Characterization of CRISPR- mediated Gene Regulation in Myxococcus xanthus

Myxococcus xanthus is a gram-negative bacterium found in soil, mainly studied for its exceptional cell signaling mechanisms. Starvation-induced development of M. xanthus is an excellent model to investigate the way bacterial communities sense and respond to environmental signals. M. xanthus is known for its multicellular response during starvation. Morphological changes include cell differentiation from rods to round spores within special structures called fruiting bodies to evade starvation. The gene regulatory network for this process includes the dev operon, which is similar to CRISPR-Cas systems that aid in phage defense. Genes of the dev operon, such as devT, devR, and devS, regulate spore formation by repressing expression of DevI, also encoded in the operon. DevI acts as a sporulation timer and inhibits spore formation when highly expressed. We will perform experiments to identify the mechanism of developmental regulation by DevTRS proteins. Cas proteins typically associate to form a “Cascade” complex that binds to target nucleic acid sequences. To test whether DevTRS proteins associate to form such a complex, we will do bacterial two-hybrid analysis. We have cloned the devTRS genes separately into bacterial two-hybrid analysis vectors, and will test them for interactions in various combinations. Positive interaction between one or more dev proteins will support the idea of complex formation. These experiments will help define the mechanism by which DevTRS proteins affect spore formation and help better understand the role of CRISPR-Cas systems in gene regulation. This allows for improved comprehension of regulatory mechanisms in bacteria, with industrial and therapeutic applications. Key words: Myxococcus xanthus, Cascade complex, sporulation, fruiting bodies, cell signaling, gene regulatio