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JOURNAL OF BACTERIOLOGY, July 1995, p. 4183–4186 Vol. 177, No.

14
0021-9193/95/$04.0010
Copyright 1995, American Society for Microbiology

Detection of XerC and XerD Recombinases in Gram-Negative


Bacteria of the Family Enterobacteriaceae
STÉPHANE SIROIS AND GEORGE SZATMARI*
Département de Microbiologie et Immunologie, Université de Montréal,
Montréal, Québec, Canada H3C 3J7
Received 23 December 1994/Accepted 6 May 1995

XerC and XerD are site-specific recombinases of the lambda integrase family which resolve multimeric
replicons to monomers by acting at specific sites such as cer, ckr, nmr, parB, and psi, which are found in
plasmids, or at the dif site found in the Escherichia coli chromosome. By using Southern hybridizations to
cloned E. coli xerC and xerD genes and a cer-nmr plasmid-based resolution assay, the presence of these genes
in several species of Enterobacteriaceae is shown.

Site-specific recombination systems are important for DNA detected. Serratia liquefaciens (Fig. 1, lane 3) displayed hybrid-
rearrangements such as deletions, inversions, and integrations ization to two HindIII fragments of 6.5 and 1.5 kb. The less-
of DNA in both prokaryotes and eukaryotes. These systems intense signal observed with the lower-molecular-weight frag-
require a specific DNA sequence and recombination enzymes ment corresponded to an uncut cryptic plasmid in this strain.
which act at that site. A well-characterized site-specific recom- This was confirmed by hybridizing xerC probes to Southern
bination system which uses two recombinases of the lambda blots of S. liquefaciens plasmid DNA (data not shown). In the
integrase family to catalyze a single recombination event is the various Proteus strains used (Fig. 1, lanes 4, 5, 8, and 9), no
xerC-xerD system of Escherichia coli. In this system, the XerC significant homology to xerC was detected.
and XerD recombinases act on a specific site to monomerize The same strains were tested for the presence of sequences
the E. coli chromosome or, in the presence of the accessory homologous to that of the xerD gene of E. coli (Fig. 1, lanes 13
factors ArgR and PepA (13–15), plasmid multimers (2–4, 9, 16, to 24). Both E. coli DS941Y17 (Fig. 1, lane 21) and E. coli
17). These accessory factors are not necessary for recombina- DS9029 (Fig. 1, lane 23) displayed hybridization to a single
tion at the chromosomal dif site (1). high-molecular-weight EcoRI chromosomal fragment of ap-
Many plasmids have sites which are similar to dif (3, 5, 7, 8, proximately 20 kb. Although E. coli DS9029 contains a 1.5-kb
16, 18). Since these plasmids are stably maintained in a variety insertion in xerD (Tpr inserted in Mlu1 site; no internal EcoRI
of gram-negative bacteria, it is quite conceivable that these site), the increased molecular weight of the xerD-hybridizing
other bacterial species harbor genes which are either structur- fragment is not evident, since this is out of the linear range of
ally or functionally equivalent to the XerC and XerD recom- the 1% agarose gel. Both M. morganii (Fig. 1, lane 13) and S.
binases. We report here the presence of sequences homolo-
typhimurium (Fig. 1, lane 20) showed xerD-specific hybridiza-
gous to the E. coli xerC and xerD genes in many members of the
tion to single genomic EcoRI fragments of 3.5 and 8.7 kb,
Enterobacteriaceae family and the possession of site-specific
respectively. K. pneumoniae (Fig. 1, lane 14) displayed strong
recombination activities analogous to those of XerC and XerD
xerD hybridization to a 6.5-kb chromosomal EcoRI fragment
among these species.
and a weaker hybridization which corresponded to partially
DNA sequences homologous to those of xerC and xerD are
digested chromosomal DNA. S. flexneri (Fig. 1, lane 22) dis-
present in Enterobacteriaceae. Chromosomal DNAs from sev-
played strong xerD-specific hybridization to 2.9- and 5.4-kb
eral different species of Enterobacteriaceae (Table 1) were ex-
EcoRI fragments, indicating the presence of an internal EcoRI
tracted (6), digested, and transferred to nylon membranes and
site in the xerD gene of this species. In S. liquefaciens (Fig. 1,
hybridized to either xerC- or xerD-specific probes. The xerC
lane 19), a 20-kb EcoRI fragment and a 1.5-kb fragment both
probe consisted of a 1.2-kb EcoRI-HindIII fragment puri-
hybridized to xerD. As was the case with xerC hybridization,
fied from plasmid pSD105 (4), and the xerD probe consisted
this lower-molecular-weight fragment corresponded to an un-
of a 1.5-kb EcoRI-HindIII fragment purified from plasmid
cut cryptic plasmid residing within S. liquefaciens.
pRM130 (2). The xerC probe hybridized to two chromosomal
Finally, in all of the Proteus species tested, only one, Proteus
EcoRI fragments of 5.6 and 4.5 kb in the positive control
vulgaris ATCC 13315 (Fig. 1, lane 15) showed a 4.6-kb frag-
strains E. coli DS941Y17 and DS9029 (Fig. 1, lanes 1 and 2). In
ment hybridizing to the xerD gene. This hybridization was
these strains, the xerC gene is disrupted by a mini-Mu PR13
shown to reside in a cryptic plasmid, since undigested chromo-
insertion (containing an internal EcoRI site). For Klebsiella
somal DNA did not hybridize to xerD (not shown).
pneumoniae (Fig. 1, lane 4), Morganella morganii (Fig. 1, lane
In vivo detection of xerC-xerD recombination activity. Under
11), Salmonella typhimurium (Fig. 1, lane 5), and Shigella flex-
our stringent hybridization conditions, only high homology be-
neri (Fig. 1, lane 10), hybridization to single EcoRI chromo-
tween the immobilized DNA and probe led to a detectable
somal fragments of 4.3, 5.0, 8.0, and 4.7 kb, respectively, were
signal. However, the apparent lack of homology to xerC or xerD
does not signify the lack of these genes. It is quite possible that
* Corresponding author. Mailing address: Département de Micro-
gene products which perform an identical function to either
biologie et Immunologie, Université de Montréal, C.P. 6128, Succur- xerC or xerD have diverged throughout evolution such that
sale Centre-ville, Montréal, Québec, Canada H3C 3J7. Phone: (514) their detection by Southern blotting may not be possible.
343-5767. Fax: (514) 343-5701. Electronic mail address: Szat@ere. To screen for these recombinational activities, we utilized an
umontreal.Ca. assay based on the abilities of these bacterial strains to delete

4183
4184 NOTES J. BACTERIOL.

TABLE 1. Bacterial strains used in this study type refers to the ability of a strain to undergo site-specific
Reference or
recombination at cer or cer-related sites), site-specific recom-
Strain Characteristics bination between nmr and cer occurs, resulting in deletion of
sourcea
the npt gene and generation of a single hybrid recombination
E. coli site (19). This can be detected in vivo (by kanamycin sensitiv-
DS941 AB1157 recF lacIQ lacZ 17
ity) and in vitro (by restriction analysis of plasmid DNA or
DM15
DS9029 DS941 xerD::Tpr xerC:: D. J. Sherratt PCR). This plasmid is more effective in detecting site-specific
mini-Mu PR13 recombination because of the reduced homology between cer
DS941Y17 DS941 xerC::mini-Mu 6 and nmr (3, 19), since plasmids with identical recombination
PR13 sites can undergo recombination in the absence of xerC, xerD,
DS956 DS941 argR::Tpr D. J. Sherratt and recA function (11). Plasmid pNCK-1, which was purified
DS957 DS941 pepA::Tn5 20 from the xerC xerD mutant strain DS9029, was first trans-
K. pneumoniae ATCC 13863 ATCC
formed into xer1 and mutant xer strains of E. coli and then
M. morganii UM 82-83 UM
P. mirabilis UM 240-82 UM
transformed into various enteric species by electroporation
P. mirabilis MH 3184 Martha Howe (12). Transformants were initially selected on kanamycin-con-
P. vulgaris ATCC 13315 ATCC taining media and subsequently subcultured into ampicillin-
P. vulgaris UM 5.71 UM containing media. This ensured that the initial transformation
S. typhimurium UM 2.69 UM event selected for intact, nonresolved pNCK-1 and that reso-
S. liquefaciens LSPQ 2422 LSPQ lution could occur after subsequent culturing of the transfor-
S. flexneri ATCC 12022 ATCC mants with media that selected for the presence of the plasmid
a
UM, Université de Montréal (collection of clinical isolates); LSPQ, Labora- only. Minipreps were performed with selected transformants,
toire de la Santé Publique du Québec; ATCC, American Type Culture Collec- and the extracted plasmid DNA was electrophoresed on 0.5%
tion. agarose gels and visualized by ethidium bromide fluorescence
(Fig. 2). Upon transformation into the xer1 E. coli strain
DS941 (Fig. 2, lane 8), pNCK-1 was efficiently resolved, gen-
a genetic marker flanked by two recombination sites. Plasmid erating a 3.1-kb plasmid. Transformation into the xerC xerD
pNCK-1, which is a derivative of the Apr plasmid pCN20 (19), mutant strain DS9029 did not result in any change in pNCK-1
possesses the Tn903 npt gene between directly repeated nmr (Fig. 2, lane 9).
and cer sites cloned in the pUC19 multiple cloning site. When pNCK-1 was then transformed into a variety of gram-nega-
this plasmid is transformed into xer1 bacteria (the xer pheno- tive Enterobacteriaceae. Figure 2 (lanes 1 to 7) shows the anal-

FIG. 1. Detection of xerC and xerD homology in several strains of Enterobacteriaceae by Southern hybridization. Chromosomal DNAs isolated from the indicated
strains were digested with EcoRI, except for S. liquefaciens and phage lambda, which were digested with HindIII. Fragments were electrophoresed on a 1%
Tris-acetate-EDTA agarose gel, transferred to a nylon membrane, and hybridized overnight with a xerC probe (lanes 1 to 12) and a xerD probe (lanes 13 to 24). The
bacterial DNAs used are as follows: E. coli DS941Y17 (lanes 1 and 21), E. coli DS9029 (lanes 2 and 23), K. pneumoniae ATCC 13863 (lanes 4 and 14), M. morganii
UM 82-83 (lanes 11 and 13), P. vulgaris ATCC 13315 (lanes 6 and 15), P. vulgaris UM 5.71 (lanes 7 and 16), P. mirabilis MH 3184 (lanes 8 and 17), P. mirabilis UM
240-82 (lanes 9 and 17), S. typhimurium UM 2.69 (lanes 5 and 20), S. liquefaciens LSPQ 2422 (lanes 3 and 19), S. flexneri ATCC 12022 (lanes 10 and 22), and phage
lambda (lanes 12 and 24).
VOL. 177, 1995 NOTES 4185

present. This could allow for each separate strand exchange


event to be more precisely controlled (10). Another possible
reason for having two recombinases is to increase the efficiency
of the site-specific recombination reaction. When XerC and
XerD act on the E. coli chromosome, the dif sites are separated
by about 4 Mb of DNA. Having two recombinases may make
it easier for the recombinases to find their sites and ultimate-
ly to align them for the recombination reaction. The observa-
tion that xerC-xerD binding to dif is cooperative (2) lends
some support to this hypothesis. In addition, the xerC-xerD-
dif system is the only known site-specific recombination system
in which the genes encoding the recombinases are located
far away from their binding sites (ranging from 1.11 to 1.44
Mb).
The similarity between the amino acid sequences of xerC and
xerD and their DNA-binding sites (2) also leads us to suggest
that these genes may have originated from a common ancestor.
This similarity may become more apparent once the sequences
of corresponding xerC and xerD genes from other bacterial
species become known.

We thank David J. Sherratt, Martha Howe, and the Département de


Microbiologie et Immunologie of the Université de Montréal for bac-
FIG. 2. Detection of cer-nmr site-specific recombination in Enterobacteri-
terial strains and plasmids; and Nicolina Zakova Pavlova and Nicolas
aceae. Plasmid pNCK-1 was isolated from transformed xer1 (lane 9) and xerC
xerD mutant (lane 8) strains of E. coli and the following Enterobacteriaceae: S. Réhel for ideas and technical support.
flexneri ATCC 12022 (lane 1), S. liquefaciens LSPQ 2422 (lane 2), S. typhimurium This work was supported by an operating grant from the Natural
UM 2.69 (lane 3), K. pneumoniae ATCC 13863 (lane 4), P. mirabilis MH 3184 Sciences and Engineering Research Council of Canada.
(lane 5), P. vulgaris UM 5.71 (lane 6), and M. morganii UM 82-83 (lane 7). The
plasmid DNA was electrophoresed in 0.5% Tris-acetate-EDTA agarose gels for
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