Association and Natural Internalization of Escherichia coli in the Rhizosphere of Lettuce Plants

This dissertation includes four main studies. The objective of the first study was to identify transcriptional changes in Escherichia coli K12 after it colonized the lettuce rhizoshpere. After three days of interaction with lettuce roots, E. coli genes involved in protein synthesis, stress responses, and attachment were up-regulated. Mutants in curli production (crl, csgA) and flagella synthesis (fliN) had a reduced capacity to attach to roots as determined by bacterial counts and by confocal laser scanning microscopy. This study indicated that E. coli K12 can efficiently colonize lettuce roots by using attachment genes and can readily adapt to the rhizosphere of lettuce plants.;The objective of a second study was to investigate the transcriptional changes induced in E. coli O157:H7 interacting with lettuce roots using the same hydroponic system. Our findings indicated that the ycfR mutant of E. coli O157:H7 was impaired in attachment and colonization of lettuce roots. Transcriptional analyses suggested that E. coli O157:H7 behaved dramatically different from E. coli K12 when interacting with the lettuce rhizosphere. The ycfR gene, which is involved in biofilm modulation, was essential for colonization of the lettuce rhizosphere by E. coli O157:H7.;The third objective was to compare the biochemical profiles of E. coli K12 and O157 when interacting with lettuce roots using surface enhanced Raman spectroscopy (SERS). The spectra of E. coli K12 and O157 exposed to the HS showed some differences in the nucleic acid, protein, and lipid regions compared with controls. The spectra of E. coli K12 HS cells exhibited differences compared to spectra from E. coli O157 HS cells in the RNA and protein regions. The overall band intensity of amide regions declined for E. coli O157 HS cells, while it increased for E. coli K12 HS cells. The intensity of the RNA bands of E. coli K12 HS cells was also found to be much higher than those of the E. coli O157 HS cells. These findings were in agreement to our previous microarray data that studied the interactions of E. coli and lettuce at the gene expression level.;The objective of the fourth study was to determine the presence and taxonomic classification of naturally-internalized bacteria from commercially grown lettuce leaves. The characterization of the endophytic microorganism residing in commercial lettuce leaves was conducted in two parts. First, a total of 30 and 25 heads of romaine and red leaf lettuce, respectively, served as the source of individual leaves which were surface sterilized, and used for isolation of microorganism using non-selective media. In the second study, 21 and 60% of romaine and red leaf lettuce heads, respectively, had internalized bacteria capable of growing on MacConkey agar. Members of the genera Pseudomonas and Pantoea were most frequently isolated gram negative strains, and Enterobacter isolates were obtained from three red leaf samples. In summary, spore-forming bacteria and traditional epiphytic bacterial genera were frequently detected in surface-sterilized commercial lettuce leaves. Despite the common occurrence of internalized bacteria, only Enterobacter was related to E. coli O157:H7 and Salmonella.;The data generated during these studies helped us to elucidate the interaction between lettuce plants and E. coli. In particular, specific genetic responses of E. coli for attachment to lettuce roots and survival in the rhizosphere were determined. Those finding could be applied for preventing the initial attachment or colonization of E. coli on lettuce root. Therefore, the following association of these bacteria with lettuce plants could be potentially eliminated. In the last part of this study, the natural incidence of internalization of bacteria into lettuce leaves was determined. Our results indicated that since human pathogens such as E. coli O157:H7 and Salmonella were not naturally present inside lettuce leaves, those bacteria would not likely leash out from the lettuce leaves during post-harvesting. (Abstract shortened by UMI.)...