Xis Protein Binding to the Left Arm Stimulates Excision of Conjugative Transposon Tn916

Conjugative transposons are extremely promiscuous genetic elements that disseminate antibiotic resistance among gram-positive and gram-negative bacteria (reviewed in references 14, 49, 52, and 54). Most conjugative transposons confer tetracycline-minocycline resistance, while resistance markers for macrolides-lincosamides-stretogramins, aminoglycosides, beta-lactams, chloramphenicol, erythromycin, mercuric chloride, and nisin have been found on larger elements (49). Their ability to move between unrelated bacterial cells through conjugation and their prevalence in drug-resistant pathogens that cause human disease (for example, cholera [61], pneumonia [4], endocarditis [47], and nosocomial [9] and suppurative [12] infections) make these elements a serious health threat. One of the most extensively studied conjugative transposons is Tn916, which is 18 kb in length and encodes resistance to tetracycline (11, 18). Excision of the element from its bacterial host occurs through staggered cleavages at the ends of the transposon, producing variable (5 to 6 bp in length) 5′ overhangs (8, 34). The overhangs, which are derived from heterologous bacterial DNA flanking the transposon, are then sealed to produce a circular transposon intermediate with a novel 5- to 6-bp heteroduplex (8, 35, 53). The circularization of Tn916 enables the expression of transfer genes (11) and its ultimate single-strand transfer to a recipient bacterial cell (53) via a functional origin of transfer (25). Tn916 integrates into the recipient chromosome with limited target preference (33, 53) and does not duplicate the target sequence (13). Two transposon-encoded proteins, integrase (Tn916Int) and excisionase (Tn916Xis), mediate transposition. The Tn916Int protein is a heterobivalent DNA-binding recombinase (32). Its C-terminal domain is related, based on primary sequence homology, to the tyrosine family of recombinases (reviewed in references 3 and 17), and it performs the strand cleavage and joining reactions through a phosphotyrosine intermediate. Footprinting studies of the N- and C-terminal domains of Tn916Int fused to the maltose binding protein have revealed their binding sites on the transposon (32). The C-terminal domain of Tn916Int interacts with core-type sites at the ends of the transposon (Fig. ​(Fig.1),1), while the N-terminal domain binds to an unrelated set of distal sites (called DR2 sites) through an unusual three-stranded beta-sheet-DNA interface (15, 62). The Tn916Xis protein enhances excisive recombination (37, 38, 48, 50, 56) and binds to sites that are proximal to the DR2 sites on each arm of the transposon (51). FIG. 1. Comparison of the protein binding sites on transposon Tn916 and bacteriophage λ. The protein binding sites on each element are shown and drawn to the same scale, which is indicated in base pairs. The attachment sites (attL and attR) are labeled. ... The general mechanism of Tn916 transposition is presumably related to the site-specific reactions that excise and integrate bacteriophage λ (54), since each encodes functionally homologous Xis and Int proteins that bind to the arms of each element (Fig. ​(Fig.1).1). In bacteriophage λ, its integrase (λInt) and excisionase (λXis) proteins, as well as the host-encoded proteins integration host factor (IHF) and the factor for inversion stimulation (FIS), cooperatively assemble onto the phage to control the directionality of recombination. λXis plays a key architectural role in recombination, dramatically bending DNA (57) and cooperatively recruiting the λInt and FIS proteins to the phage arm (59). However, the relative importance of host-encoded factors and specific protein-DNA and protein-protein interactions in conjugative transposition remains unknown and cannot be inferred from studies of bacteriophage λ. This is because the Int and Xis proteins of each element share no significant primary sequence homology outside of the integrase catalytic active site, and because there are fundamental differences in the arrangement of their binding sites on the phage and transposon (Fig. ​(Fig.1).1). In this study we have determined the functional significance of specific Tn916Xis-transposon interactions and host-encoded protein factors in the excisive recombination of the Tn916 transposon.