Bacteria have developed sophisticated mechanisms to communicate and compete with each other in diverse environments including water, soil, plants, and animals. The overall objective of this project is to understand the mechanism of contact dependent growth inhibition (CDI) in bacteria. CDI is a new form of intercellular communication between bacteria involving direct cell-to-cell contact. It is widespread amongst gram negative bacteria including Escherichia coli, Yersinia pestis and Burkholderia pseudomallei, but little is known about its mechanism or its role(s) in bacterial biology. CDI is orchestrated by a two-partner secretion system consisting of an outer membrane protein CdiB (~60 KDa), which facilitates export of CdiA (~320 KDa) to the cell surface. An additional protein, CdiI (~8 KDa) confers immunity to cells expressing CdiA/CdiB. Recent work indicates that the receptor for CdiA on E. coli is the essential outer membrane protein BamA (a.k.a. YaeT). The focus of this proposal is on understanding how the CdiA effector protein is assembled and presented on inhibitor cell surfaces, interacts with BamA receptor, and then transmits a growth inhibitory signal to target cells. CDI is a dynamic process since binding between CdiA and BamA is reversible and involves proteolytic processing of CdiA. The first aim of the project is to determine the topology of CdiA, including whether the N-terminus or C-terminus extends out from the cell surface. To test the hypothesis that the CdiI immunity protein may also act as a chaperone for CdiA during export, CdiI will be epitope tagged to examine its fate together with CdiA using immunofluorescence microscopy and co-immunoprecipitation experiments. The second aim is to examine the interactions between CdiA and the BamA receptor. The region of CdiA that binds to BamA will be determined by screening in-frame deletion mutations in BamA for binding to target cells. The BamA region(s) required for binding of CdiA will be identified by mutation of potential extracellular domains of BamA and the effects of these mutations on inhibitor-target cell binding will be quantified. The third aim is to test the hypothesis that the C-terminal region of CdiA, called CdiA-CT, is injected into target cells after binding to BamA. Using a genetic approach, the cdiA gene region coding for CdiA-CT will be mutated and the effects of these mutations on binding to BamA and proteolytic cleavage of CdiA will be measured. Antibodies to CdiA-CT will be used to directly determine if CdiA-CT enters target cells. Because functional homologues of the cdiBAI genes are present in a wide range of bacteria found in soil, plants, and animals, this work has the potential for broad impacts in understanding how diverse bacterial species interact and compete with one another. Broader Impacts This CDI project is being carried out by both undergraduate and graduate students, including underrepresented groups. Since many basic aspects of molecular and cell biology are being used in the project, students will have an excellent opportunity to develop tools and approaches in basic research. Students will receive training from the P.I. and from postdoctoral fellows in the laboratory, and will present their findings in laboratory meetings as well as local and national scientific meetings. For example, this past year students presented their work at the Undergraduate Research Colloquium and the Analytical Genetics Conference, and students have been authors on scientific papers. Work on the CDI project will be presented by the P.I. at scientific meetings and invited seminars, and also at Santa Barbara City College as part of a course to introduce community college students to scientific research. Collaborations with other scientists including structural biologists, biochemists, bioinformaticists and systems engineers is ongoing with the potential for impacts in these fields.
