Cyclic di-GMP Regulation of PilB in Motility and Biofilm

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To go, or to stay, that is the decision a bacterium must make in any environment it encounters. To go, it must assemble a motility apparatus and disable structures for attachment to surfaces. To stay, it needs to establish a protected settlement by producing building materials of its own to form a bacterial biofilm on a solid surface. This decision and its execution by bacteria profoundly impact our lives and society because biofilms can form on so many critical environmental surfaces. This research project probes at the molecular level how a bacterium makes and executes this decision to either form biofilms or move by enabling its motility. Besides advancing basic microbiology, this project will allow integrated teaching and learning opportunities as well as interdisciplinary training of both graduate and undergraduate students. In addition, a variety of outreach activities will be performed to help diversify our STEM workforce.This project focuses on the regulation of PilB by the universal bacterial second messenger cyclic-di-GMP (cdG). PilB is an ATPase that powers the assembly of bacterial type IV pilus (T4P) as a motility apparatus for movement on solid surfaces. Additionally, Myxococcus xanthus T4P has been demonstrated to stimulate the production of exopolysaccharide (EPS) which is the major building materia of bacterial biofilms. M. xanthus PilB (MxPilB) would thus appear to promote both motility and biofilm formation, which are two alternate and mutually exclusive lifestyles of bacteria. Evidence indicates that MxPilB and its Chloracidobacterium thermophilum orthologue (CtPilB) both bind cdG using a conserved cdG-binding domain. The investigators propose a model wherein cdG inversely regulates the two seemingly incompatible and opposing functions of PilB. One part of this project uses M. xanthus to test this model in vivo combined with cdG binding assays in vitro. The rest of the project employs CtPilB as the model protein to analyze the interactions of PilB with cdG by quantitative and structural analysis in vitro as well as advanced imagining platforms including single-particle cryo-EM and high-speed atomic force microscopy. The successful completion of this project will provide insight into the lifestyle decision-making by T4P-motile bacteria that are present in numerous ecosystems and on many branches of the phylogenetic tree.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.