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Journal of Bacteriology, October 1998, p. 5437-5442, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Point Mutations in the Integron Integrase IntI1
That Affect Recombination and/or Substrate Recognition
Annie
Gravel,
Nancy
Messier, and
Paul H.
Roy*
Centre de Recherche en Infectiologie, Centre
Hospitalier de l'Université Laval and Département de
Biochimie, Faculté des Sciences et de Génie,
Université Laval, Sainte-Foy, Québec, Canada
Received 13 July 1998/Accepted 5 August 1998
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ABSTRACT |
The site-specific recombinase IntI1 found in class 1 integrons
catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system. The integron integrase belongs to the tyrosine recombinase (phage integrase) family. The members of this family, exemplified by the lambda integrase, do not share extensive amino acid
identities, but three invariant residues are found within two regions,
designated box I and box II. Two conserved residues are arginines, one
located in box I and one in box II, while the other conserved residue
is a tyrosine located at the C terminus of box II. We have analyzed the
properties of IntI1 variants carrying point mutations at the three
conserved residues of the family in in vivo recombination and in vitro
substrate binding. We have made four proteins with mutations of the
conserved box I arginine (R146) and three mutants with changes of the
box II arginine (R280); of these, MBP-IntI1(R146K) and
MBP-IntI1(R280K) bind to the attI1 site in vitro, but
only MBP-IntI1(R280K) is able to excise cassettes in vivo. However,
the efficiency of recombination and DNA binding for
MBP-IntI1(R280K) is lower than that obtained with the
wild-type MBP-IntI1. We have also made two proteins with mutations of
the tyrosine residue (Y312), and both mutant proteins are similar to
the wild-type fusion protein in their DNA-binding capacity but are
unable to catalyze in vivo recombination.
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TEXT |
Integrons are DNA elements that
capture genes, especially antibiotic resistance genes, by a
site-specific recombination system (32). The
recombination system consists of a DNA integrase (Int) and two types of
recombination sites, attI and attC (59-base
element). The integrase gene (int) is located in the 5'
conserved segment of the integron structure (Fig.
1) and is a member of the tyrosine recombinase family (1, 4, 13, 23, 24). Three types of
integrases, sharing around 50% identity among themselves, have been
identified; they define integron classes 1, 2, and 3 (30). The 5' conserved segment found in class 1 integrons also contains a promoter region responsible for the
expression of inserted cassettes (11, 21) and the
recombination site attI1 (31). The 3' conserved segment of the class 1 integrons includes an ethidium bromide resistance determinant (qacE
1), a sulfonamide resistance
gene (sulI), an open reading frame (ORF5) of unknown
function, and further sequences that differ from one integron to
another (5, 6, 28). The 3' conserved segment of class 2 integrons includes transposition genes (20) while that of
class 3 integrons has not yet been studied (2). The variable
region, located between the two conserved segments, usually contains
antibiotic resistance genes; In0 contains no inserted genes while In21
possesses eight cassettes with ten genes (or ORFs) in this region
(5, 16). These genes are part of mobile cassettes which
include a recombination site, attC, that differs from one
gene to another (18, 33). Incoming genes must be associated
with an attC to be recognized by the integron integrase and
are preferentially inserted at the recombination site attI1
(11). Cassettes are excised as circular intermediates and
integrated at core sites by the action of the integrase
(8-10). The core site, defined as GTTRRRY, makes up the 3'
end of attI1 and attC, with the crossover taking
place between the G and the first T (19). Antibiotic
selection pressure can reveal cassette rearrangements in which a given
resistance is nearest the promoter and thus most strongly expressed
(10).
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FIG. 1.
General structure of class 1 integrons. Cassettes are
inserted in the integron variable region by a site-specific
recombination mechanism. The attI1 site is shown by a black
circle, core sites are represented by ovals, the attC site
is indicated by a black rectangle, and promoters are denoted by P. intIl, integrase gene; qacE1, antiseptic
resistance gene; sulI, sulfonamide resistance gene; orf5,
gene of unknown function.
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Site-specific recombination, unlike homologous recombination, is
characterized by relatively short, specific DNA sequences and requires
only limited homology of the recombining partners (12). Site-specific recombination is an entirely
conservative process since all DNA strands that are broken (two per
exchange site) are rejoined in a process that involves neither ATP nor DNA synthesis. Homology alignments of site-specific recombinases assign
them to two families: the resolvase family, named after the TnpR
proteins encoded by the transposons 
and Tn3, and the integrase family. The integrase family includes over 140 members to
date, but they are highly diversified proteins (13, 23). Members of this family, which include the well-studied 
integrase, recombine DNA duplexes by executing two consecutive strand
breakage and rejoining steps and a topoisomerization of the
reactants. The first pair of exchanges form a four-way Holliday
junction and the second pair resolve the junction to complete the
recombination. The integrase nucleophile is a conserved tyrosine that
becomes associated with a phosphate group on DNA. The
cleavage sites on each DNA duplex are separated by 6 to 8 bp
with a 5' stagger, and the tyrosine joins to the 3' phosphate
(17).
The initial definition of the integrase family was based on comparisons
of seven sequences, and three invariant residues were identified: an
HXXR cluster and a Y residue (4). Alignment of 28 sequences
identified a fourth invariant position, occupied by an arginine residue
(1). These four conserved residues are found in two
boxes located in the second half of the protein. A recent analysis has
shown that the conserved histidine is present in 136 of the 147 members
(93%); this residue is then not conserved in all members of the family
(13). Another recent analysis has identified three patches
of residues located around box I, which seem to be important in the
secondary structure of these proteins (23). In this study,
we analyzed the properties of several mutants of the conserved residues
R146, R280, and Y312 of the integron integrase IntI1 in in vivo
recombination and in vitro substrate binding.
Construction of plasmids overexpressing mutant MBP-IntI1
fusion proteins.
The plasmids encoding various mutants of
MBP-IntI1 were constructed by PCR using pLQ369 (50 ng) as a
template (15). Two primer pairs, designed with the OLIGO
software package (version 4.1; National Biosciences,
Plymouth, Minn.), were used to construct each set of mutants. The
R146 mutants were constructed with an XcmI-BamHI
primer pair [IntI1(R146)-XcmI,
5'-TTCACCAGCTTCTGTATGGAACGGGCATG(A/G)(A/T)AATCAG-3'; IntI1(R146)-BamHI,
5'-CCGGATCCCTACCTCTCACT-3'], the R280 mutants were constructed with an NruI-XmnI primer
pair [IntI1(R280)-NruI, 5'-AGCCGTCGCGAACGAGTGC(C/T)(C/T)GAGGG-3';
IntI1(R280)-XmnI,
5'-ACCCCTAATGAAGTGGTTCGTATCC-3'], and the Y312
mutants were constructed with a AatII-ScaI primer pair [IntI1(Y312)-AatII,
5'-ATTCCGACGTCTCTACTACGATGATTT(C/T)CACGC-3'; pLQ369-ScaI, 5'-ATGCTTTTCTGTGACTGGTG-3'] (restriction
sites within primer sequences are underlined). PCR conditions
were 10 min at 94°C, three cycles consisting of 45 s at 94°C,
45 s at 47°C, and 90 s at 72°C, 30 cycles consisting of
45 s at 94°C, 45 s at 60°C (50°C for Y312 mutants), and
90 s at 72°C, and a final elongation step of 10 min at 72°C.
The XcmI, NruI, and AatII primers were degenerate in one or two positions, so that a single primer could give
all mutants. Mutant PCR fragments were digested and cloned directly
into pLQ369 digested with the same enzymes, except for the R146 mutant
fragments that were subcloned into pLQ364 at first. New mutant
PCR fragments were then amplified on these subclones, using
IntI1(R146)-BamHI and IntI1(R280)-XmnI primers. These
mutant PCR fragments were cleaved with BamHI and
XmnI, and the resulting fragments were cloned into
pLQ369. This avoids the necessity of partial digestion of pLQ369 with
XcmI. Mutant clones were digested with restriction
endonucleases and sequenced to determine the mutation.
In vivo recombination.
Mutant MBP-IntI1 clones were
introduced into Escherichia coli TB1 {F'
ara(lac-proAB) rpsL
(Strr)
[
80dlac(lacZ)M15]
hsdR(rK
mK)}
containing pLQ428 by transformation (Fig.
2 and Table
1). E. coli TB1 cells
containing pLQ428 and one of the MBP-IntI1 mutants were grown at
37°C for 3 h in Luria-Bertani medium. Excision of the
aacA1-ORFG and/or ORFH cassettes was induced by the
overexpression of the malE-intI1 gene by using 0.3 mM
isopropyl-
-D-thiogalactopyranoside (IPTG; Sigma
Chemical Co.) and by incubation at 37°C for another 3 h.
Cell culture was done in the presence of 50 µg of ampicillin per ml,
15 µg of amikacin per ml, and 50 µg of chloramphenicol per ml.
Plasmid DNA was then prepared from 5-ml cultures with the Perfect Prep
DNA extraction kit (Mandel Corporation). In order to determine
the capacity of mutant MBP-IntI1 proteins to excise aacA1-ORFG and/or ORFH cassettes of In21, we used PCR
primers pACYC184-5' (5'-TGTAGCACCTGAAGTCAGCC-3') and
pACYC184-3' (5'-ATACCCACGCCGAAACAAG-3') (Fig. 2, primers
1 and 2) to detect the reduction of pLQ428 length. PCR conditions
were 10 min at 94°C, 30 cycles consisting of 1 min at 94°C, 1 min
at 60°C, and 5 min at 72°C, and a final elongation step of 10 min
at 72°C. A major PCR fragment can be seen in each lane containing a
DNA preparation from a mutant clone (Fig.
3, lanes 2 to 9). This band is 2,499 bp
long and, as determined by restriction enzyme digestions, represents
the pLQ428 clone without any cassette excision (data not shown). This
band is also observed in the negative control, which is the pMAL-c2
vector without any gene fused to malE (Fig. 3, lane 12).
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FIG. 2.
Representation of plasmids used in this study. The
positions of the three invariant residues of the integrase family are
indicated, along with restriction sites used to construct mutant
proteins. Core sites are represented by black circles, and
attCs are shown by white boxes. The numbered arrows
represent the PCR primers used to detect excision events,
pACYC184-5' (1) and pACYC184-3' (2). bla, gene
encoding -lactamase; cat, gene encoding chloramphenicol
acetyltransferase; intIl, gene encoding the integron
integrase (IntI1); malE, gene encoding the maltose binding
protein (MBP); ori, origin of replication; Ptac,
tac promoter; Ptet, tetracycline promoter. Only relevant
restriction sites are indicated.
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FIG. 3.
Electrophoresis of PCR products obtained with the
pACYC184 primer pair and 100 ng of DNA
preparations from overexpressed cultures on a 1% agarose gel. Lane 1, 1-kb DNA ladder (Gibco BRL); lane 2, DNA preparation of pLQ428-pLQ377
(R146E); lane 3, pLQ428-pLQ378 (R146I); lane 4, pLQ428-pLQ376 (R146K);
lane 5, pLQ428-pLQ379 (R146V); lane 6, pLQ428-pLQ390 (R280E); lane 7, pLQ428-pLQ388 (R280G); lane 8, pLQ428-pLQ391 (R280K); lane 9, pLQ428-pLQ394 (Y312F); lane 10, pLQ428-pLQ393 (Y312S); lane 11, pLQ428-pLQ369 (wild type); lane 12, pLQ428-pMAL-c2 (MBP).
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The 2,499-bp PCR product was not obtained in the reaction containing
the wild-type MBP-IntI1-expressing clone pLQ369 (Fig.
3, lane 11),
indicating that there were no remaining full-length
pLQ428 molecules.
This shows that the wild-type fusion protein
is very efficient in
site-specific recombination and that all
pLQ428 clones have undergone
an excision of one or both cassettes.
In this PCR, we observed two
major bands of 1,341 and 889 bp.
The 1,341-bp PCR product was digested
with restriction enzymes
to show that it represents a pLQ428 clone
which has lost the
aacA1-ORFG
cassette (data not shown). The
889-bp band was also digested with
restriction enzymes to show that it
represents a pLQ428 clone
which has lost both
aacA1-ORFG and
ORFH cassettes (data not shown).
These two PCR products are also
observed in the reaction containing
the mutant clone pLQ391, which
expresses the MBP-IntI1(R280K)
fusion protein. This mutant
protein is, however, less efficient
than the wild-type protein, as seen
by the intensity of the PCR
products (Fig.
3, lane 8). We were not able
to detect a PCR product
of 2,047 bp, corresponding to the excision of
the ORFH cassette
alone; this is not surprising since this event has
been shown
in another study to be rare (
16). It is possible
to observe
another band in pLQ428-pLQ391 (R280K) and pLQ428-pLQ369
(wild
type) PCRs (Fig.
3, lanes 8 and 11); this PCR product is 1,100
bp
long and probably represents a recombination event at a secondary
site.
Restriction enzyme digestions were done on this product,
but
we were unable to identify its origin. This product results
from
an event mediated by the integron integrase since it is seen
only in
reactions containing active proteins. An 1,800-bp PCR
band is also
present in the negative control and in all PCRs containing
a mutant
clone. This product appears to be nonspecific, and the
fact that it is
not seen in the PCR containing the pLQ428-pLQ369
(wild-type) clones
probably results from the PCR being more favorable
to smaller PCR
products.
In vitro substrate binding.
The experiments described above
demonstrate that only one of our mutants of IntI1 protein is able to
catalyze in vivo recombination. Can all mutant proteins recognize and
bind to the IntI1 recombination site in a manner similar to the
wild-type protein? To investigate this question, we used purified
fusion proteins and a gel retardation assay with the complete
attI1 site (5' site) of the integron. MBP-IntI1 fusion
proteins were purified as suggested by New England Biolabs. The
concentration of the purified fusion protein was determined by using
the Bradford protein assay (Bio-Rad). The protein solution was then
made 20% in glycerol and stored at 
80°C. The purity of
MBP-IntI1 was evaluated as >90% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown).
Binding reactions were done with labeled 5'-site DNA fragments
(20,000 cpm, 0.25 pmol), incubated with different concentrations of
MBP-IntI1 in a 10-µl volume containing 10 mM HEPES
(K+, pH 8.0), 60 mM KCl, 4 mM MgCl2, 100 µM
EDTA (pH 8.0), 100 µg of bovine serum albumin per ml, 250 µM
dithiothreitol, 100 ng of poly(dI-dC), and 10% glycerol. Reaction
mixtures were incubated at room temperature for 15 min prior to
electrophoresis through 4 or 5% prerun, nondenaturing polyacrylamide
gels buffered with 0.5× Tris-borate-EDTA. Dried gels were subjected to
autoradiography. The wild-type fusion protein and native IntI1
were shown to lead to the same four distinct complexes (I, II,
III, and IV) with this DNA substrate (Fig.
4) (15). These complexes
represent the binding of four IntI1 molecules to four different
sites in the attI1 site (15). Figure 4
shows results obtained with nine mutants of the MBP-IntI1 fusion
protein. We observed that MBP-IntI1(R146E), MBP-IntI1(R146I), and MBP-IntI1(R146V) lost their
ability to bind to the attI1 site, as no complexes are seen
in the gel retardation experiment (Fig. 4A). However,
MBP-IntI1(R146K) formed four IntI1-DNA complexes with the 5'
site DNA fragment. The band pattern and the intensity observed
with this mutant protein are similar to those observed with the
wild-type protein, suggesting that MBP-IntI1(R146K) and
MBP-IntI1 bind DNA with similar affinities.
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FIG. 4.
Binding of mutant MBP-IntI1 fusion proteins purified
from E. coli TB1 to the 5'-site DNA fragment containing the
complete attI1 site of the In2 integron (from nucleotide
96 to nucleotide +71, relative to the G residue of the core site as
position 0). (A) MBP-IntI1(R146) mutants; (B)
MBP-IntI1(R280) mutants; (C) MBP-IntI1(Y312) mutants. A
purified labeled fragment was incubated with different concentrations
of mutant fusion proteins. Free DNA (F) and protein-DNA complexes (I,
II, III, and IV) were separated on 4 or 5% polyacrylamide gels and are
indicated by arrows. Lanes 1, free DNA; lanes 2 through 7, purified
fusion protein at 250, 375, 500, 12.5, 37.5, and 62.5 nM, respectively.
The wild-type (WT) lanes in panel C were from a separate gel.
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Competition with a specific fragment with a 30-fold excess of unlabeled
DNA competed away all four complexes, while a 100-fold
excess of a
nonspecific unlabeled DNA fragment did not compete
away any complexes,
indicating their specificity (data not shown)
(
15). We
observed that MBP-IntI1(R280G) and MBP-IntI1(R280E)
lost their ability to bind the 5'-site DNA fragment, while the
MBP-IntI1(R280K) could still bind the
attI1
site (Fig.
4B). However,
the band pattern obtained with this
mutant protein is weaker than
that obtained with the wild-type
integrase. For example, at a
protein concentration of 250 nM
MBP-IntI1(R280K) (lane 2), we
observed the formation of
complexes I, II, and III, with a stronger
intensity for the
fastest-migrating complexes, while the intensity
of the fourth complex
was very weak. At the same concentration
of the wild-type protein, we
observed the formation of all four
complexes, with a stronger intensity
for the slowest-migrating
complexes and no unbound DNA. These results
show that MBP-IntI1(R280K)
binds the
attI1 site with
a lower affinity than the wild-type
fusion protein. As shown in Fig.
4C, both MBP-IntI1(Y312F) and
MBP-IntI1(Y312S) lead to
the formation of four complexes that
migrate similarity to those
obtained with wild-type MBP-IntI1,
as judged by the gel migration
of these complexes. The band pattern
observed shows that the binding
affinity of these mutant proteins
is the same as or even better than
that of the wild-type protein.
Relationships with other members of the family.
We found
that MBP-IntI1 recombinase in which Arg-146 has been changed
to lysine [MBP-IntI1(R146K)] by PCR mutagenesis
cannot excise cassettes but can bind to the attI1 site
with the same efficiency as the wild-type fusion protein. However,
MBP-IntI1(R146I), MBP-IntI1(R146E), and
MBP-IntI1(R146V) mutant proteins have completely lost both
phenotypes. These findings are different from those for other members
of the family. The only mutant protein of the lambda integrase at this
residue [(R212Q)] binds the core site partially and is not
able to catalyze in vivo or in vitro recombination (22). Mutants of the Cre recombinase with a change at this
residue [Cre(R173K)] bind DNA as well as the wild-type protein but
cannot catalyze in vivo or in vitro recombination (1).
Mutants of Flp [Flp(R191K) and Flp(R191E)] bind FRT
recombination sites as well as the wild-type protein but cannot carry
out in vivo or in vitro recombination, except for the Flp(R191K)
protein, which has shown slight activity in in vivo recombination
(Table 2) (7, 14,
25). Therefore, the Cre(R173K) and Flp(R146K) mutants have
the same phenotype as the MBP-IntI1(R146K) protein. However, the Flp(R191E) mutant protein shows efficient DNA binding while MBP-IntI1(R146E) does not bind to the attI1
site. We interpret these results according to the charge of the
Arg-146 residue. The positively charged side chain of this
residue makes contact with the DNA, which is negatively charged.
This contact is probably important for the good conformation of the
protein molecule in positioning the tyrosine residue to perform
recombination. When this residue is exchanged for a lysine, DNA
contacts are still able to take place because of the charge of
the residue, but the side chain is smaller and the lysine is
probably not able to position the tyrosine to catalyze
recombination. We think that the charge of this residue is very
important in the formation of DNA-protein complexes in the integron
system, since all other MBP-IntI1 mutants tested are unable to bind
DNA. This observation differs from those for Flp, because even
when the wild-type residue was replaced by a negatively charged one, it
could still bind DNA as well as the wild-type protein (Table 2).
We have also made proteins with mutations at position 280; these
were MBP-IntI1(R280E), MBP-IntI1(R280G), and
MBP-IntI1(R280K).
We found that the
MBP-IntI1(R280K) mutant protein binds the
attI1 site
and excises integron cassettes with a lower efficiency than
the
wild-type MBP-IntI1, while MBP-IntI1(R280E) and
MBP-IntI1(R280G)
have completely lost both phenotypes. The
Flp(R308K) mutant protein
has been shown to bind DNA as well as the
wild-type protein, but
it recombines DNA with a lower efficiency than
wild-type Flp (
27).
Another mutant protein of Flp
[Flp(R308G)] has also been shown
to bind DNA as well as the
wild-type protein, but it was unable
to catalyze in vivo or in vitro
recombination (
27). These results
show that Flp(R308K)
and MBP-IntI1(R280K) act similarly but that
the other Flp
mutant [Flp(R308G)] can bind DNA while the MBP-IntI1
mutant
[MBP-IntI1(R280G)] cannot (Table
2). We also think that
the
positive charge of this residue is important for the binding
of
the recombinase to DNA, but Arg-280 does not seem to be
implicated
in the positioning of the tyrosine residue, since the
MBP-IntI1(R280K)
mutant protein can perform recombination.
We found that MBP-IntI1(Y312S) and MBP-IntI1(Y312F)
mutant proteins bind the
attI1 site with the same efficiency
as the wild-type
protein but are not able to catalyze in vivo
recombination. As
expected, these results are the same as those
obtained with the
lambda integrase [
(Y342F)], the XerC and XerD
recombinases [XerC(Y275F)
and XerD(Y279F)], and the Flp recombinases
[Flp(Y343S) and Flp(Y343F)]
(Table
2) (
3,
22,
26,
29). The loss of the catalytic
activity of the
MBP-IntI1(Y312F) mutant protein is not surprising,
since the
hydroxyl group of the tyrosine, which is responsible
for the
nucleophilic attack of the DNA at the recombination site,
is not
present on the phenylalanine residue. The phenotype of
MBP-IntI1(Y312S) indicates that the conformation of the
tyrosine
residue is important for the good activity of the recombinase,
because even if the serine residue has a hydroxyl group, it is
not able
to catalyze recombination. These results indicate that
the integron
integrase IntI1 uses the hydroxyl group of the conserved
tyrosine
(Y312) to catalyze site-specific recombination, like
other members of
the family. However, in vitro recombination using
this mutant protein
needs to be done to confirm this.
These results of point mutations show that mutations of the conserved
arginines by nonpositively charged residues abolish
substrate
recognition, unlike the corresponding mutants of other
members of the
family. However, further mutational analysis, such
as of residues
around and in patch III, would be interesting,
since only integron
integrases contain more residues in this region
than other members of
the family (
23). In vitro recombination
assays with purified
mutant proteins also need to be done in order
to study thoroughly the
mechanism of site-specific recombination
in integrons.
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ACKNOWLEDGMENTS |
We thank France Gagnon for the construction of pLQ428 and technical
assistance. We thank Jean Renaud for automatic sequencing and
Bénédicte Fournier for helpful discussions. We thank
Dominic Esposito for the maintenance of the Tyrosine Recombinases Web Site (http://orac.niggk.nih.gov/www/trhome.html), which has helped us
in the statistical analysis of the Int family.
This work was supported by grant MT-13564 from the Medical Research
Council (MRC) of Canada to P. H. Roy. A. Gravel and N. Messier
held fellowships from MRC Canada.
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ADDENDUM IN PROOF |
A recent article by C. M. Collis, M.-J. Kim, H. W. Stokes, and R. M. Hall (Mol. Microbiol. 29:477-490, 1998) shows that
FLAG-IntI1 mutant proteins R280G and Y312F cannot catalyze cointegrate
formation. These results correspond to our results found in in vivo
recombination experiments for the same mutant MBP-IntI1 proteins.
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FOOTNOTES |
*
Corresponding author. Mailing address: Centre de
Recherche en Infectiologie, CHUL, Local RC-709, 2705 Boul. Laurier,
Sainte-Foy, Québec, Canada, G1V 4G2. Phone: (418) 654-2705. Fax:
(418) 654-2715. E-mail: Paul.H.Roy{at}crchul.ulaval.ca.
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Journal of Bacteriology, October 1998, p. 5437-5442, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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