Final Report Summary - MOLSTRUCTTRANSFO (Molecular and Structural Biology of Bacterial Transformation)
Apart from the accumulation of random point mutations, bacteria are only able to generate clonal populations of themselves. Therefore, they should be extremely vulnerable to changes in their environment. Instead, these organisms are extremely adaptable and able adjust their lifestyle very quickly when theses changes occur. One dramatic illustration of this capacity is the spread of antibiotic resistance among bacterial pathogens due to antibiotics overuse in human healthcare and livestock. In this context, it is crucial to fully understand the molecular mechanism of bacterial adaptability to ultimately target and limit this ability. This project focused on the human pathogen Streptococcus pneumoniae (the pneumococcus). Despite our antibiotic arsenal and the introduction of the pneumococcal vaccine in the 80s, infections by S. pneumoniae remain a major cause of mortality worldwide with 1.6 million deaths annually. S. pneumoniae is considered as a paradigm for recombination-mediated genetic plasticity and adaptabilty. Indeed, in this species, the horizontal gene transfer mechanism of natural transformation is central to the bacterial physiology and virulence. This intricate multistep process is mediated by a group of dedicated molecular machines called the transformasome. While it is well established that these machines mediate the binding and internalization of external DNA and, ultimately, its recombination in the bacterial genome, the molecular details of these processes are unknown.
During this project, we aimed at acquiring a detailed understating of the molecular mechanism of each of these steps. We studied the composition, architecture and function of the molecular machines involved in these processes. More than core 20 proteins were genetically identified as being required for this process. We isolated these proteins, studied their network of interaction among them and systematically studied their function in vitro and in vivo. For some of them, we managed to obtain crucial insights about their structure and function. In particular, we discovered a new appendage at the surface of S. pneumoniae cells and showed that this appendage is similar in morphology and composition to appendages called Type IV pili commonly found in Gram-negative bacteria. We demonstrated that this new pneumococcal pilus is essential for transformation and that it directly binds DNA. We also identified a new key ATPase called RadA involved in the recombination process. We determined its crystal structure and identified its function in vitro and in vivo. We could show that this protein is a DnaB-type helicase that is recruited by the recombinase RecA at the recombination site to promote strand exchange and therefore promote the recombination process. RadA is crucial for an efficient transformation process but is also involved in genome maintenance is all bacterial species.
Overall, this project laid the foundations of a longer-term project that will eventually solve the central question of the molecular mechanism(s) of bacterial transformation and more broadly of bacterial adaptability processes.
During this project, we aimed at acquiring a detailed understating of the molecular mechanism of each of these steps. We studied the composition, architecture and function of the molecular machines involved in these processes. More than core 20 proteins were genetically identified as being required for this process. We isolated these proteins, studied their network of interaction among them and systematically studied their function in vitro and in vivo. For some of them, we managed to obtain crucial insights about their structure and function. In particular, we discovered a new appendage at the surface of S. pneumoniae cells and showed that this appendage is similar in morphology and composition to appendages called Type IV pili commonly found in Gram-negative bacteria. We demonstrated that this new pneumococcal pilus is essential for transformation and that it directly binds DNA. We also identified a new key ATPase called RadA involved in the recombination process. We determined its crystal structure and identified its function in vitro and in vivo. We could show that this protein is a DnaB-type helicase that is recruited by the recombinase RecA at the recombination site to promote strand exchange and therefore promote the recombination process. RadA is crucial for an efficient transformation process but is also involved in genome maintenance is all bacterial species.
Overall, this project laid the foundations of a longer-term project that will eventually solve the central question of the molecular mechanism(s) of bacterial transformation and more broadly of bacterial adaptability processes.