ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2008, p. 1472–1480
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 52, No. 4
Different Pathways to Acquiring Resistance Genes Illustrated by the
Recent Evolution of IncW Plasmids?†
Carlos Revilla,1# M. Pilar Garcilla ´n-Barcia,1# Rau ´l Ferna ´ndez-Lo ´pez,1Nicholas R. Thomson,2
Mandy Sanders,2Martin Cheung,3Christopher M. Thomas,3and Fernando de la Cruz1*
Departamento de Biologı ´a Molecular e Instituto de Biomedicina y Biotecnologı ´a de Cantabria (IBBTEC), Universidad de
Cantabria-CSIC-IDICAN, C. Herrera Oria s/n, 39011 Santander, Spain1; The Pathogen Sequencing Unit,
Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge CB10 1SA,
United Kingdom2; and School of Biosciences, University of Birmingham, Edgbaston,
Birmingham B15 2TT, United Kingdom3
Received 28 July 2007/Returned for modification 23 September 2007/Accepted 3 February 2008
DNA sequence analysis of five IncW plasmids (R388, pSa, R7K, pIE321, and pIE522) demonstrated that they
share a considerable portion of their genomes and allowed us to define the IncW backbone. Among these
plasmids, the backbone is stable and seems to have diverged recently, since the overall identity among its
members is higher than 95%. The only gene in which significant variation was observed was trwA; the changes
in the coding sequence correlated with parallel changes in the corresponding TrwA binding sites at oriT,
suggesting a functional connection between both sets of changes. The present IncW plasmid diversity is shaped
by the acquisition of antibiotic resistance genes as a consequence of the pressure exerted by antibiotic usage.
Sequence comparisons pinpointed the insertion events that differentiated the five plasmids analyzed. Of
greatest interest is that a single acquisition of a class I integron platform, into which different gene cassettes
were later incorporated, gave rise to plasmids R388, pIE522, and pSa, while plasmids R7K and pIE321 do not
contain the integron platform and arose in the antibiotic world because of the insertion of several antibiotic
Naturally occurring plasmids tend to fall into coherent ge-
netic clusters commonly known as incompatibility groups (21).
This is in contrast to the genetic organization of bacterio-
phages, which show extreme modularity, generating great di-
versity by the permutation of modules (82). Plasmids of a given
incompatibility group normally share the mechanisms of rep-
lication (1), copy number control (25), and other maintenance
functions (27; for reviews see references 5, 26, and 55). They
thus function as a single pool in the bacterial cell and due to
random selection for replication or partitioning will segregate
cells with one or the other plasmid but not both. They are thus
said to be incompatible. From the many incompatibility groups
described, a few have been analyzed in detail. Some of the
best-known incompatibility groups are those within the IncF
complex (IncFI and IncFII) (13, 58), the IncPs (2, 9), the IncQs
(62, 63), and the IncH group (33). Although incompatibility is
not always associated with sequence identity, some incompat-
ibility groups show a conserved genetic backbone. Their mem-
bers generally share an essential gene set for plasmid survival,
differing in the presence of mobile genetic elements (MGEs)
harbored in their genomes. Plasmids can thus be considered
vehicles carrying cargos of transposons, integrons, and inser-
tion sequences that move across bacterial populations. IncP,
IncN, and IncW groups have a broad host range, so they might
serve as MGE shuttles between different species. The role of
horizontal gene transfer in bacterial evolution is well docu-
mented (23, 56). A planetary-scale experiment in bacterial
evolution took place when antibiotics were introduced by hu-
mans around 1950 (49). The result was that antibiotic-sensitive
bacteria were rapidly replaced by bacteria that had acquired
mobile pieces of DNA containing antibiotic resistance genes.
Among them, transposons and integrons account for a large
portion of the antibiotic resistance genes carried by plasmids
and other MGEs (31).
The IncW group takes its name from T. Watanabe, who
described the first member of the group, pSa (81). The IncW
group includes three “classical” members: pSa (81), R388 (22),
and R7K (17). Electron microscope heteroduplex studies (34)
and restriction enzyme maps of these plasmids (6, 45, 78, 80)
underscored the high DNA sequence homology between them.
Physical and genetic maps of these plasmids were drawn based
on information from many authors (79). R388 has been studied
in the most detail, and its complete sequence has been used to
infer many features of the family (28). In this work, we ana-
lyzed the DNA sequences of five IncW plasmids in order to
ascertain their diversity and evolution. These are pSa, R388,
R7K, and two more recent isolates, pIE321 and pIE522 (35).
IncW plasmids are less than 40 kb in size (Table 1) and have
been isolated from different species of Enterobacteriaceae; pSa
was obtained from Shigella (81), R388 from Escherichia coli
(41), R7K from Providencia rettgeri (17), pIE321 from Salmo-
nella dublin, and pIE552 from Klebsiella pneumoniae (35). The
host range of IncW plasmids, under laboratory conditions, has
* Corresponding author. Mailing address: Departamento de Biolo-
gı ´a Molecular e Instituto de Biomedicina y Biotecnologı ´a de Cantabria
(IBBTEC), Universidad de Cantabria-CSIC-IDICAN, C. Herrera
Oria s/n, 39011 Santander, Spain. Phone: 34-942-201942. Fax: 34-942-
201945. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://aac
# These authors are equal contributors.
?Published ahead of print on 11 February 2008.
been shown to comprise most of the species from the phylum
Proteobacteria that have been tested so far (for a review, see
In spite of their common genetic backbone, IncW plasmids
confer different antibiotic resistance profiles, indicating that
these plasmids differ in the antibiotic resistance determinants
they carry. Based on the comparison of the DNA sequences of
pSa, R388, R7K, pIE321, and pIE522, this work analyzes the
recent evolution and diversification of the IncW backbone.
MATERIALS AND METHODS
Standard DNA techniques. For cloning, plasmid DNA was extracted using a
Qiaprep spin miniprep kit (Qiagen). For DNA array, plasmid DNA was ex-
tracted using a GenElute plasmid miniprep kit (Sigma), and total genomic E. coli
DNA was obtained according to the method described in reference 61. DNA was
extracted from agarose gels with a Qiaquick gel extraction kit (Qiagen). Restric-
tion enzyme digestions, agarose gel electrophoresis, DNA cloning, and the trans-
formation of E. coli were all carried out according to the methods described in
Conjugation assays. Matings were carried out using derivatives of strain
DH5? [F?endA1 recA1 gyrA96 thi-1 hsdR17 supE44 relA1 ?(argF-lacZYA)U169
?80dlacZ?M15] (38) carrying various plasmids as donors and UB1637 (F???lys
his trp rpsL recA56) (24) as a recipient strain. Saturated cultures of donor and
recipient strains were mixed in a 1:1 ratio and mated on an LB agar surface for
1 h at 37°C as described previously (48). Transconjugants were selected on
trimethoprim (20 ?g/ml) for R388, tetracycline (10 ?g/ml) for pIE321, chloram-
phenicol (25 ?g/ml) for pSa, ampicillin (100 ?g/ml) for R7K, and gentamicin (10
?g/ml) for pIE522. Donors were counterselected on streptomycin (300 ?g/ml).
DNA macroarray hybridization. (i) Probe DNA labeling by random priming.
Plasmid DNA (25 ng) was diluted to a 45-?l final volume in Tris-EDTA buffer
(10 mM Tris-HCl, pH 8.0, 1 mM EDTA). DNA was denatured by heating at 95°C
for 5 min and placed on ice for an additional 5 min. Denatured DNA was
incubated in a reaction solution containing Rediprime II random prime labeling
system (Amersham) and 50 ?Ci of [32P]dCTP (Redivue Amersham) for 10 min
at 37°C. The reaction was stopped by adding 0.2 M EDTA (5 ?l), heating at 95°C
for 5 min, and cooling on ice for an additional 5 min.
(ii) Membrane spotting. Target DNAs were obtained by PCR amplification of
the relevant DNA fragments from either R388 or E. coli DNA. A total of 2.5 ?g
of each amplified DNA was denatured in alkali and spotted onto a nylon mem-
brane (Zeta-Probe GT; Bio-Rad) by using the high-density replicating tool
(HDRT-96) of robot Biomek 2000 (Beckman) and 0.045-in.-wide metal pins.
The spotted membrane was neutralized by washing it in Tris-HCl (20 mM, pH
7.0) for 20 min and UV-cross-linking it at 1,200 mJ in a UV Stratalinker 1800
(iii) Array hybridization. The spotted membrane was prehybridized for 2 h
with 5 ml buffer (0.25 M sodium phosphate, pH 7.2, 7% sodium dodecyl sulfate
[SDS]) at 65°C. Then, 55 ?l labeled probe DNA was added and hybridized for
20 h under the same conditions. After hybridization, the membrane was washed
twice with 5 ml buffer A (sodium phosphate, 20 mM, pH 7.2) plus 5% SDS for
30 min at 65°C and washed twice again with 5 ml buffer A plus 1% SDS in the
same conditions. Treated membranes were scanned using a Molecular Imager
DNA sequencing, sequence analysis, and annotation. The R388 and pIE321
plasmid DNAs, as well as fragments of plasmids pIE522 and pSa, were se-
quenced by progressive DNA walking, using amplified DNA fragments. The
BigDye Terminator v3.0 kit (Applied Biosystems) was used in sequencing reac-
tions. Sequencing products were analyzed in an automatic multicapillary 3700
DNA sequencer with ?4-fold DNA coverage. The program suite Vector NTI 5.5
was used for analyzing and annotating the plasmid sequences. ContigExpress was
used for assembling the sequences. GC content analysis was carried out with
BioPlot. Multiple alignments were done with AlignX. Open reading frames
(ORFs) encoding at least 50 amino acids after a translation start codon were
identified. DNA and deduced protein sequences were searched by BLAST anal-
ysis. ORFs were manually annotated using evidence from BLAST analyses (4)
and experimental or other evidence from previous literature. For R7K, the DNA
was fragmented by sonication and size fractionated before constructing libraries
in pUC19. The sequence was generated from paired-end reads from two pUC19
libraries with insert sizes of 2 to 4 kb and 4 to 6 kb. These sequence reads were
performed with ABI BigDye Terminator chemistry on ABI3730 sequencing
machines and gave a total coverage for the plasmid of approximately eightfold.
All identified repeats were bridged with read pairs or end-sequenced PCR
products. Error checking and the finishing of the sequence were performed
according to standard criteria using Phrap to assemble the sequence and Gap4
Nucleotide sequence accession numbers. Complete DNA sequences for R7K
and pIE321 are available at EMBL-EBI Bank and GenBank, respectively, under
accession numbers AM901564 (R7K) and EF633507 (pIE321). Partial sequences
of pIE522 and pSa are available under GenBank accession numbers EU247928
and EU419764, respectively.
RESULTS AND DISCUSSION
A DNA array for the analysis of IncW plasmids. An array of
79 spots was prepared on nylon membranes by including each
of the 43 R388 genes, 14 intergenic regions, 12 E. coli chro-
mosomal genes, and total genomic DNA from E. coli and R388
(see Fig. S1 in the supplemental material). Among other pro-
jected applications, this array is useful as a first characteriza-
tion tool to analyze the diversity of IncW plasmids.
When R388 DNA was hybridized against the array, the in-
tensity of the signals correlated well with the size of the DNA
in the spot (data not shown), indicating that association kinet-
ics was the limiting step. No hybridization signal appeared with
spots from E. coli ORFs, except an unexplained signal in gene
rpoB. When the same array was hybridized against each of the
four IncW plasmids shown in Table 1, hybridization to most
R388 probes (ORFs and intergenic regions) was observed (see
Fig. S1 in the supplemental material). Differences in the hy-
bridization profiles of pIE522 and pSa concerned just the gene
cassettes carried by the respective integrons, a consequence of
their different antibiotic resistance profiles. Besides, plasmids
pIE321 and R7K lacked the six spots (see Fig. S1, 9E to 10D,
TABLE 1. General features of IncW plasmids
Original host (year of isolation)
Size (bp) GC content (%)
No. of indels in
E. coli (1972) (22)
Salmonella enterica (1996) (35)
Providencia rettgeri (1972) (17)
Klebsiella pneumoniae (1996) (35)
Shigella sp. (1968) (82)
Apr, Smr, Spr
Gmr, Kmr, Sur, Tobr
Cmr, Gmr, Kmr,
Smr, Spr, Sur
aTpr, resistance to trimethoprim (20 ?g/ml); Sur, resistance to sulfonamide (10 ?g/ml); Tcr, resistance to tetracycline (10 ?g/ml); Smr, resistance to streptomycin (20
?g/ml); Apr, resistance to ampicillin (100 ?g/ml); Spr, resistance to spectinomycin (100 ?g/ml); Gmr, resistance to gentamicin (10 ?g/ml); Kmr, resistance to kanamycin
(50 ?g/ml); Cmr, resistance to chloramphenicol (25 ?g/ml); Tobr, resistance to tobramycin (10 ?g/ml).
bSince these plasmids have not been completely sequenced, it is possible (but not likely according to the array results and calculated overall size of the plasmids)
that they contain additional indels.
VOL. 52, 2008RECENT EVOLUTION OF IncW PLASMIDS1473
in the supplemental material) related to the integron, indicat-
ing that these two plasmids lack an integron platform. Finally,
gene osa was absent in plasmid R7K. The use of the array
allowed us to include plasmids pIE321 and pIE522 (from
which nearly nothing was known) within the realm of IncW
plasmids. Furthermore, based on the array results, we decided
to sequence plasmids pIE321 and R7K, which showed the most
interesting differences with respect to R388. We did not com-
plete the sequences of the other two because they are practi-
cally identical to the R388 backbone.
Analysis of the DNA sequences of five IncW plasmids. The
complete sequences of plasmids pIE321 (accession number
EF633507) and R7K (accession number AM901564) were ob-
tained. Plasmid R388 (accession number BR000038) was rese-
quenced, and the 50? sequence differences were submitted to
GenBank for amending. The revised R388 sequence was used
in all later analyses. In addition, we used the following pSa
sequences available in the databases: L06822 (18, 40),
AF143206 (10), X00060 (71), X68227 (11), U04277 and
U04278 (68, 77), U30471 (16, 57), and M11444 (72). Addition-
ally, we sequenced 5,600 bp of plasmid pIE522, comprising the
backbone region oriT-trwA and the integron insertion (acces-
sion number EU247928) as well as the stbA-oriT-trwA region of
pSa (accession number EU419764).
Both the DNA sequences and the previous array data
indicate that all five IncW plasmids show extensive genetic
conservation. Using the sequence information, the IncW
genetic backbone was extrapolated as the genomic sequence
in common between its five members and is represented in
Fig. 1. The IncW backbone is 29,684 bp long and contains 37
ORFs. Some comparative data among backbones are shown
in Table 2. Plasmids R388, pIE321, and R7K diverged sig-
nificantly (pIE321 and R7K were 97.0 and 97.5% identical
to R388, respectively) in comparison to pSa and pIE522
(both had almost 100% identity to R388 in the available
backbone sequences). The five IncW plasmids contain one
or more indels containing antibiotic resistance genes in an
otherwise practically identical backbone (Table 1). They are
FIG. 1. Gene load in the IncW backbone. The insertions of the different MGEs in the IncW backbone that originated the five plasmid isolates
are represented. Coding sequences (CDSs) and other sequence features belonging to the IncW backbone are gray, white, or gray-white
combinations, depending on their assignment to functional modules. Plasmid-specific genes are represented by the color scheme indicated in the
row of rectangles (at right) with inscribed plasmid names. The 5? and 3? conserved sequences of the integron platform are brown. When the 5?
or 3? portion of a given CDS has been deleted, it is indicated by a prime (?) before or after the CDS name, respectively. The deletion sites within
transposons are marked by vertical lines.
1474 REVILLA ET AL.ANTIMICROB. AGENTS CHEMOTHER.
depicted in Fig. 1. Since all IncW plasmid isolations were
carried through biparental matings, based on direct detec-
tion of antibiotic resistance markers, IncW cryptic plasmids
devoid of transposable elements or integrons have yet to be
Sequence divergence in the IncW backbone. The high degree
of sequence conservation and the evidence of recent MGE
insertions indicate that IncW is a homogeneous and relatively
“young” plasmid cluster. This assertion can be confirmed by
using relaxase protein domains as a molecular clock for esti-
mating divergence rates in plasmid backbones, following the
seminal work of Francia et al. (30). For instance, IncP plasmids
also share an extensive backbone (73). Relaxases of IncP sub-
group ? are 89% identical. Identity drops to 64% if the relax-
ases of subgroup ? plasmids are considered. Meanwhile, IncW
relaxase domains share 100% amino acid identity. The only
alterations in the IncW backbone are indels caused by the
MGEs described above. No evidence of backbone disruption
was observed (see Table S1 in the supplemental material),
contrary to what happened in the IncP-? plasmid pB10 (66). It
can be assumed that the described IncW plasmids diverged
from a common ancestor more recently than IncP plasmids.
Perhaps due to their recent origin, IncW plasmids are found
less frequently in natural isolates than IncPs (35, 36).
However, ?95% identity was observed in four individual
genes (see Table S1 in the supplemental material), of which
trwA, encoding a relaxosomal protein, shows the highest vari-
ation (74 to 87% amino acid identity). These differences ex-
plain the lack of PCR and hybridization signals when using
trwA probes in several IncW isolates that were positive using
oriT and oriV probes (35). trwA sequence divergence indicates
that this gene suffered a substitution rate higher than that of
other backbone genes. The nonsynonymous-to-synonymous
substitution ratio (Ka/Ks) between all pairs of trwA sequences is
lower than 1 (R388 versus pIE321, 0.1601; R388 versus R7K,
0.3773; and R7K versus pIE321, 0.2956; calculated with the
K-Estimator 6.1 program [http://www.biology.uiowa.edu
/comeron/index_files/Page432.htm]) (19, 20). That is, trwA, as
well as the remaining IncW genes, is under purifying selection.
As can be seen in the alignment depicted in Fig. 2A, amino
acid differences between TrwAR388and TrwApIE321are dis-
tributed along the protein lengths. Conversely, TrwAR388and
TrwAR7Kexhibit their amino acid differences mainly in the
N-terminal part of the protein (Ka? 0.15038 for the 72 N-
terminal residues; Ka? 0.00861 for the 48 C-terminal resi-
dues). All three TrwA variants retain residue R10, proved to
be the most important amino acid for TrwA binding to oriT
(51). Interestingly, TrwAR7Kshows mutations in two appar-
ently important residues, Q8 and S12, that are involved in
DNA recognition (50). Perhaps even more interesting, the oriT
region also shows striking differences among these plasmids
(Fig. 2B). The sequence recognized by the relaxase domain of
TrwC (identical for all these plasmids), comprising the in-
verted repeat IR2and the nic site, remains unchanged. Vari-
ations fundamentally affect the regions recognized by the pro-
teins integration host factor (IHF) and TrwA (51, 52), as well
as the trwA promoter sequence. oriTR7Kexhibits the highest
variation. IHF binding inhibits nic cleavage catalyzed by TrwC
(52), while TrwA binding performs a dual function: stimulation
of conjugation by enhancing TrwC nicking and transcriptional
repression of the trwABC operon (51). No significant differ-
ences were found in conjugation frequencies of these plasmids.
It is likely that oriT and TrwA coevolved to conform plasmid-
specific interactions. As a result, they could influence nic cleav-
age and trwABC transcription rates, modeling differences in
the conjugation properties in different environments.
Natural history of each MGE insertion. The IncW backbone
was loaded with different MGEs in each of the five plasmids
(Fig. 1). The integron platform was inserted in R388, pSa, and
pIE522. Class II transposons were inserted in R7K (Tn1 and
Tn5393?1) and pIE321 (Tn1721?1 and Tn5393?2,3). Interest-
ingly, an integron gene cassette, aadA13, was inserted in a
secondary site of plasmid R7K. All insertions occurred outside
the conjugation modules. MGEs in IncP plasmids show signif-
icant hot spots in oriV-klcA and tra-trb (44, 74). Judging from
the representations in Fig. 1 and 3, there are not preferred
regions for MGE insertion in the IncW backbone. It may also
be an indication of the recent origin of IncW plasmids that all
MGE insertions can be explained in one or a few steps. The
inserted MGEs themselves are not composite transposons.
Figure 1 shows that insertions did not produce extensive reor-
ganizations in the IncW backbone and synteny was maintained.
Transposon and cassette insertions in plasmid R7K. In
R7K, a 4,949-bp insertion (Fig. 1) disrupts the repressor gene
klcB (28). The inserted element is 99.8% identical to Tn1,
located in Birmingham IncP-? plasmids RP1/RP4 (accession
number L27758) (59), 98.2% to Tn2 (accession numbers
X54607 and AY123253) (60), and 98% to Tn3 (accession num-
ber V00613). Flanking the inserted element are duplications of
the target pentanucleotide AAATT (Fig. 3, panel A), pointing
to a clean transposon insertion. Tn1 insertion in plasmid RP4
occurs also within gene klcB, although the insertion site is not
identical. Tn1R7Kshows perfect 38-bp terminal IRs and retains
its three functional genes: tnpA (transposase), tnpR (re-
solvase), and blaTEM-1c(encoding TEM-1 ?-lactamase), as well
as the resolution res site. Tn1RP4and Tn1R7Kdiffer in only 10
nucleotides, resulting in a three-residue change in their TnpAs
(A457R, D797G, and D801N) and one residue mutation in
TEM proteins (K37Q). Gene blaTEM-1c, as present in R7K,
constitutes a new allele, and differs from blaTEM-1a, blaTEM-1b,
and blaTEM-2, present in transposons Tn3, Tn2, and Tn1, re-
spectively, by the mutations shown in Table S2 in the supple-
mental material. Two regions are delimited: one comprising
the promoter and the coding region up to position 345, which
is identical to that in Tn3, and the other spanning from posi-
tion 346 to the stop codon, which is identical to that in Tn1.
TABLE 2. Percent nucleotide identity between five IncW backbonesa
Overall DNA sequence identity (%)
R388 pIE321 R7KpSab
100 97 (91.2)
aThe values in parentheses refer to DNA sequence identities (%) in the
bFor pSa, since the complete DNA sequence is not known, comparisons were
made using the region between resP and ardC (8,384 bp).
VOL. 52, 2008RECENT EVOLUTION OF IncW PLASMIDS1475
This fact suggests homologous recombination between blaTEM-1a
and blaTEM-2, originating blaTEM-1c. The resulting protein, as well
as its putative weak promoter, is identical to those encoded by
Tn2 and Tn3 (37).
A second transposon, Tn5393?1, is inserted in gene nuc,
leaving its last 14 codons contiguous to the transposon 3? end
(Fig. 1 and see Fig. S2 in the supplemental material).
Tn5393?1 is 99% identical to Tn5393 from plasmid pEa34
(accession number M95402) (21) but has an internal deletion
(1,982 bp) comprising the resolvase gene, insertion element
IS1133, and the first 50 codons of strA. The deletion is clean,
without relics of other transposable elements. The transposon
is flanked by imperfect IRs (77 nucleotides out of 81) and a
direct repeat of GACAG, corresponding to the target dupli-
cation at the insertion site (Fig. 3, panel B).
Gene aadA13, coding for aminoglycoside-3?-adenylyltrans-
ferase that confers resistance to streptomycin and spectinomy-
cin, is located proximal to Tn5393?2 in plasmid R7K (Fig. 1).
The presence of 14 codons of the 3? region of nuc between the
insertions of Tn5393?1 and aadA13 demonstrates that both
elements inserted independently (see Fig. S2 in the supplemen-
tal material). Protein AadA13R7Kshows 98% identity to reported
versions of AadA13 (accession numbers AAV49321, AAY18576,
ABG76948, ABG76949, and BAF73713). The AadA family
alignment (from AadA1 to AadA15), which contains the N-
terminal nucleotidyltransferase domain, shows 33.5% consen-
sus identity. AadA1 is most related to AadA13 (85.3% iden-
tity), while AadA14 is the least similar (54.3%).
The aadA13 insertion looks like an integron gene cassette
(855 bp long), containing a 3? attC element as well as a recom-
bination site (G/TTAGAC) upstream of the aadA13 start
codon (Fig. 3, panel C). It lacks attI or any other integron
component. The attC sequence of the hypothetical circularized
cassette (60 bp long) is 100% identical to previously reported
aadA13-related attC sequences (accession numbers AY940492,
DQ779001, DQ779002, and AB332415) (43). All these homo-
logues are contained in class I integrons. Thus, we assume that
the aadA13 cassette integrated in an IntI1 secondary site (G/
TTAGCG; the consensus being GWTMW ) located in the
complementary strand of gene nuc, just downstream of its stop
codon (see Fig. 3, panel C). Since the aadB cassette was shown
to excise from a secondary site with a completely conserved
GTT triplet (67), the composite core site of cassette aadA13
may also be active in IntI1-mediated recombination.
FIG. 2. Alignments of the TrwA and oriT sequences. Alignments of the TrwA (panel A) and oriT (panel B) sequences of R388/pIE522/pSa,
pIE321, and R7K are shown. Invariant nucleotides or amino acids in all plasmids are shadowed in dark gray, while those identical in most plasmids
are shadowed in light gray. (A) Residues 8, 10, and 12, which are important for oriT recognition by TrwAR388, are indicated by vertical arrows.
(B) The oriT DNA strand complementary to that cleaved by the relaxase is shown. The nic site is represented by a black triangle. Structural motif
IR2is represented by convergent horizontal arrows. The sites of interaction of oriTR388with proteins IHF (ihfA and ihfB) and TrwA (sbaA and
sbaB) are marked by horizontal lines. The start codon of gene trwA (trwA start), its putative ribosome binding site (rbs), and the trwA promoter
?10 and ?35 sequences are indicated.
1476 REVILLA ET AL.ANTIMICROB. AGENTS CHEMOTHER.
The integration of cassette aadA13 produced a deletion in
the osa gene, leaving 114 bp of its 3? region. Interestingly, this
truncated segment shows only 83% identity to the functional
osaR388, perhaps underscoring the rapid shift of nonfunctional
sequences (see Fig. S2 in the supplemental material). Osa
impairs transport of Vir proteins (46) and DNA transfer (14)
to plant cells. The repercussion of osa deletion in the ecology
of plasmid R7K remains to be investigated.
Gene cassettes do not contain their own promoters and thus
are transcribed from the Pantpromoter when inserted in the
integron, or from a suitable oriented promoter when inserted
in a secondary site (64). For example, cassette aadB was inte-
grated in a secondary site of plasmid pRAY and is transcribed
from a plasmid promoter (67). In R7K, aadA13 is expressed
since the host bacteria are resistant to spectinomycin, a phe-
notype that is not attributable to the str genes present in trans-
poson Tn5393?1. A putative promoter is located upstream of
aadA13 in the ?osa region (detected using the bacterial pro-
moter prediction BPROM program at http://www.softberry
.com) (see Fig. S2B in the supplemental material). Finally, a
13-bp string of DNA appears between cassette aadA13 and
?osa that is neither part of the gene cassette nor part of the
Insertions in plasmid pIE321. Plasmid pIE321 contains in-
sertions of truncated class II transposons Tn1721 and Tn5393c
(Fig. 1). The 6,295-bp-long Tn1721?1 element is inserted be-
tween coding sequence 14 (CDS14) and stbC. Compared to the
canonical Tn1721 (11,139 bp long) (accession number X61367)
(3), this derivative shows a 4,844-bp deletion that includes the
left terminal IR, the gene orfI, the resolution site res, the
resolvase gene tnpR, and part of the transposase gene tnpA. No
DNA target duplication was observed (Fig. 3, panel D). The
transposon keeps the last 256 codons of tnpA, tetR (encoding a
tetracycline resistance repressor), tetA(A) (encoding a tetracy-
cline efflux protein that confers resistance to tetracycline),
pecM (related to a permease repressor), pncA (a cystein-hy-
drolase), the second truncated copy of tnpA (encoding the 583
C-terminal residues), and the right IR (IRR2), which appears
duplicated in the middle of the element (IRR1), as happens in
the original transposon. A simple one-ended transposition
event explains the Tn1721?1 insertion. Single-ended deriva-
tives of Tn3-like transposons Tn1721, Tn21, and TnA were
FIG. 3. Junction sequences of specific insertions. Each insertion is represented at the right by a diagram following the color scheme used in Fig.
1 and is preceded by two DNA sequences. The top one represents the backbone sequence (gray) where the insertion takes place. The exact point
of insertion is depicted by a vertical arrow. The bottom sequence contains the corresponding insertion colored according to the same scheme.
Target duplications of the backbone sequence are shown in bold italics. Individual base changes with respect to the backbone sequence are
underlined. In panel C, two green triangles indicate suggested recombination crossover points flanking the aadA13 integrated cassette. Gene
aadA13 start and stop codons as well as the complementary sequence of the nuc stop codon are boxed. A putative ribosome binding site is shown
in bold. Core (R?), inverse core (R?), and internal IR (L? and L?) sequences of the potentially recircularized aadA13 cassette are signaled by
horizontal green arrows, while the composite core site of the integrated cassette is indicated by a horizontal gray arrow. ?osa, 5? portion of osa has
been deleted; ?nuc, 5? portion of nuc has been deleted.
VOL. 52, 2008RECENT EVOLUTION OF IncW PLASMIDS 1477
found to cleanly insert into a recipient plasmid (7, 42, 53).
Insertion products contain the entire donor plasmid plus a
duplication of the IR (7, 42, 53, 54), or only variable-length
portions of the transposable derivative (8, 42, 53). So, trans-
position termination at a non-IR sequence can yield a simple
insertion of a truncated transposon without duplication of the
target sequence, like Tn1721?1 in plasmid pIE321. This is not
an isolated example. A single-end Tn1721 derivative found in
the IncP plasmid pB10 (accession number AJ564903) has al-
most the same truncation (66). Given the fact that single-
ended TnpA?Tn21 and TnA derivatives transpose when the
cognate transposase is provided in trans (7, 42), the Tn1721?1
copy in pIE321 could potentially be mobilized.
The 2,148-bp Tn5393?2,3 derivative is inserted between
ardK and LDR1 (Fig. 1), duplicating the target sequence
ATCAA (Fig. 3, panel E). The element retains the Tn5393
81-bp terminal IRs and genes strA and strB encoding strepto-
mycin resistance (aminoglycoside-3?-phosphotransferase and
aminoglycoside-6-phosphotransferase) but has two internal de-
letions. The first (?2; 2,316 bp) includes the N-terminal por-
tions of the transposase (tnpA and tnpR) and resolvase genes,
leaving their last 77 and 37 codons, respectively. The second
(?3; 1,233 bp) comprises IS1133. The three remaining por-
tions, IR-?tnpA, ?tnpR, and strA-strB-IR, are juxtaposed, as
happens in the R7K element, and are 100%, 98%, and 100%
identical, respectively, to Tn5393 (accession number M95402)
(15). Blast analysis of Tn5393?2,3 showed 100% nucleotide
identity and coverage with an element contained in plasmid
pMBSF1, which has exactly the same deletions (accession
number AJ518835). This plasmid was obtained from E. coli
isolated from pigs and, as with pIE321, also contains a trun-
cated Tn1721 copy but inserted in a different location (12).
Insertion of the integron platform in plasmids R388, pSa,
and pIE522. Complete class I integrons are present in R388,
pSa, and pIE522 at exactly the same backbone location (Fig. 3,
panel F). They contain the same 5? and 3? conserved regions.
In all three cases, the integron disrupts orphan gene CDS36,
present in integron-free plasmids R7K and pIE321. As a result
of the integron insertion, an internal 112-nucleotide deletion
was produced in CDS36, leaving 22 codons 5? and 77 codons 3?
of the inserted integron. Thus, we assume the integron inserted
just once in an ancestral IncW backbone, as proposed by Gorai
et al. (34) and Stokes et al. (68), as will be discussed later.
Upon integration, gene CDS36 is split in such a way that an
in-frame GTG start codon near the integron border gives rise
to a chimeric gene in plasmids containing the integron plat-
form. We annotated it as tnpM in R388 (28) by analogy with
tnpM of Tn21 (also located after intI1, as shown previously
). However, there is practically no similarity with the Tn21
gene, so it should not be called tnpM, to avoid confusion. The
DNA sequence that CDS36 and tnpM have in common is
responsible for hybridization of the R388 tnpM probe to
plasmids R7K and pIE321 in the DNA array (see Fig. S1 in
the supplemental material).
The IncW integron platform does not retain the transposi-
tion functions responsible for integron movement but con-
serves the terminal sequence IRi (25 bp long; close to the 3?
end of intI1) of Tni, the vehicle transposon present in Tn21
(47). The integron 3? conserved region, besides the common
qacE?1 gene and sul1, the sulfonamide resistance gene, har-
bors orf5 and a truncated orphan gene, orf6 (coding for its first
57 residues). The integron variable regions contain different
gene cassettes. In R388, cassette dhfr codes for dihydrofolate
reductase that confers resistance to trimethoprim (70, 83),
while cassette orfA (69, 70) has no assigned phenotype. In
pIE522, cassette aadB encodes 2-aminoglycoside (2?) adenylyl-
transferase and confers resistance to gentamicin/kanamycin/
tobramycin. In pSa, the inserted integron contains two gene
cassettes, aacA4 and aadA2, which confer resistance to genta-
micin/kanamycin and streptomycin/spectinomycin, respectively
The existence of different gene cassettes as well as the high
conservation of the integrase with that of Tn21 (they differ only
in residue 39: His in IntTn21and Asn in IntIncWplasmids)
suggests that the IncW integrase is active in the genesis of new
IncW plasmids. According to theory, the first acquired cassette
is located in the last position of the integron variable region.
Consistent with this, the first inserted cassette in each integron
contains the same sequence (GTTAGAT) in its attC core site,
derived from the primary attI recombination site in the empty
In the integron version of plasmid pSa, the 3? conserved
genes qacE?1 and sul1 have been duplicated (68). In between,
there is a chloramphenicol resistance gene, catA2, which does
not correspond to a cassette insertion, followed by an ISCR1
element. This insertion sequence contains orf513, a gene found
close to many integrons, which encodes a protein related to the
IS91 transposase (32, 76). Three copies of the putative 5? end
of the ISCR1 element oriISCR1 appear downstream of orf513.
The pSa integron distal 3? conserved sequence is identical to
that in R388. Based on the proposed three-step mechanism
(75, 76), a possible scenario for the formation of the pSa
integron includes rolling-circle transposition of ISCR1 fused to
the 3? conserved region of a class I integron (containing orf513,
sul1, and qacE?1) to the vicinity of gene catA2. Subsequent
one-ended rolling-circle transposition of this element, now
containing catA2, gives rise to circular intermediates that can
recombine with the 3? conserved region of a simple integron
platform, like that present in R388.
Concluding remarks. Several horizontal gene transfer
events shaped currently known IncW plasmids. The most par-
simonious assumption is to consider that the integron platform
was inserted only once in an ancestral IncW plasmid, since its
genetic location is exactly the same for the three IncW plas-
mids harboring it. Different gene cassettes were then incorpo-
rated into the integron by integrase-catalyzed reactions, pro-
ducing the present versions in R388, pIE522, and pSa. A
subsequent acquisition of an ISCR1 element in the pSa inte-
gron occurred. The diversifications in the integron arrange-
ments must be very recent, considering the 100% nucleotide
identity in the integron platform of the three plasmids. Inde-
pendent events of MGEs loaded in the ancestral plasmid gave
rise to the IncW variants pIE321 and R7K. Divergence of
those plasmids is older than that of R388/pIE522/pSa, since
pIE321 and R7K exhibit small but significant differences in the
backbone (see Table S1 in the supplemental material). The
IncW group thus provides an example of the recent evolution
of a plasmid backbone which acquired different MGEs that
were selected by the heavy use of antibiotics.
1478REVILLA ET AL.ANTIMICROB. AGENTS CHEMOTHER.
Work in the FdlC lab was supported by grant BFU2005-03477/BMC
from the Spanish Ministry of Education and Science and contract
LSHM-CT-2005_019023 from the E.U. VI Framework Programme.
The help of the core sequencing group at the PSU and The Wellcome
Trust (grant 063083) are also acknowledged.
We are grateful to K. Smalla for providing plasmids pIE522 and
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