The concentration of messenger molecules in bacterial cells needs to be tightly regulated. This can be achieved by either controlling the synthesis rate, degradation, or export by specific transporters, respectively. The regulation of the essential second messenger c-di-AMP is achieved by modulation of the diadenylate cyclase activity as well as by specific phosphodiesterases that hydrolyze c-di-AMP in the cell. We provide here structural and biochemical data on the DHH-type phosphodiesterase TmPDE (TM1595) from Thermotoga maritima. Our analysis shows that TmPDE is preferentially degrading linear dinucleotides, such as 5'-pApA, 5'-pGpG, and 5'-pApG, compared with cyclic dinucleotide substrates. The high-resolution structural data provided here describe all steps of the PDE reaction: the ligand-free enzyme, two substrate-bound states, and three post-reaction states. We can furthermore show that Pde2 from Streptococcus pneumoniae shares both structural features and substrate specificity based on small-angle X-ray scattering data and biochemical assays.

Summary Conjugative transposition drives the emergence of multidrug resistance in diverse bacterial pathogens, yet the mechanisms are poorly characterized. The Tn 1549 conjugative transposon propagates resistance to the antibiotic vancomycin used for severe drug-resistant infections. Here, we present four high-resolution structures of the conserved Y-transposase of Tn 1549 complexed with circular transposon DNA intermediates. The structures reveal individual transposition steps and explain how specific DNA distortion and cleavage mechanisms enable DNA strand exchange with an absolute minimum homology requirement. This appears to uniquely allow Tn 916 -like conjugative transposons to bypass DNA homology and insert into diverse genomic sites, expanding gene transfer. We further uncover a structural regulatory mechanism that prevents premature cleavage of the transposon DNA before a suitable target DNA is found and generate a peptide antagonist that interferes with the transposase-DNA structure to block transposition. Our results reveal mechanistic principles of conjugative transposition that could help control the spread of antibiotic resistance genes.

Transposons are mobile genetic elements that drive evolution and adaptation throughout the tree of life. In bacteria, they often transfer antibiotic resistance genes and contribute to the emergence of multidrug‐resistant pathogens. In this talk, I will present our recent discoveries on a group of transposons that efficiently propagate antibiotic resistance genes across diverse microbial communities. Using a dedicated computational pipeline, we mapped the most wide‐spread transposons in bacterial genomes and characterized their diversity, genetic cargo, and transfer dynamics. Selected elements were experimentally reconstituted to describe their molecular mechanisms and biochemical pathways. We determined numerous high‐resolution crystal and cryo‐EM structures of the protein‐DNA assemblies involved in transposon movement. The structures capture various stages of transposition and reveal an intricate interplay between the core transposition machinery and host‐encoded proteins. Protein interactions shape transposon and genomic DNA in unique ways to cut, exchange and re‐join strands in a tightly regulated manner. Importantly, our data also explain how specific DNA distortion and cleavage mechanisms enable the transposons to move between diverse genomic sites, expanding gene transfer across many species. These results shed new light onto the molecular strategies of antibiotic resistance carrying transposons and show how they evolved to effectively spread key genetic traits. Our insights also open new opportunities to develop strategies for blocking transposition and thereby help control resistance spreading.

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