed
to form this complex. This was examined in more detail by
mutagenizing
one of the half sites at IRL-on.
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FIG. 6.
Comparison of wild-type and mutant R144Q binding to
IRL-on in vitro. Shown is an electrophoretic mobility shift assay in
which increasing concentrations of lysates containing MBP, wild-type
MBP-FimB, and the MBP-FimB mutant R144Q were incubated with
radiolabeled DNA amplified from the IRL component of fimS.
Species corresponding to complexes with two bound recombinase protomers
(DS) and a single bound protomer (SS) are shown. P is the unbound DNA
probe (111 bp).
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A 4-bp deletion was engineered in the outside half site at
IRL-on. This was done with plasmid pSGS501 (
38),
which contains
an 8.5-kb fragment that includes inactive copies
of the
fimB and
fimE genes (interrupted by a
kanamycin resistance cassette and
IS
1 insertion element,
respectively) and
fimS in the off orientation,
inserted into
the
EcoRV site of pACYC184 (Table
2). This plasmid
was
cotransformed into strain CH1483 with multicopy
fimB
(pSLD203)
to invert
fimS in plasmid pSGS501 to the on
orientation. Transformants
were screened using the PCR switch assay
(
38), and plasmid DNA
was isolated from one transformant
that contained
fimS in the
on orientation. This DNA was
digested with
SacI and
KpnI, which
cut within
plasmid pSLD203 but not within plasmid pSGS501. The
digested DNA was
transformed into the IHF-deficient strain CH1483.
DNA from one
transformant was isolated (pLSB121), and analysis
by PCR switch
assay showed that it contained
fimS in the on orientation.
(All plasmid constructs were maintained in the strain CH1483,
as the
absence of IHF was found to prevent random DNA rearrangements.)
Plasmid
pLSB121 was digested with
MfeI, which cuts only once
in
plasmid pLSB121, within the IRL. The digested DNA was treated
with
mung bean nuclease to remove the 4-bp overhang resulting
from
MfeI digestion, ligated, and transformed into CH1483.
Plasmid
DNA (pLSB122) was isolated and sequenced (MWG Biotech) to
confirm
that it contained a 4-bp deletion within the IRL and that
fimS was locked in the on orientation (Fig.
1).
The mutant IRL was crossed onto the chromosome of a strain
lacking a functional
fimE gene (VL386
recD).
Plasmid pLSB122 was
digested with
EcoRV, and an 8.5-kb
fragment, encompassing DNA
upstream of the
fimB gene
(interrupted by a kanamycin resistance
gene cassette) through to the
lacZ gene, was isolated from a gel.
Approximately 2 µg of
this fragment was electroporated into strain
VL386
recD
(
38), and transformants were selected on MacConkey
lactose
agar containing kanamycin (25 µg ml
1). Candidate allele
replacement mutants were then screened to
confirm that they did not
contain plasmid DNA. PCR was used to
confirm that they were phase on,
and it was confirmed that the
mutant IRL sequence could not be cleaved
with
MfeI. Finally, the
4-bp deletion within the IRL and the
integrity of the rest of
fimS were confirmed by sequencing.
Site-specific inversion of
fimS mediated by either FimE or
FimB could not be detected in
this background (Fig.
7). Plasmids expressing the four mutant
FimB derivatives were introduced, and the strains were tested
for
inversion of
fimS by the PCR assay. The results obtained
showed
that in no case could any of the mutant FimB proteins invert
fimS (Fig.
7).
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FIG. 7.
The mutant fimS element is not invertible.
Shown is a DNA inversion assay in which the fimS derivative
with a 4-bp deletion in the outer half site at IRL-on is amplified by
PCR from cells expressing wild-type and mutant FimB proteins. pUC18 is
the vector-only control; FimE is wild-type FimE recombinase, a protein
that inverts the multicopy wild-type switch rapidly from the on to the
off phase. The switch remained in the on orientation in all samples.
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Each of the half sites at the IRL (and IRR) probably binds one Fim
recombinase protomer. It is possible that the R144Q mutation
has
altered the way that FimB binds to the IRL half sites, making
binding
at one site contingent on occupation of the other. This
could account
for the lack of single-half-site occupation demonstrated
for the R144Q
mutant at the IRL. Thus, the R144Q mutant would
be unable to produce
the SS mobility-shifted species. The requirement
for two half sites at
IRL-on for R144Q to bind was confirmed when
mobility shift assays were
performed with the mutant IRL. The
wild type and mutants R47A, H141A,
and Y176F could all form the
SS shifted species with the mutated IRL
sequence (Fig.
8). This
showed that these
proteins did not require both halves of the
IRL site in order to bind.
In contrast, mutant FimB R144Q could
not bind to this mutant IRL at
all, showing that it required both
halves to be intact in order for it
to bind to the IRL.
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FIG. 8.
Binding of wild-type and mutant MBP-FimB fusion proteins
to the mutated fimS element. Shown is an electrophoretic
mobility shift assay of interactions of lysates containing FimB and its
mutant derivatives with a radiolabeled IRL sequence with a 4-bp
deletion in the outer half site. The first two lanes are negative
controls. SS is the shifted species that corresponds to IRL with one
half site occupied by a FimB protomer. UC and P are an unknown complex
found in all lanes and the unbound radiolabeled probe, respectively.
The IRL DNA fragment was 107 bp.
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Although the integrase family of tyrosine site-specific recombinases
contains over 100 members, only a small number have been
examined in
detail by site-directed mutagenesis. These proteins
bind to distinct
sequences and recombine short repeated motifs
that are unique to each
integrase (
30). Most of the mutations
made in these proteins
affect catalytic-motif residues, making
the data interesting to compare
with those obtained in this study
of FimB. When the first arginine of
the lambda integrase catalytic
motif is converted to a glutamine
(R212Q), the protein retains
partial DNA binding activity but loses DNA
recombination activity
(
28), a situation similar to that of
R47A in FimB. Substituting
glutamate for the corresponding arginine in
the Flp recombinase
in
Saccharomyces cerevisiae leaves DNA
binding activity intact
while abolishing recombination
(
9). When the first arginine
of the catalytic motif in the
integron Intl1 was converted to
an isoleucine, valine, or glutamate,
all DNA binding activity
and recombination activity was lost
(
19). These data show that,
in addition to its role in
recombination, the first arginine of
the motif also contributes to DNA
binding activity in at least
some
integrases.
Converting the second catalytic-motif arginine of the yeast Flp protein
to glycine disrupted recombination but only weakened
rather than
abolished DNA binding activity (
33). In contrast,
the
corresponding substitution in the integron Intl1 integrase
abolished
both DNA binding and recombination (
19). Thus, different
members of the integrase family show different responses to an
equivalent substitution within the motif. In the case of FimB,
the
R144Q substitution had a very precise effect on the activity
of the
protein: it altered its interaction with a particular component
of the
DNA substrate and abolished DNA recombination
activity.
The tyrosine residue of the catalytic motif forms a covalent bond with
the cleaved DNA at the recombination site, making it
an essential amino
acid for recombination. Substituting the related
aromatic amino acid
phenylalanine for this tyrosine abolished
recombinase activity in the
lambda, the integron Intl1, and the
Flp integrases (
19,
28,
32,
34). The Y176F substitution
in FimB resulted in an equivalent
phenotype. In common with other
well-characterized integrases, FimB was
not impaired in DNA binding
by mutagenesis of this tyrosine
residue.
The results obtained in vitro with the R144Q mutant of FimB suggest
that this integrase interacts with itself, at least at
IRL-on, and that
this is a DNA-dependent interaction. This specific
DNA dependence may
explain why we have been unable to demonstrate
FimB-FimB
protein-protein interactions (or FimE-FimE or FimB-FimE
interactions)
using the yeast two-hybrid system (S. G. J. Smith
and C. J. Dorman, unpublished data). To date, the crystal structures
of four
integrases have been solved. These are HP1 (
22), Cre
(
20), XerD (
39), and Int (
27). At
least for XerD, the implied
interactions for the XerC-XerD heterodimer
bound at the
dif site
are consistent with DNA-dependent
protein-protein interactions.
Perhaps significantly, XerC and XerD are
the integrases most closely
related to FimB (and FimE) (
15).
Further work is in progress
to elucidate in more detail the relation of
structure to function
in the FimB
recombinase.
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ACKNOWLEDGMENTS |
We thank present and past members of the Dorman laboratory for many
useful discussions.
This work was supported by grants 046233/Z/95/Z from the Wellcome Trust
and SC96/301 from Enterprise Ireland (Forbairt).
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Moyne Institute of Preventive Medicine, University of
Dublin, Trinity College, Dublin 2, Republic of Ireland. Phone: 353 1 608 2013. Fax: 353 1 679 9294. E-mail: cjdorman{at}tcd.ie.
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Abremski, K. E., and R. H. Hoess.
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Evidence for the second conserved arginine residue in the integrase family of site-specific recombination proteins.
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Journal of Bacteriology, May 2000, p. 2953-2959, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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