We are currently working
on three different aspects of the
The distribution of super-integrons and their evolutionary
The inventory of the cassette encoded functions,
The recombination process and cassette genesis.
The development of antibiotic resistance has
lead to the discovery of many natural mobile elements,
including transposons and conjugative plasmids.
Comparative sequence analysis of these elements
ultimately led to the discovery of integrons, natural
cloning and expression systems that incorporate
open-reading frames (ORF) and convert them to functional
genes (for review see (8, 20)). The functional
integron platform consists of a gene coding for
an integrase (intI) of the tyrosine-recombinase
family and a proximal primary recombination sequence
called an attI site. The integrase mediates recombination
between the attI site and a secondary target called
an attC site (or 59-base elements (59be)). The attC
site is generally associated with a single ORF in
a structure termed a gene cassette, and the gene
cassette constitutes the mobile component of the
system (9, 16, 21, 22) (Figure 1). Insertion of
the gene cassette at the attI site, which is located
downstream of a resident promoter internal to the
intI gene, drives expression of the encoded proteins
Three classes of resistant integrons (RI) have
been defined based on the divergence among their
integrase genes and each class appears to be able
to acquire the same gene cassettes (7). The integron
platform itself is defective for self-transposition
but they are often found associated with insertion
sequences (ISs), transposons and / or conjugative
plasmids that can serve as vehicles for the intra
and inter-species transmission of genetic material.
The Tn21 and Tn7 families provide examples of this
(11, 23). As such, they have been found in a variety
of genetic contexts and among a large number of
phylogenetically diverse Gram-negative and Gram-positive
More than 70 different antibiotic resistance
gene cassettes have been characterized in the three
classes of RI (Mazel & Davies, 1999) and most
of their attC sites are unique (Table 1). The length
and sequence of the attC sites vary considerably
(from 57 to 141 bp) and their similarities are primarily
restricted to their boundaries, which correspond
to the inverse core-site (ICS; RYYYAAC) and the
core site (CS; G(TTRRRY; (, recombination point
(Collis et al., 1998; Stokes, et al., 1997)). The
studies of Mazel et al. (12) and Manning and colleagues
(1) examining the relationship between RI gene cassette
arrays and the Vibrio cholerae repeats (VCRs) cluster
led to the discovery of the Vibrio cholerae super-integron.
This distinct type of integron is now known to be
an integral component of many ?-proteobacterial
The super-integrons and their distribution
The integron discovered in chromosome 2 of V.
cholerae has two characteristics that distinguish
it from known RIs; the large number of cassettes
that are gathered and the high homology observed
between the attC sites of these cassettes, the VCRs
in the case of V. cholerae (12). These are the key
features that define a super-integron (SI) (Figure
2). Both the V. cholerae SI and RIs possess specific
and related integrases that are responsible for
the insertion of coding sequences (ORFs) into a
unique chromosomal attachment site, leading to the
formation of tandem arrays of genes. In the case
of V. cholerae, the cluster of VCR-associated ORFs
represents at least 216 unidentified genes in an
array of 179 cassettes which starts from the VchintIA
gene and occupies about 3% of the genome (10). The
extent to which this system has impacted genome
evolution has emerged with the discovery of SIs
in several diverse proteobacterial genera and the
various species include human, animal and plant
pathogens as well as non-pathogenic bacteria. SI
structures have been identified among the Vibrionaceae
and their close relatives, the Shewanella, the Xanthomonads
(3, 18), and the Pseudomonads (18, 25) (Table 1).
They share the same general characteristics (i.
e. a large size and a high homology between their
endogenous cassette attC sites) and clearly predate
the antibiotic era, as they are present in isolates
from the last century (12). Integron integrase-like
genes have also been identified in the genomes of
other proteobacteria including Acidithiobacillus
ferroodoxans, Nitrosomonas europaea, Geobacter sulfurreducens,
and Treponema denticola (Table 2), but they have
not been further characterized (13, 18).
PCR primers directed against conserved regions of
the integron-integrase genes and attC sites, Stokes
and colleagues were able to identify three new classes
of integrons from four markedly different environmental
DNA samples that had no known previous exposure
to antibiotics (13). The protocol they used to retrieve
the integron-integrase loci allowed recovery of
the majority of the intI gene, the sequence covering
the attI site and the first cassette up to the ICS
of its associated attC site. Unfortunately, such
a short sequence does permit determination of the
source of these integrons, be it the endogenous
SI of a soil bacterium or an integron located in
a mobile structure. However, these findings support
the hypothesis developed by the discovery of SIs;
that integrons are widespread among bacterial populations
either as components of mobile DNA elements or the
chromosome and that they are not confined to pathogenic
or multidrug-resistant bacteria.
Integrons are ancient evolutionary apparatuses.
All characterized integron-integrases clearly
group together and form a specific clade within
the tyrosine recombinase family (Figure 3). Furthermore,
it has been noticed that all contained a specific
stretch of 16 amino acids (13) located between conserved
patchs II and III of the tyrosine recombinase family
(14). The role of this integron-integrase specific
sequence is unknown. The integron platform is undoubtedly
ancient as attested to by the species-specific clustering
of the respective SI integrase genes in a pattern
that adheres to the line of descent among the bacterial
species in which they are found (Figure 3) (18). Thus,
the establishment of SIs likely predates speciation
within the respective genera, indicating that integrons
are ancient structures that have been steering the
evolution of bacterial genomes for hundreds of millions
of years. It is however possible that transfer of
either a part or all of a SI occured (from an Hfr-type
strain, for example) during such a long period of
evolution. This could be the origin of the discrepency
observed in the SI-integrase and 16S rRNA gene trees
for the V. fischeri branching points among the other
bacterial species (18). However, the phylogenetic
analysis performed on the rplT genes from the same
species, which encode the conserved ribosomal L20
protein, produced a branching order identical to
the one found for the intI dendogram. This supports
co-evolution of the V. fischeri SI integrase gene
with the rest of the V. fischeri genome.
The SIs identified to date collectively equal
a small genome in size, suggesting that the process
of cassette genesis is constant and efficient. The
majority of the cassettes examined thus far appeared
to be unique to the host species. Furthermore, most
of their encoded genes have no counterparts in the
database or the sole homologues are unassigned ORFs
of viral, bacterial or eukaryotic origins, indicating
their recruitment from all kingdoms of life (10,
17). A precise inventory of the functions encoded
by the cassettes remains to be established, however,
a preliminary study indicates that many of the SI
cassettes encode adaptive functions, sensu lato,
beyond pathogenicity and antibiotic resistance.
In V. cholerae, two pathogencity genes, the heat-stable
toxin gene (sto) (15) and the mannose-fucose-resistant
haemagglutinin gene, mrhA (26), and a lipoprotein
gene have been found to be cassette encoded (2).
We have determined the metabolic function of three
SI cassettes: a sulfate binding protein in a V.
cholerae SI cassette, a psychrophilic lipase in
a Moritella marina SI cassette and a restriction
enzyme (Xba I) and its cognate methylase, in a Xanthomonas
campestris pathovar badrii SI cassette (18). Genes
with homology to DNA methylases, immunity proteins,
restriction endonucleases, dNTP triphophohydrolases,
periplasmic sulphate binding proteins, lipases and
8-oxoguanine triphosphatases (MutT), among others,
have been found (3, 10, 17, 18). Although a known
antibiotic-resistance gene cassette has not yet
been identified within a SI, several potential progenitor
cassettes with significant homology to aminoglycoside,
phosphinotricin, fosfomycin, streptothricin and
chloramphenicol resistance genes are present. The
determination of the metabolic activities of several
SI cassettes, whose activities are not related to
antibiotic resistance or virulence, confirms that
integrons operate as a general gene capture system
in bacterial adaptation (2, 18). If each bacterial
species harboring a SI has its own cassette pool,
the resource in terms of gene cassette availability
will be immense and the functions of the encoded
genes have fantastic potential from both genetic
and biotechnological standpoints.
Intra-species cassette content variations
The activity of integron cassettes offers a fast-track
to bacterial innovation. The size of SIs and the
ancient and dynamic nature of the system is a reminder
that the cassettes that currently occupy the SI
represent only a fraction of those that may have
participated in the evolution of the host, since
the cassettes will presumably be subject to episodic
selection. The more than 165 different O serotypes
of V. cholerae are represented by species of ecological,
geographical and temporal diversity. Thus, comparison
of SI organization from recent and earlier isolates
as well as between recent isolates from different
geographical locations and ecological niches may
yield valuable information. Clark et al. examined
the global SI organization of 65 different V. cholerae
O serotypes by PCR and Southern hybridization (3).
Extensive restriction polymorphism was observed
even among closely related isolates, suggesting
an appreciable plasticity for that these structures
and their microevolution through integrase-mediated
gene acquisition and gene loss (24) as well as via
cassette rearrangement events (5, 6, 22). Concerning
this latter occurrence, an important question is
can cassettes be mobilized in clusters? The SI organization
of two V. cholerae strains suggests they can; the
cassettes in positions 1-4 of the SI of strain 569B
are found in the same order in the SI of strain
N16961 (10) but they occupy positions 79 to 82 (a
displacement of more than 40 Kb) (17). This has
also been seen for other cassettes (3). These observations
could represent a true group mobilization event
or simply temporal differences in cassette acquisition.
There is evidence that not all repeated sequences
are equally functional; some are known to contain
mutations or deletions within the CS that could
render them non-functional (Rowe-Magnus, Guerout
and Mazel unpublished results). Therefore, their
movement would have to be co-ordinated with those
of other cassettes. Collis and Hall demonstrated
that integron gene cassettes are excised as covalently
closed circles and observed differences in the resulting
recombination products (4). Some cassettes could
be mobilized as individual units while others were
only excised in tandem with another cassette. Whether
such cassette hitch-hiking is by design to ensure
simultaneous transmission of genes, or is just a
matter of happenstance is not known.
The integron system is remarkably versatile with
its ability to recognize highly variable target
recombination sequences and apparently limitless
capacity to exchange and stockpile cassettes. Such
flexibility permits rapid adaptation to the unpredictable
flux of environmental niches by allowing bacteria
to scavenge foreign genes that may ultimately endow
increased fitness to the host. Likewise, genes that
fail to provide a meaningful function can be readily
eliminated. It is also quite likely that many of
the cassettes that presently occupy the SI are not
expressed, but they may persist in the absence of
selective pressure nevertheless and provide a genetic
basis for the evolution and subsequent retention
of novel derived activities. In addition to the
plethora of antibiotic resistance genes, two virulence
genes of V. cholerae are also structured as gene
cassettes, underscoring the potential of this system
to participate in the establishment of pathogenicity
islands. It is conceivable, that any ORF can be
structured as a gene cassette and it is vital to
decipher the mechanism governing cassette genesis.
According to the GC % and codon usage differences
observed in the ORFs found in the cassettes, they
must have many different origins, while their associated
recombination elements, are highly homologous (e.g.
74 % identity for the VCRs). This last characteristic
suggests that the VCRs were added to the ORFs inside
the Vibrio cell. Therefore, it is very likely that
the capture process occurs in vivo, but the nature
of this process remains unknown.
As such, integron-driven gene capture is likely
to be an important factor in the more general process
of horizontal gene transfer in the evolution of
bacterial genomes. It appears that multi-resistant
integrons have evolved from super-integrons, through
entrapment of intI genes and their cognate attI
sites into highly mobile structures like transposons.
The combination of this mobility and the selection
pressure exerted by antibiotic use may have driven
the specific capture of resistance cassettes from
the many different kinds of super-integron cassette
pools through multiple lateral transfers. To support
this hypothesis, we showed that MRIs can randomly
recruit genes directly from the cache of V. cholerae
SI cassettes. By applying a selective constraint
for the development of antibiotic resistance, we
demonstrated bacterial resistance evolution through
the recruitment a novel, but phenotypically silent,
chloramphenicol acetyltransferase gene from the
V. cholerae SI and its precise insertion into the
MRI(19). The resulting resistance profile could
then be disseminated by conjugation to other clinically
relevant pathogens at high frequency.
1.Barker, A., C. A. Clark,
and P. A. Manning. 1994. Identification of VCR, a repeated sequence
associated with a locus encoding a hemagglutinin in Vibrio cholerae O1. Journal of Bacteriology. 176:5450-5458.
2.Barker, A., and P. A.
Manning. 1997. VlpA of Vibrio
cholerae O1: the first bacterial member of the alpha 2-microglobulin
lipocalin superfamily. Microbiology. 143(Pt
3.Clark, C. A., L. Purins, P.
Kaewrakon, T. Focareta, and P. A. Manning. 2000. The Vibrio cholerae O1 chromosomal integron. Microbiology. 146:2605-12.
4.Collis, C. M., and R. M.
Hall. 1992. Gene cassettes from the insert region of integrons are excised
as covalently closed circles. Mol Microbiol. 6(19):2875-85.
5.Collis, C. M., and R. M.
Hall. 1992. Site-specific deletion and rearrangement of integron insert
genes catalyzed by the integron DNA integrase. Journal of Bacteriology. 174(5):1574-85.
6.Collis, C. M., G. D.
Recchia, M. J. Kim, H. W. Stokes, and R. M. Hall. 2001. Efficiency of
recombination reactions catalyzed by class 1 integron integrase IntI1. J
7.Hall, R. M., and C. M.
Collis. 1998. Antibiotic resistance in gram-negative bacteria: the role of
gene cassettes and integrons. Drug Resistance Updates. 1(2):109-119.
8.Hall, R. M., and C. M.
Collis. 1995. Mobile gene cassettes and integrons: capture and spread of genes
by site-specific recombination. Molecular Microbiology. 15(4):593-600.
9.Hall, R. M., and H. W.
Stokes. 1993. Integrons: novel DNA elements which capture genes by
site-specific recombination. Genetica. 90(2-3):115-32.
10.Heidelberg, J. F.,.
2000. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature. 406:477 - 483.
11.Liebert, C. A., R. M.
Hall, and A. O. Summers. 1999. Transposon Tn21, flagship of the floating
genome. Microbiol Mol Biol Rev. 63(3):507-22.
12.Mazel, D., B. Dychinco, V.
A. Webb, and J. Davies. 1998. A distinctive class of integron in the Vibrio cholerae genome. Science. 280(5363):605-608.
13.Nield, B. S., A. J.
Holmes, M. R. Gillings, G. D. Recchia, B. C. Mabbutt, K. M. Nevalainen, and H.
W. Stokes. 2001. Recovery of new integron classes from environmental DNA.
FEMS Microbiol Lett. 195(1):59-65.
14.Nunes-Duby, S. E., H. J.
Kwon, R. S. Tirumalai, T. Ellenberger, and A. Landy. 1998. Similarities and
differences among 105 members of the Int family of site-specific recombinases.
Nucleic Acids Research. 26(2):391-406.
15.Ogawa, A., and T. Takeda.
1993. The gene encoding the heat-stable enterotoxin of Vibrio cholerae is flanked by 123-base pair direct repeats.
Microbiology and Immunology. 37(8):607-616.
16.Recchia, G. D., and R. M.
Hall. 1995. Gene cassettes: a new class of mobile element. Microbiology. 141:3015-3027.
17.Rowe-Magnus, D. A., A.-M.
Guerout, and D. Mazel. 1999. Super-Integrons. Research in Microbiology. 150:641-651.
18.Rowe-Magnus, D. A., A.-M.
Guerout, P. Ploncard, B. Dychinco, J. Davies, and D. Mazel. 2001. The
evolutionary history of chromosomal super-integrons provides an ancestry for
multi-resitant integrons. Proceedings of the National Academy of Sciences of
the United States of America. 98:652-657.
19.Rowe-Magnus, D. A., A. M.
Guerout, and D. Mazel. 2002. Bacterial resistance evolution by recruitment
of super-integron gene cassettes. Mol Microbiol. 43(6):1657-69.
20.Rowe-Magnus, D. A., and D.
Mazel. 1999. Resistance gene capture. Current Opinion in Microbiology. 2:483-488.
21.Stokes, H. W., D. B.
O'Gorman, G. D. Recchia, M. Parsekhian, and R. M. Hall. 1997. Structure and
function of 59-base element recombination sites associated with mobile gene
cassettes. Molecular Microbiology. 26(4):731-745.
22.Sundstrom, L. 1998.
The potential of integrons and connected programmed rearrangements for
mediating horizontal gene transfer. APMIS. Supplementum. 84:37-42.
23.Sundstrom, L., P. H. Roy,
and O. Skold. 1991. Site-specific insertion of three structural gene cassettes
in transposon Tn7. Journal of Bacteriology. 173(9):3025-8.
24.Takeda, T., Y. Peina, A.
Ogawa, S. Dohi, H. Abe, G. B. Nair, and S. C. Pal. 1991. Detection of
heat-stable enterotoxin in a cholera toxin gene-positive strain of Vibrio
cholerae O1. FEMS Microbiol Lett. 64(1):23-7.
25.Vaisvila, R., R. Morgan,
and E. Raleigh. 1999. The pacIR gene resides within a potential
super-integron in the Pseudomonas
alcaligenes genome. IXth international congress of bacteriology and applied
microbiology, Sidney, 1999.
26.van Dongen, W. M. A. M.,
M. M. A. van Vlerken, and F. K. De Graaf. 1987. Nucleotide sequence of a
DNA fragment encoding a Vibrio cholerae haemagglutinin.
Mol. Gen. (Life Sci. Adv.). 6:85-91.
Figure 1 :
Schematic representation of a
circularized cassette (A) and model for cassette
exchange (B). A, The key features of attC sites (yellow) are
the complementary inverse core-site (ICS) and core-site
(CS) consensus sequences (red arrows) and the imperfect
palindromic variable region (red dotted arrows).
B, Outline of the process by which circular antibiotic
resistance gene cassettes (antR) are repeatedly
inserted at the specific attI site in a class1 integron downstream
of the strong promoter Pant. The vertical line in the
attC symbol (triangle) represents the recombination
point in the CS sequence. intI, integrase encoding
gene; Int, integrase IntI; attC, 59-be. See text
Figure 2 :
Structural comparison of
a "classical" multi-resistant integron (A)
and the V. cholerae super-integron (B). A, Schematic
representation of In40 (22). The various resistance genes are associated
with different 59-be (see text) whose consensus
sequence is shown. B, The ORFs are separated by
highly homologous sequences, the VCRs, for which
the consensus sequence is shown. Recombination occurs between
the G and the first T of the core-site sequence.
Figure 3 :
relationship of the integron intI genes among the
proteobacteria and chromosomal location of the Vibrio
Super-integrons. Unrooted dendrogram based on known
intI gene sequences. The integrases from the five
classes of RI are boxed. Organism abbreviations
for the SIs are as follows: Vibrio cholerae (Vch),
V. metschnikovii (Vme), V. parahaemolyticus (Vpa),
V. fischeri (Vfi), Listonella pelagia (Lpe), Shewanella
oneidensis (Son), S. putrefaciens (Spu), Xanthomonas
campestris pathovar camperstris (Xca pv ca) or badrii
(Xca pv ba), X. species (Xsp), Nitrosomonas europaea
(Neu). The sources of intI6-2, intI7-2, intI8-2
and the intI of plasmid pRVS1 (GB accession number
AJ277063) are unknown. For the integrase and recombinase
proteins: XerC/D (of E. coli). Representation of
the genetic context in which each of the SIs were
found relative to their phylogenetic distribution
according to their IntIA genes is shown. SIs in
identical locations are group within the same phylogenetic
clade. The corresponding orthologues in V. cholerae
are marked with VC(A) followed by the number designation
for the ORF. The attI site and VXRs are also indicated.
For clarity, only the first cassette within each
SI is shown. rplT and rpmL, ribosomal genes; dark
gray or black boxes, adjacent gene(s) which are
not part of the SI structure.