Many bacterial species are now known to communicate with each other via small molecules released into the extracellular environment (quorum sensing) and by cell contact, controlling diverse cellular processes such as DNA uptake, cell movement, and biofilm formation. Recently, a new form of intercellular communication expressed by non-pathogenic Escherichia coli was described, known as contact-dependent growth inhibition or "CDI." This is the first description of a system in bacteria by which one cell binds to another and inhibits target cell growth. A large cell-surface protein designated CdiA and two-partner secretion family member CdiB are required for growth inhibition. The overall goal of this project is to determine the mechanism of CDI. The first aim is to identify the cellular targets of the CDI system using genetic approaches and determine the protein-protein interactions occurring during contact-dependent growth inhibition. CDI-resistant mutants will be isolated to identify genes essential in the growth inhibition pathway. Another strategy will be to take advantage of the finding that certain E. coli contain a cdiI gene encoding a small protein that confers immunity to CDI. CdiI could bind to CdiA itself or the cellular target of CdiA. Potential targets of CdiA and CdiI will be identified using a bacterial two-hybrid system. The interactions between CdiA and its cellular targets will be analyzed by co-precipitation using epitope-tagged proteins. An alternative in vitro approach will be to blot proteins onto nitrocellulose and probe with partner proteins radiolabeled in vitro (Far Western analysis). To identify potential partners of CdiA and CdiI biochemically, as an adjunct to the genetic approaches in Aim 1, these proteins will be tethered to agarose beads and used for affinity purification. The second aim will be to determine how the interaction of CdiA with cellular targets affects cellular metabolism and growth. Preliminary results indicate that a possible target is the AcrB multidrug channel in the inner membrane, which uses the proton electrochemical gradient to transport molecules/toxins out of the cell. Based on these data, the hypothesis that CdiA interacts with AcrB, causing leakage of protons and dissipation of the proton electrochemical gradient across the membrane, will be tested. An autoinhibitory system for CDI has been developed for analysis of the effects of CDI on membrane transport, the proton electrochemical gradient, intracellullar ATP level, and other cellular metabolic processes.
The intellectual merit of this research project is that contact-dependent growth inhibition is a recently described cellular communication mechanism that we know very little about beyond basic phenomenology. Because CDI inhibits cellular growth, understanding the mechanism of CDI may shed new light on the regulation of cellular metabolism, analogous to the use of bacteriocins to probe bacterial cell physiology. Functional CdiA and CdiB homologues exist in uropathogenic E coli strains and potential homologues are also present among a wide variety of bacteria, including plant pathogens. Thus, information about CDI obtained using E coli as a model system is likely to be directly relevant to understanding how this form of intercellular interaction contributes to the biology of a broad range of bacterial species.
Broader impacts of these studies include enhancing the infrastructure of research and training through support and laboratory training of undergraduates and graduate students. The graduate students will help mentor two undergraduate researchers, chosen from the top students in a MCDB101A Genetics class who have a burning interest in research. A number of outstanding undergraduate students trained in this laboratory in the past have gone on to graduate schools including most recently U.C. San Francisco, U.C. Los Angeles, Duke University, and Washington University (St. Louis). The work proposed here will generate high quality papers such as the original paper in Science last year on the discovery of Contact-dependent growth Inhibition, and that the graduate student supported by this project will present her or his work at local (e.g. Bacterial Physiology meeting at Asilomar, CA) and national meetings (e.g. American Society for Microbiology, Gordon Conference).
