The Integration and Excision of CTnDOT.

Bacteroides species are one of the most prevalent groups of bacteria present in the human colon. Many strains carry large, integrated elements including integrative and conjugative elements (ICEs). One such ICE is CTnDOT, which is 65 kb in size and encodes resistances to tetracycline and erythromycin. CTnDOT has been increasing in prevalence in Bacteroides spp., and is now found in greater than 80% of natural isolates. In recent years, CTnDOT has been implicated in the spread of antibiotic resistance among gut microbiota. Interestingly, the excision and transfer of CTnDOT is stimulated in the presence of tetracycline. The tyrosine recombinase IntDOT catalyzes the integration and excision reactions of CTnDOT. Unlike the well-characterized lambda Int, IntDOT tolerates heterology in the overlap region between the sites of cleavage and strand exchange. IntDOT also appears to have a different arrangement of active site catalytic residues. It is missing one of the arginine residues that is conserved in other tyrosine recombinases. The excision reaction of CTnDOT is complex, involving excision proteins Xis2c, Xis2d, and Exc, as well as IntDOT and a Bacteroides host factor. Xis2c and Xis2d are small, basic proteins like other recombination directionality factors (RDFs). Exc is a topoisomerase; however, the topoisomerase function is not required for the excision reaction. Exc has been shown to stimulate excision frequencies when there are mismatches in the overlap regions, suggesting that it may play a role in resolving Holliday junctions (HJs) containing heterology. Work is currently under way to elucidate the complex interactions involved with the formation of the CTnDOT excisive intasomes.

Interactions of NBU1 IntN1 and Orf2x proteins with attachment site DNA.

NBU1 is a mobilizable transposon found in Bacteroides spp. Mobilizable transposons require gene products from coresident conjugative transposons for excision and transfer to recipient cells. The integration of NBU1 requires IntN1, which has been identified as a tyrosine recombinase, as well as Bacteroides host factor BHFa. Excision of NBU1 is a more complicated process, involving five element-encoded proteins (IntN1, Orf2, Orf2x, Orf3, and PrmN1) as well as a Bacteroides host factor and a cis-acting DNA sequence. Little has been known about what role the proteins play in excision, although IntN1 and Orf2x have been shown to be the only proteins absolutely required for detectable excision. To determine where IntN1 and Orf2x bind during the excision of NBU1, both proteins were partially purified and tested in DNase I footprinting experiments with the excisive attachment sites attL and attR. The results demonstrate that IntN1 binds to four core-type sites that flank the region of cleavage and strand exchange, as well as six arm-type sites. A unique feature of the system is the location of DR2a and DR2b arm-type sites immediately downstream of the attL core. The DR1a, DR1b, DR3a, and DR3b arm-type sites were shown to be required for in vitro integration of NBU1. In addition, we have identified one Orf2x binding site (O1) on attL as well as a dA+dT-rich upstream element that is required for Orf2x interactions with O1.

CTnDOT integrase interactions with attachment site DNA and control of directionality of the recombination reaction.

IntDOT is a tyrosine recombinase encoded by the conjugative transposon CTnDOT. The core binding (CB) and catalytic (CAT) domains of IntDOT interact with core-type sites adjacent to the regions of strand exchange, while the N-terminal arm binding (N) domain interacts with arm-type sites distal to the core. Previous footprinting experiments identified five arm-type sites, but how the arm-type sites participate in the integration and excision of CTnDOT was not known. In vitro integration assays with substrates containing arm-type site mutants demonstrated that attDOT sequences containing mutations in the L1 arm-type site or in the R1 and R2 or R1 and R2' arm-type sites were dramatically defective in integration. Substrates containing mutations in the L1 and R1 arm-type sites showed a 10- to 20-fold decrease in detectable in vitro excision, but introduction of multiple arm-type site mutations in attR did not have an effect on the excision frequency. A sixth arm-type site, the R1' site, was also identified and shown to be required for integration and important for efficient excision. These results suggest that intramolecular IntDOT interactions are required for integration, while the actions of accessory factors are more important for excision. Gel shift assays performed in the presence of core- and arm-type site DNAs showed that IntDOT affinity for the attDOT core was enhanced when IntDOT was simultaneously bound to arm-type site DNA.