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Antimicrobial Agents and Chemotherapy, October 1999, p. 2538-2541, Vol. 43, No. 10
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Reconstruction of an Active Integron Recombination
Site after Integration of a Gene Cassette at a Secondary Site
Heidi
Segal,1
M.
Victoria
Francia,2
Juan M. García
Lobo,2 and
Gay
Elisha1,3,*
Department of Medical Microbiology,
University of Cape Town,1 and Groote
Schuur Hospital,3 Cape Town, South Africa, and
Departamento de Biología Molecular, Facultad de
Medicina, Universidad de Cantabria, 39011 Santander,
Spain2
Received 21 January 1999/Returned for modification 24 May
1999/Accepted 22 July 1999
 |
ABSTRACT |
As the site of insertion of the aadB gene cassette on
pRAY, from a clinical isolate of Acinetobacter, is almost
identical to the preferred site on integrons, the composite 59-base
element (59-BE) associated with this cassette is potentially
recombinationally active. By using a conduction assay to quantitate
site activity, the 59-BE was recognized by integrase with high
frequency, indicating that the composite site is recombinationally active.
| |
TEXT |
The acquisition of antibiotic
resistance gene cassettes by integrons and the role of integrons in the
mobilization of antibiotic resistance genes to and from members of the
family Enterobacteriaceae have been reported extensively
(17). There is much less information on these elements in
species of Acinetobacter, and only recently have gene
cassettes and integrons been reported in this genus (1, 9, 20,
22).
Integrons encode a site-specific recombinase, integrase (IntI1), that
catalyzes the mobility of gene cassettes (10, 14, 15, 19).
Although IntI1 preferentially recognizes the core sites (GTTRRRY) of
attI1 and of 59-base element (59-BE) flanking gene
cassettes, it also recognizes secondary sites, conforming to the
consensus GWTMW (8).
A gene cassette encoding a 2"-adenylyltransferase, which has activity
against aminoglycoside antibiotics, including gentamicin, tobramycin,
and kanamycin, was identified at a secondary site on plasmid pRAY in a
clinical isolate of Acinetobacter (20). The
aadB gene is regulated by a promoter on pRAY
(21). The boundaries of the cassette suggest that insertion
of the cassette was catalyzed by IntI1 (20). Furthermore,
the putative insertion site (GTTAGGA) on pRAY is similar to
the preferred site on integrons, and it may be that the cassette is
flanked by two putatively active recombination sites (20),
which is unusual for a cassette at a secondary site (18).
Plasmids and bacterial strains.
The plasmids and bacterial
strains used in this study are listed in Table
1. A 1.7-kb
BamHI-HindIII fragment, containing the
aadB gene cassette from pRAY, was restricted from pHS100
(20) and ligated to pSU18 to generate pSU1817.
Escherichia coli DH5
was used as a host for plasmid
construction and in gene cassette excision experiments. E. coli UB5201 (F
pro met recA56 gyrA
Nalr) and UB1637 (his lys trp recA56 rpsL) were
used as recipient and donor strains, respectively, in mating
experiments.
Conduction assays.
Gene cassettes at secondary sites are
thought to be stably integrated because the composite 59-BE formed on
insertion is recombinationally inactive (18). Following the
insertion of an aadB cassette at a secondary site on
RSF1010, generating pIE723, a 59-BE core sequence (GATCAAA)
which differs from the consensus core sequence site (GTTRRRY) at
one conserved position and two consensus positions was formed. Studies
have shown that a T
A transversion in the second position of the core
sequence reduced the frequency of recombination 1,000-fold
(23). Changes in the first and third positions of the GTT
triplet and a transition at the second position reduced recombinational
activity 40- to 170-fold, showing that the central T is critical for
site activity (23). That the aadB 59-BE from
pIE723 was essentially recombinationally inactive is consistent with
these data, and it was concluded that the cassette is stably integrated
in pIE723 (18).
As the insertion site on pRAY (GTTAGGA) is almost identical
to the consensus core and, importantly, contains the critical GTT, the
core site of the composite 59-BE of the cassette on pRAY is potentially
recombinationally active (20). To test whether the putative
recombination sites could be recognized by integrase, the frequency of
conduction of pSU1817 by R388 was determined. The integron In3 on R388
contains two integrated gene cassettes, dfrB2 and
orfA, and consequently three recombination sites, the two
cassette-associated 59-BEs and an attI1 site. Integrase
was supplied in trans by including pSU2056, which expresses
high levels of integrase (15). Conduction experiments were
carried out as described previously (8). E. coli
UB1637(pSU2056)(R388) was transformed with pSU1817 and conjugated with
UB5201. Donor and recipient cells were mixed on a 0.22-µm-pore-size
Millipore filter on Luria-Bertani (LB) agar and were left undisturbed
at 37°C for 3 to 4 h. Filters were washed with 1 ml of
physiological saline, and 10-fold dilutions of the suspension were
plated onto M9 minimal medium (16) containing trimethoprim
(20 µg/ml) and LB agar containing chloramphenicol (25 µg/ml) and
nalidixic acid (20 µg/ml). Trimethoprim (20 µg/ml) was incorporated
into the M9 minimal medium to select for UB5201 transconjugants
containing R388. LB agar containing chloramphenicol (25 µg/ml) and
nalidixic acid (20 µg/ml) was selective for UB5201 containing
pSU1817/R388 cointegrates. The conduction frequency in each assay was
calculated as the ratio of Cmr to Tpr
transconjugants. The average frequency of conduction of pSU1817 (2.3 × 101) was more than 5 orders of magnitude
higher than that of the vector pSU18, which does not contain any
specific sites, and similar to that of pSU18R2 (1.3 × 102), which contains a 59-BE (Table
2).
Mapping of the recombination sites.
To determine the site of
insertion of pSU1817 into R388, plasmid DNA from 24 transconjugants
from two independent crosses was digested with BamHI, and
the fragments were separated by agarose gel electrophoresis. Plasmid
R388 contains four BamHI fragments of ~29, 2.06, 1.75, and
0.23 kb (2). The potential site-specific sites of
recombination of In3 on R388 are located in the 2.06-kb (attI1 and 59-BE of dfrB2) and 1.75-kb (59-BE of
orfA) fragments. Recombination involving any of the sites in
the 2.06- or 1.75-kb fragments would alter the sizes of these
fragments. BamHI digestion of plasmid DNA from 24 Cmr transconjugants generated three profiles. Seventy
percent (17 of 24) of the cointegrates had a profile consistent with a
recombination event involving attI1 on R388 and the 59-BE of
aadB on pSU1817 (Fig. 1a). The
second profile (4 of 24) resulted from a similar recombination event
between attI1 on R388 and the 59-BE on pSU1817 and excision
of the orfA gene cassette (Fig. 1b). A recombination event
between R388 attI1 and the 59-BE on pSU1817, combined with the excision of the orfA and dfrB2 cassettes,
resulted in a third profile (Fig. 1c) in 3 of the 24 cointegrates
analyzed. Antibiotic susceptibility testing confirmed that the
Tpr gene cassette had been excised.
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FIG. 1.
Cointegrate formation involving R388 attI1
site ( ) and aadB 59-BE
( ) on pSU1817. Note that the
attI1 site flanking aadB on pSU1817 is
incomplete. The double-headed arrow indicates sites involved in the
formation of cointegrates. BamHI (B) and
HindIII (H) sites are shown. (a) Recombination between
R388 attI1 site and 3' core site of 59-BE on pSU1817; (b)
recombination between R388 attI1 site and 3' core site of
59-BE on pSU1817 and excision of orfA gene cassette; (c)
recombination between R388 attI1 site and 3' core site of
59-BE on pSU1817 and excision of orfA and dfrB2
gene cassettes. Note that the same result would also be produced by
recombination between the 59-BE of orfA of R3888 and
aadB of pSU1817, combined with the excision of the
orfA and dfrB2 cassettes. pSU18 is indicated by a
broken line. Sizes below maps are in kilobases.
|
|
A primer from the 5' side of attI1 in R388 was used to
determine the nucleotide sequence of the sites involved in cointegrate formation. The oligonucleotide, 5'-GGGAATTCAGCAACGATGTTACGCA-3', was labelled with [
-32P]ATP and included in a
sequencing reaction as described previously (8). DNA
sequencing of one junction from a cointegrate of R388 and pSU1817 of
each profile described above confirmed that recombination was site
specific and involved attI1 on R388 and the 59-BE on pSU1817
(Fig. 2).
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FIG. 2.
Sequence of boundary of the aadB gene
cassette on pSU1817 and the attI1 insertion site on R388
involved in cointegrate formation. The secondary site and 59-BE core
site flanking the aadB gene cassette and the
attI1 site on R388 are indicated in boldface type. The
arrows indicate the sites involved in the formation of the recombinant
junction.
|
|
These data show that unlike the 59-BE from pIE723, the 59-BE from pRAY
is highly active. Cointegrations involving the 59-BE on pSU1817 and
attI1 on R388 occurred at frequencies comparable to those of
recombination events involving two 59-BEs (i.e., 3.7 × 102) (4, 5), whereas the 59-BE from pIE723 was
conducted at a frequency only slightly above background
(18).
Cassette excision analysis.
The transfer of gene cassettes may
proceed through a route that does not involve a cointegration event.
Gene cassettes are excised from integrons as free circles. Following
excision, a cassette can be inserted by a single IntI1-mediated event
between the 59-BE of a circular cassette and either a specific site on an integron or a secondary site (4-6, 8, 10, 11, 13, 19).
There is a strong requirement for two primary recombination sites in
order for gene cassettes to be excised (4, 5). As the
aadB gene in this study is flanked by a 59-BE and an
incomplete attI1 site, excision of the cassette was
anticipated to be a rare event. Two separate methods were used to
detect IntI1-mediated excision of the aadB gene cassette
from pSU1817. In the first methods, the plasmid was propagated in the
presence of high levels of integrase encoded by pSU2056
(15). Plasmid DNA was isolated from DH5 cells containing
pSU2056 and pSU1817 and reintroduced into DH5 by transformation.
Transformants were selected on medium containing chloramphenicol (25 µg/ml). To screen for the loss of the aadB gene cassette
from pSU1817, transformants were subsequently replica plated onto LB
medium containing either chloramphenicol (25 µg/ml) or kanamycin (25 µg/ml). Cmr transformants that were Kms were
indicative of loss of the aadB gene cassette from pSU1817. A
total of 300 and, in a separate experiment, 700 Cmr
E. coli DH5 (pSU2056)(pSU1817) transformants were replica
plated onto medium containing either chloramphenicol (25 µg/ml) or
kanamycin (25 µg/ml). No Kms transformants were isolated,
indicating either that the aadB gene cassette could not be
excised from pSU1817 or that excision occurred at a low frequency
(<1.4 × 103).
In the second method, a PCR assay was used to check for cassette
excision. Plasmid pSU1817 was completely digested with a restriction
endonuclease that cleaves this plasmid at a single site within the
aadB cassette. The cleaved DNA was used as a template in a
PCR assay with two primers (CAGGAAACAGCTATGAC and
GTAAAACGACGGCCAGTG) that correspond to the vector-located
forward and reverse primer sites flanking the aadB cassette
in pSU1817. After 30 cycles of sequential 30-s incubations at 94, 50, and 72°C in a Perkin-Elmer 2400 apparatus, the products were analyzed
by electrophoresis. We were unable to obtain any amplification product,
indicating again that cassette deletion was either a rare event or a
nonexistent one.
We can conclude that upon integration of a gene cassette at a secondary
site, the resistance gene in the cassette becomes flanked by a
secondary site and a hybrid 59-BE. As shown here, the hybrid 59-BE may
be fully recombination proficient. This quality in itself confers
mobility on the resistance gene cassette, since IntI1-mediated replicon
cointegration may occur at a high frequency with just the hybrid 59-BE
present in the mobilized replicon and a functional integron in the
driver. However, cassette excision is a reaction that requires further
specificity and will probably not occur at all in the case of the
cassettes integrated at secondary sites.
| |
ACKNOWLEDGMENTS |
This work was supported by a UNESCO-MCBN short-term fellowship to
H.S. and by grants from the University of Cape Town to B.G.E.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology, Medical School UCT, Anzio Rd., Observatory 7925, Cape Town, South Africa. Phone: (27) (21) 406 6378. Fax: (27) (21) 448 8153. E-mail: gelisha{at}medmicro.uct.ac.za.
| |
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Antimicrobial Agents and Chemotherapy, October 1999, p. 2538-2541, Vol. 43, No. 10
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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