Many bacteria exist in two alternative states: either they explore their environment as motile planktonic individuals or produce a sessile multicellular aggregate called a biofilm. The transition between the two forms is relevant because it represents an early stage in biofilm formation and is a natural target for biofilm control. In the undomesticated Bacillus subtilis strain 3610, the DNA binding protein SinR governs the motility to biofilm transition by directly repressing the fifteen gene eps operon, which both directs the biosynthesis of a structural extracellular polysaccharide (EPS) and encodes EpsE, an inhibitor of motility. This project will explore the regulation of biofilm formation, the mechanism of motility inhibition, and the structural basis of biofilm assembly. Aim 1 studies the regulation of the eps operon. Dr. Kearns has identified various genes which when mutated alter the expression of the eps operon and affect biofilm formation. The genes and their corresponding products will be genetically and biochemically characterized to assemble a biofilm regulatory hierarchy relative to the SinR master regulator. Aim 2 will explore the mechanism by which EpsE, a bifunctional putative glycosyltransferase, inhibits motility by acting as a brake on flagellar rotation. This aim will not mechanistically explore how motility is inhibited during the transition to biofilms but also illuminate a new form of flagellar regulation. Aim 3 will characterize the structure of the EPS that stabilizes B. subtilis biofilms. The sugar composition of purified EPS will be determined and genes within the eps operon will be mutated to evaluate their roles in EPS synthesis.
Intellectual Merit: For many bacteria, the transition from motility to EPS synthesis represents the earliest stage in biofilm formation and is a natural target for biofilm control. Dr. Kearns's project explores biofilm regulation in an undomesticated Gram positive organism that adds phylogenetic breadth to a field dominated by Gram-negative model systems. In addition, EpsE may represent a second example of a new class of bifunctional glycosyltransferases, the two functions of which directly couple EPS biosynthesis to motility inhibition. The first function of EpsE is to enzymatically synthesize the structural EPS required for biofilm assembly. The second function of EpsE is unrelated to its enzymatic activity and EpsE inhibits motility by acting as a molecular break on flagellar rotation. Combined, the two functions of EpsE ensure that cells become immobilized simultaneously with the activation of EPS biosynthesis. Bifunctional glycosyltransferases may be more common than people realize and may be responsible for the pleiotropic effects associated with biofilm formation in other bacterial systems. In addition, the discovery of a protein that acts as a brake on flagellar rotation constitutes a potentially important new form of motility regulation. Finally, the location of the gene encoding the EpsE glycosyltransferase is especially relevant to the motility-to-biofilm transition as it is encoded within the greater eps operon. Thus, the transition between motility and biofilm formation is governed at a single locus under the direct control of the master regulator, SinR.
Broader Impacts: This project impacts the robust B. subtilis community as new biology is explored in the context of an undomesticated strain. This project will also impact student training as the work is suitable for two or three graduate student dissertation projects. Furthermore, the genetic analyses will provide opportunities amenable to research participation by undergraduates. Indiana University has a number of programs in place to foster undergraduate research with particular emphasis on groups that are underrepresented in science. Preliminary data from this project was generated as part of an undergraduate research experience course designed by the PI. These protocols were successful at introducing undergraduates to molecular biological techniques and have already been implemented as an Advanced Molecular Genetics Laboratory (M485) at Indiana University.
Bacteria exist in the environment in one of two forms, either motile individuals or non-motile aggregates called biofilms. While many biofilms are beneficial in the environment, some biofilms, like dental plaque, can cause disease. Furthermore, biofilms are a frequent source of persistent biocontamination that promote biocorrosion or the spread of antibiotic resistance. The goal of our work is to discover how bacteria transition from the motile to the biofilm state so that we could target that step and prevent biofilms from forming. Through or work in the harmless soil bacterium Bacillus subtilis, we discovered a protein called EpsE that acts like a molecular clutch on the bacterial flagellum. The flagellum rotates at high speed to propel bacteria and EpsE disconnects the drive train from the power source to arrest flagellar rotation. EpsE is the first clutch discovered for a biological motor and could be a useful model for manipulating the function of nanomachines. Our work was published in the journal Science and featured on the front page of the NSF website. Other work in the lab has discovered important activator proteins that stimulate the production of biofilms. We have studied how motility is turned on and off in bacteria at the level of flagellar assembly. Finally, we have explored fundamental aspects of how cells grow, divide, and separate from one another.
