EDGE CMT: RUI: Roles of Toxin-Antitoxin Systems in Biofilm Formation and Horizontal Gene Transfer in the genus Variovorax

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This proposal will address the connection between the movement of DNA in bacterial populations and the formation of complex multi-cellular structures called biofilms. This project is focused on evaluating the role of these elements in the important soil bacterium Variovorax paradoxus. This bacterium plays a crucial role in the association of bacterial populations with plant roots, as well as in many other soil processes. Biofilm formation and DNA transfer in the environment are connected by the release of genetic material into the environment by cells within the biofilm. This material forms part of the structure of the biofilm, as well as a potential source of genetic variation. The bacteria within the biofilm are lysed to release their cellular contents, including their DNA, which then becomes part of the structure. The movement of DNA between bacterial cells is critical factor in understanding how bacterial populations respond to changes in their environment. These processes are thought to be connected in many bacterial species from diverse environments, and the mechanism proposed to be at work here may be widely responsible for connecting the two phenotypes. Using cutting edge DNA and RNA sequencing approaches, combined with laboratory experiments, the researchers, which include many undergraduates, aim to functionally link the structure and function of specific genetic elements to the formation of biofilms and the transfer of genetic material between cells via the biofilm matrix. Via course based undergraduate research experiences, students will have the opportunity to formulate their own research questions, perform state of the art genomic sequencing and bioinformatic analyses, and contribute to the writing of peer-reviewed publications. Recently finished genome assemblies show that Variovorax isolates have diverse genomic architectures including chromosomes, chromids, and plasmids. These genomes include numerous putative Toxin-Antitoxin systems in all three types of replicon, that are proposed to function as addiction or anti-addiction modules depending on expression level and location. This is hypothesized to be connected with biofilm formation through a global regulatory system controlling transcript stability during biofilm formation. An alternative RNA degradosome component is highly expressed specifically in Variovorax paradoxus EPS biofilms, which is proposed to decrease TA system RNA stability to promote cell lysis, releasing eDNA critical for dense biofilm formation and horizontal gene transfer. Differentially expressed TA operons are potential determinants of both biofilm formation and horizontal gene transfer. The TA systems in the genus Variovorax will be identified in silico, expression will be evaluated using RNA-seq, and addiction module function will be tested using tightly regulated expression systems. Saturation mutagenesis (RB-TnSeq) will be used to identify accessory genes that influence the function of TA systems in regulating plasmid stability. Chromosomal TA systems as anti-addiction modules will be evaluated similarly with TA systems integrated in single copy using mini-Tn7. The connection between TA system function, biofilm formation, and HGT will be examined using marked strains. These experiments will demonstrate that eDNA generation by TA system toxin activity is necessary and sufficient to allow for natural transformation in Variovorax biofilms. They will also show that these phenotypes are inextricably linked through biofilm growth dependent alteration of RNA stability.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.