One-step inactivation of rpoS to investigate its role on Escherichia coli O157:H7 biofilm formation and survival

Homologous linear recombination using the recombinogenic capabilities of lambda phage red genes exo, bet and gam, has been used increasingly to generate targeted gene replacements in Gram-negative bacteria. While lambda Red recombination has been widely applied in nonpathogenic strains of Escherichia coli, the system has been employed to a lesser extent in pathogenic strains of E. coli, and the frequency of these successes is rarely reported. In this thesis, lambda Red recombination was used to generate a disruption of rpoS in pathogenic E. coli O157:H7. Four gene cassettes carrying either a green fluorescent protein - kanamycin resistant gene cassette (gfp-aph(3')I) or a gentamycin resistance determinant (accC1, herein gm) flanked by 40 or 500 bp of rpoS homology were tested for their ability to facilitate gene replacement using this system. A singular success resulted from these efforts using gm flanked by 500 bp homologous to rpoS in H32-gfp, a strain of E. coli O157:H7 H32 labeled with gfp gene through random insertion of a Tn5 transposon. The frequency of this success was 80 gene disruptions per 108 cells surviving transformation (cell survivors), with an efficiency of 4x102 gene replacements per mug of linear DNA (1.97 x 1011 linear cassette copies). The frequency and efficiency of the gene replacement event, coupled with the non-reproducible nature of the rpoS disruption suggests that the activity of the lambda Red system is hindered in E. coli O157:H7.;Over a 13-day period of starvation in deionized water, the presence or absence of rpoS did not have an apparent influence in the survival of E. coli O157:H7 biofilms, whereas rpoS enhanced the overall survival of E. coli O157:H7 under these conditions when both attached and detached biofilm cells were considered. The rpoS-knockout also exhibited decreased numbers of detached viable cells, suggesting that rpoS may enhance detachment, survival of detached cells, or both. H32-gfp biofilm cells exhibited decreased survival and increased biofilm detachment relative to H32, suggesting that the gfp-labeling of H32 influenced survival and detachment of this strain. The transposon insertion site was identified as uhpC, a regulatory protein in sugar phosphate transport UhpC is therefore implicated in the detachment and subsequent survival of E. coli cells under conditions of starvation and osmotic stress.;The findings of this thesis suggest that while Lambda Red recombination does not represent an ideal means of generating gene disruptions in pathogenic E. coli, the generation of these targeted manipulations is invaluable to the elucidation of gene functions such as rpoS. While the full role of rpoS has not been revealed, it is evident that rpoS plays an important role in biofilm formation and in the survival of E. coli O157:H7. The influence of culture media on the impact of rpoS- on biofilm formation highlights the importance of consistency between investigations and helps to reconcile the contradictory findings in the literature.;The stationary phase sigma factor, RpoS, has been implicated both in the survival of planktonic E. coli during periods of stress and in biofilm formation. The effect of rpoS deletion on E. coli O157:H7 biofilm formation and survival was examined through the establishment of biofilms on glass coverslips and subsequent examination through confocal laser scanning microscopy and viable cell counts, respectively. The influence of rpoS on biofilm formation was found to be nutrient-dependent, where rpoS mutants displayed a negligible reduction in biofilm formation relative to wild-type strains in nutrient-rich media (TSB and 1/5th strength TSB), but enhanced biofilm formation in nutrient-poor media (MSM + 0.04% glucose). The rpoS mutants exhibited 3.5 to 7-fold greater biovolume relative to H32 and H32- gfp, respectively, and 7-fold greater substratum coverage relative to both H32 and H32-gfp. Biofilm formation in both the wild-type and rpoS-knockout (H32-gfpDeltarpoS::gm) strains was found to be negatively correlated with nutrient availability, where all strains displayed enhanced biofilm formation in MSM + 0.04% glucose.