Christina M. Collis

Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination

An integron is a genetic unit that includes the determinants of the components of a site‐specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes. An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors. The essential components of an integron are an int gene encoding a site‐specific recombinase belonging to the integrase family, an adjacent site, attl, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette‐encoded genes. The cassettes are mobile elements that include a gene (most commonly an antibiotic‐resistance gene) and an integrase‐specific recombination site that is a member of a family of sites known as 59‐base elements. Cassettes can exist either free in a circularized form or integrated at the attl site, and only when integrated is a cassette formally part of an integron. A single site‐specific recombination event involving the integron‐associated attl site and a cassette‐associated 59‐base element leads to insertion of a free circular cassette into a recipient integron. Multiple cassette insertions can occur, and integrons containing several cassettes have been found in the wild. The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an integron and generate free circular cassettes. Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the plasmids and transposons that contain them. However, a more general role in evolution is also likely. Events involving recombination between a specific 59‐base‐element site and a nonspecific secondary site have recently been shown to occur. Such events should lead either to the insertion of cassettes at non‐specific sites or to the formation of stable cointegrates between different plasmid molecules, and a cassette situated outside the integron context has recently been identified.

Site-specific insertion of gene cassettes into integrons

Site‐specific insertion of gene cassettes into the insert region of integrons has been demonstrated. Insertion was only observed if the integron DNA integrase was expressed in the recipient cell and if the cassette DNA was ligated prior to transformation. The essential ligation products were resistant to treatment with exonuclease III, indicating that they were closed circular molecules. Insertion of cassettes into integron fragments containing either no insert (one recombination site), or one gene cassette (two recombination sites), was demonstrated. In the latter case, insertion occurred predominantly at the core site located 5′ to the resident cassette, which corresponds to the only site available when no insert is present in the recipient. When DNA molecules including two gene cassettes were used, insertion of only one of the gene cassettes was generally observed, suggesting that resolution of the circular molecule to generate two independent circular cassettes occurred more rapidly than insertion into the recipient integron.

Gene cassettes from the insert region of integrons are excised as covalently closed circles

Integrons are DNA elements which generally include one or more discrete gene cassettes inserted at a specific site. We have recently proposed a model for the acquisition and dissemination of genes found in the insert region of integrons, which requires the existence of circularized gene cassettes. Evidence for the existence of covalently closed circular molecules consisting of one or more gene cassettes has now been obtained. Low levels of small molecules which hybridize to probes specific for individual gene cassettes were detected in plasmid DNA isolated from cells containing a plasmid which includes an integron fragment with three gene cassettes aacC1, orfE and aadA2. These molecules were only detected when the gene encoding the integron DNA integrase was also present and are thus products of site‐specific cassette excision. The excised cassettes have been shown to be in the form of covalently closed supercoiled circles, by digestion with restriction enzymes exonuclease III and DNase I. The circular excision products detected included either one cassette, aadA2 or orfE, two cassettes, aacC1 and orfE or all three cassettes. The predicted sequence of the recombinant junction in the excised aadA2 cassette confirmed that excision was precise. The predicted unique sequences of the 59‐base elements associated with individual genes in the circular cassette form were compiled, and the sequences of the seven‐base core sites which flank 59‐base elements are now, with few exceptions, exact inverted repeats.

Mobile Gene Cassettes and Integrons in Evolution

ABSTRACT: Integrons and the site‐specific recombination systems encoded by them provide a simple mechanism for the addition of new genes to bacterial chromosomes. Although there is substantial divergence among the four known integron‐encoded integrases, they all recognize the recombination sites, known as 59‐base elements, that are associated with genes that are packaged in gene cassettes. In contrast, the integron‐associated recombination sites, attI sites, are preferentially recognized by the cognate integrase.

Definition of the attI1 site of class 1 integrons

Integron-encoded integrases recognize two distinct types of recombination site: attI sites, found in integrons, and members of the 59-base element (59-be) family, found in the integron-associated gene cassettes. The class 1 integron integrase, IntI1, catalyses recombination between attI1 and a 59-be, two 59-be, or two attI1 sites, but events involving two attI1 sites are less efficient than the reactions in which a 59-be participates. The full attI1 site is required for high-efficiency recombination with a 59-be site. It is 65 bp in length and includes a simple site, consisting of a pair of inversely oriented IntI1-binding domains, together with two further directly oriented IntI1-binding sites designated strong and weak. However, a smaller region that contains only the simple site is sufficient to support a lower level of recombination with a complete attI1 partner and the features that determine the orientation of attI1 reside within this region. An unusual reaction between the attI1 site and a 59-be appears to be responsible for the loss of the central region of a 59-be to create a potential fusion of two adjacent gene cassettes.

Binding of the purified integron DNA integrase Intl1 to integron- and cassette-associated recombination sites.

The site‐specific recombinase IntI1, encoded by class 1 integrons, catalyses the integration and excision of gene cassettes by recognizing two classes of sites, the integron‐associated attI1 site and the 59‐base element (59‐be) family of sites that are associated with gene cassettes. IntI1 includes the four conserved amino acids that are characteristic of members of the integrase family, and IntI1 proteins with single amino acid substitutions at each of these positions had substantially reduced catalytic activity, consistent with this classification. IntI1 was purified as a fusion protein and shown to bind to isolated attI1 or 59‐be recombination sites. Binding to attI1 was considerably stronger than to a 59‐be. Binding adjacent to the recombination cross‐over point was not detected. A strong IntI1 binding site within attI1 was localized by both deletion and footprinting analysis to a 14 bp region 24–37 bp to the left of the recombination cross‐over point, and this region is known to be critical for recombination in vivo (Recchia et al., 1994). An imperfect (13/15) direct repeat of this region, located 41–55 bp to the left of the recombination cross‐over point, contains a weaker IntI1 binding site. Mutation of the stronger binding site showed that a single base pair change accounted for the difference in the strength of binding.

Efficiency of Recombination Reactions Catalyzed by Class 1 Integron Integrase IntI1

The class 1 integron integrase, IntI1, recognizes two distinct types of recombination sites, attI sites, found in integrons, and members of the 59-be family, found in gene cassettes. The efficiencies of the integrative version of the three possible reactions, i.e., between two 59-be, between attI1 and a 59-be, or between two attI1 sites, were compared. Recombination events involving two attI1 sites were significantly less efficient than the reactions in which a 59-be participated, and the attI1 x 59-be reaction was generally preferred over the 59-be x 59-be reaction. Recombination of attI1 with secondary sites was less efficient than the 59-be x secondary site reaction.

Integron-encoded IntI integrases preferentially recognize the adjacent cognate attI site in recombination with a 59-be site.

Integrons have the capacity to capture small mobile elements known as gene cassettes, and this reaction is catalysed by integron‐encoded IntI integrases. IntI integrases form a distinct family within the tyrosine recombinase superfamily and include a characteristic additional domain that is well conserved. Two different IntI enzymes were used to examine their ability to recognize heterologous attI sites in both integration and excision assays. IntI1 and IntI3 are 59% identical and catalyse both integrative and excisive recombination between a cassette‐associated 59‐be site and the cognate attI1 or attI3 site. Integrative recombination events involving a 59‐be and a non‐cognate attI site, attI2 and attI3 for IntI1 or attI1 and attI2 for IntI3, were detected extremely rarely. In cassette excision assays, the non‐cognate attI3 site was recognized by IntI1, but attI1 was not well recognized by IntI3. The purified IntI1 and IntI3 proteins bound strongly only to their cognate attI site.

Breakage of double-stranded DNA due to single-stranded nicking*

Enzymes such as pancreatic deoxyribonuclease (DNase I) nick the single strands of double-stranded DNA. Two nicks sufficiently close on opposite strands will lead to breakage of the DNA molecule. This paper gives a mathematical model for the breakage of circular, supercoiled DNA under the action of an enzyme which nicks at random sites (or at preferred sites, these being in abundance and randomly positioned around the circle). After the first nick the DNA loses its supercoiled structure; after many nicks it breaks to become topologically linear; further nicks lead to fragmentation of this linear form. Formulae are given for the proportions of DNA molecules in each of the four classes: supercoiled; nicked but still circular; linear; fragmented. Formulae are also presented for the case when there is, in addition to nicking, simultaneous action of an endonuclease which produces direct double-stranded breaks in the DNA. Finally, a general theory is given for the case where a third type of enzyme, topoisomerase I, is operative, with all three DNA modifications taking place simultaneously.

Class 1 Integron Containing a New Gene Cassette, aadA10, Associated with Tn1404 from R151

ABSTRACT The carbenicillin, gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin resistance determinants found on Pseudomonas aeruginosa plasmid R151 have previously been shown to translocate to another plasmid, R388, and it was inferred that a transposon, Tn1404, carried the resistance determinants. Sequencing of the cassette array from the plasmid known as R388::Tn1404 revealed two known gene cassettes, oxa10 and aadB, and a previously unidentified cassette determining resistance to streptomycin and spectinomycin, here designated aadA10, in the order oxa10-aadB-aadA10. These cassettes replaced the dfrB2-orfA cassette array of R388, indicating that movement of the resistance determinants from R151 to R388 resulted from recombinational exchange between two class 1 integrons rather than transposition. The AadA10 protein is most closely related to AadA6 (85% identical) and AadA7 (80% identical). The aadA10 cassette found here has only a simple site containing a 7-bp spacer derived from attI1 in place of a 59-base element and is likely to represent a derivative of the complete cassette. IntI1-mediated deletion of the aadA10 cassette was not detected, indicating that this single simple site is either inactive or only weakly active.