Initiation of bacterial type IV secretion

Bacterial type IV secretion systems (T4SS) mediate the transfer of protein and DNA molecules into various target cells, e.g., the conjugative transfer of DNA into other bacteria or the transfer of virulence proteins into human, animal or plant cells. T4 secretion is medically significant due to interbacterial spread of antibiotic resistance genes, enhanced adherence, biofilm formation, and disease induction in infected hosts. The export process from bacterial cells requires multiple proteins to create a membrane-spanning secretion channel and often a cell surface structure to support physical contacts between cells. An associated receptor protein, known as the type IV coupling protein (T4CP) is a key regulator of this process, because the receptor protein selects the bacterial proteins to be exported and controls their access to the secretion channel. In the last funding period we used genetics, structural biology and bioinformatics to gain a very detailed picture of how a T4CP recognizes secretion proteins. Current models propose that T4CPs also function to pump protein and genetic material through the secretion channel. Events taking place at the substrate /T4CP receptor interface have generally not been integrated spatially or temporally with steps occurring before and after substrate recognition. The molecular mechanisms governing how proteins or DNA enters the translocation channel remain unknown. In this application we now focus on the intracellular localization of proteins and DNA molecules, spatial coordination of transporter components, productive docking interactions and the early steps leading to the start of transfer. We exploit a different genetic model of whole cell activity that depends on the T4 secretion machinery. Importantly this viral infection model relies on a slightly different assembly of proteins. Remarkably, we observed that proteins of the R1 plasmid`s actin-like DNA segregation system are both physically colocalized and functionally integrated in the T4 secretion initiation pathway. R1 is the best characterized DNA segregation system but this functional convergence was not detected previously. The discovery opens a whole new investigation of these proteins as regulators of biochemical reactions important to secretion initiation. We will use our very detailed understanding of relaxosome and T4CP biochemistry in combination with the advanced molecular techniques established for the R1 ParMRC system to reveal how the plasmid partitioning and gene transfer systems are functionally integrated. Fluorescence light microscopy is proposed to determine whether, in addition to Par protein control of substrate docking and transporter activation, this segregation machinery has also been adapted to control dynamic movement of the T4 components.

Antibiotics were developed to treat bacterial infections. Many are effective because they kill pathogens or at least stop them from growing. At the same time, however, evolution drives changes in bacterial genes, which creates resistance to commonly prescribed antibiotics. Growth and spread of highly resistant organisms, which easily survive most or all of the clinically available treatments, has become a massive problem worldwide. One reason that bacteria are increasingly resistant to antibiotics is that they can exchange genes including those that provide resistance. To do this bacteria produce a needle-like transport system, which crosses the donor and recipient cell membranes, and an enzyme called relaxase, which prepares specific genes for export from one cell to another. This FWF financed research project brought new knowledge of the activity, cellular location, and control of the centrally important relaxase protein. A major breakthrough was achieved by determining the first three dimensional structure of a relaxase enzyme bound to its genetic cargo. The structure reveals the unique molecular basis for the function of this DNA exporting enzyme and seminal insights into the mechanisms allowing bacteria to share genes. The study was a joint effort of several international teams and our results were published in the renowned journal Cell. Detailed knowledge of this key bacterial protein creates new options to block its activity, and thereby supports efforts to slow down the spread of antibiotic resistance in bacteria.