2007 Annual Report 1a.Objectives (from AD-416) 1. Develop strains of Escherichia coli O157:H7 and non-pathogenic E. coli that contain the gfp gene inserted into the bacterial chromosome. 2. Determine the survival and growth of gfp-labeled wild type and rpoS-deficient E. coli O157:H7 populations in internalized tissues. 3. Determine differences in the expression of virulence factors in E. coli O157:H7 grown on leafy greens and ground beef. 1b.Approach (from AD-416) Strains of pathogenic E. coli O157:H7 and non-pathogenic will be transformed so that the green fluorescent protein is inserted on the bacterial chromosome. These strains will then be applied to soil where lettuce and spinach plants are growing, and these plants will be analyzed by microscopic and culture methods to determine the extent and duration that internalized cells can survive in plant tissues. Changes in virulence of E. coli O157:H7 will also be compared when grown in ground beef and lettuce to determine if these cells are primed to cause illness. 3.Progress Report This report documents research conducted under a Reimbursable Agreement between ARS and Fresh Express, Inc. Additional details of the research can be found in the report for the parent project 1265-42000-014-00D "Microbiology and Control of Pathogen Contamination on Fresh and Fresh-cut Produce" which has been recently replaced. The amplified gfp gene with a consensus sigma-70 promoter was cloned into the pGRG36 attTn7 vector to create pXLW45. The plasmid pXLW45 was introduced, by conjugation, into 3 nalidixic acid resistant strains thus far. Those being the commensal flora control strain HS, an O157:H7 strain isolated from a bag of leafy greens associated with an outbreak in 2006, and O157:H7 strain RM5279 isolated from an outbreak associated with bagged vegetables. The insertion of the gfp gene into the attTn7 site in the chromosome in each strain was confirmed by PCR. This work was performed at the University of Maryland Baltimore by an FSL collaborator. Surface decontamination and enumeration methods have been developed to kill bacteria on the surface of lettuce leaves, and these may be applied to enumerating internalized bacteria in spinach and lettuce plants. Two genes, gnd and eae, have been detected by reverse transcriptase RT-PCR from lettuce inoculated with E. coli O157:H7 and incubated at 37 deg C and from inoculated laboratory media. To the investigators’ knowledge, this is the first reported transformation of E. coli with gfp using the method described above.