Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly.

The Tn3 family is a widespread group of replicative transposons that are notorious for their contribution to the dissemination of antibiotic resistance and the emergence of multiresistant pathogens worldwide. The TnpA transposase of these elements catalyzes DNA breakage and rejoining reactions required for transposition. It also is responsible for target immunity, a phenomenon that prevents multiple insertions of the transposon into the same genomic region. However, the molecular mechanisms whereby TnpA acts in both processes remain unknown. Here, we have developed sensitive biochemical assays for the TnpA transposase of the Tn3-family transposon Tn4430 and used these assays to characterize previously isolated TnpA mutants that are selectively affected in immunity. Compared with wild-type TnpA, these mutants exhibit deregulated activities. They spontaneously assemble a unique asymmetric synaptic complex in which one TnpA molecule simultaneously binds two transposon ends. In this complex, TnpA is in an activated state competent for DNA cleavage and strand transfer. Wild-type TnpA can form this complex only on precleaved ends mimicking the initial step of transposition. The data suggest that transposition is controlled at an early stage of transpososome assembly, before DNA cleavage, and that mutations affecting immunity have unlocked TnpA by stabilizing the protein in a monomeric activated synaptic configuration. We propose an asymmetric pathway for coupling active transpososome assembly with proper target recruitment and discuss this model with respect to possible immunity mechanisms.

The Tn3-family of Replicative Transposons.

Transposons of the Tn3 family form a widespread and remarkably homogeneous group of bacterial transposable elements in terms of transposition functions and an extremely versatile system for mediating gene reassortment and genomic plasticity owing to their modular organization. They have made major contributions to antimicrobial drug resistance dissemination or to endowing environmental bacteria with novel catabolic capacities. Here, we discuss the dynamic aspects inherent to the diversity and mosaic structure of Tn3-family transposons and their derivatives. We also provide an overview of current knowledge of the replicative transposition mechanism of the family, emphasizing most recent work aimed at understanding this mechanism at the biochemical level. Previous and recent data are put in perspective with those obtained for other transposable elements to build up a tentative model linking the activities of the Tn3-family transposase protein with the cellular process of DNA replication, suggesting new lines for further investigation. Finally, we summarize our current view of the DNA site-specific recombination mechanisms responsible for converting replicative transposition intermediates into final products, comparing paradigm systems using a serine recombinase with more recently characterized systems that use a tyrosine recombinase.

Separate structural and functional domains of Tn4430 transposase contribute to target immunity.

Like other transposons of the Tn3 family, Tn4430 exhibits target immunity, a process that prevents multiple insertions of the transposon into the same DNA molecule. Immunity is conferred by the terminal inverted repeats of the transposon and is specific to each element of the family, indicating that the transposase TnpA is directly involved in the process.However, the molecular mechanism whereby this protein promotes efficient transposition into permissive targets while preventing transposition into immune targets remains unknown. Here, we demonstrate that both functions of TnpA can be uncoupled from each other by isolating and characterizing mutants that are proficient in transposition (T+) but impaired in immunity (I-). The identified T+/I- mutations are clustered into separate structural and functional domains of TnpA, indicating that different activities of the protein contribute to immunity.Combination of separate mutations had synergistic effects on target immunity but contrasting effects on transposition. One class of mutations was found to stimulate transposition, whereas other mutations appeared to reduce TnpA activity. The data are discussed with respect to alternative models in which TnpA acts as a specific determinant to both establish and respond to immunity.

Target immunity of the Tn3-family transposon Tn4430 requires specific interactions between the transposase and the terminal inverted repeats of the transposon.

Specificity of the Tn4430 target immunity signal was examined by fusing the transposase TnpA to the LacI repressor of Escherichia coli. The resulting chimeric proteins failed to impose immunity to DNA targets carrying copies of the lacO operator, though they were proficient in lacO binding in vivo and remained responsive to wild-type immunity conferred by the Tn4430 inverted repeat end. Intriguingly, the presence of lacO repeats within the target was found to strongly influence target site selection by Tn4430, but in a LacI-independent manner.