2008 Annual Report 1a.Objectives (from AD-416) I. Investigate the population dynamics of Escherichia coli O157:H7 on plant roots grown under various irrigation water salinity regimes in the field and growth chamber. II. Investigate persistence of E. coli O157:H7 in soil, irrigation water, and rhizophere under different environmental conditions. III. Utilize fluorescence-activated cell sorter (FACS) technologies to determine genes that are specifically expressed by E. coli O157:H7 cells on plant surfaces. IV. Construct knock-out mutations in E. coli O157:H7 genes that are specifically expressed in the root and test their involvement in survival and interactions with indigenous microflora. V. Develop predictive models for E. coli O157:H7 persistence/survival in soil and on plants grown under field conditions. 1b.Approach (from AD-416) We will use fluorescence microscopy methods to quantitatively study the population dynamics of enteric bacteria on plant surfaces that will show the interactions of O157:H7 strains with indigenous microorganisms and the location of this pathogen in particular microsites and high-throughput fluorescence-activated cell sorter (FACS) to identify genes up-regulated by E. coli O157:H7 cells on plant. We will also use specific real time PCR probes to quantitatively monitor survival/persistence of E. coli in soil, rhizosphere, and on leaf surfaces. Specific evaluations of persistence of E. coli O157:H7 in the lab, growth chamber, and lysimeters at different irrigation water qualities, salinity and other environmental conditions will be determined. Finally, we will model the interaction between environmental factors, population changes, and genes that are expressed during growth of bacterial pathogens in the rhizosphere and on leaf surfaces. Documents Reimbursable NRI Grant. Log 32696. 3.Progress Report This report serves to document research conducted under a Specific Cooperative Agreement between ARS and CSREES. This research is being conducted in collaboration with investigators at the USDA-ARS-U.S Salinity laboratory (Dr Ibekwe) and University of Wisconsin-Milwaukee (Dr Yang) that was funded in July, 2007 by CSREES. Funding for this project was actually received in February, 2008 and since that time we have hired personnel to work on this project. A short summary of research efforts to date follows below. We have significant progress in generating a constitutive green fluorescent protein (GFP)-expressing E. coli O157:H7 that will make it easier to visualize cells on the rhizosphere, internal tissues of lettuce and on leaf surface using confocal laser scanning microscope. Additionally, this strain will be used for bacterial survival and population assay in soil, phyllosphere or in rhizosphere by digitalizing the GFP intensity of bacterial cell by flow cytometry. From the result of preliminary experiments, it was shown that a small fragment containing GFP with short homology extension was amplified. This approach provides us with the avenue to study long term survival and interaction of E. coli O157:H7 strain 933 with other microflora since the GFP is not on the plasmid. Research activities were monitored through weekly telephone calls and emails. Both PIs met for about two hours during the annual meeting of the American Society for Microbiology in Boston to discuss progress on the project and share other information and interesting results. Dr. Ibekwe will continue to monitor this project weekly through e-mails and telephone calls to Dr. Yang and the graduate student.