K-121. Contribution of rpoS and rpoN to Virulence and Stress-Fitness Gene Regulation in Escherichia coli O157:H7

J. T. Riordan, A. C. Springman, T. S. Whittam;
Michigan State Univ., East Lansing, MI.

Sigma factors, the dissociable subunits of core RNA polymerases, contribute to pathogenicity through the regulation of virulence and stress-fitness genes. Two alternative sigma factors, rpoS and rpoN, are essential to both nutrient scavenging and stress resistance phenotypes of E. coli. To understand the role of these sigma factors in the pathogenesis of enterohemorrhagic E. coli, we examined gene expression profiles of rpoS and rpoN isogenic mutants of E. coli O157:H7 strain Sakai. RNA was purified from exponential and stationary phase cultures (n=5) of Sakai, Sakai ΔrpoS, and Sakai rpoN::kan, grown in DMEM with 0.45% glucose. Cy-dye labeled cDNA from WT and isogenic strains were paired for hybridization to 70-mer oligonucleotide DNA microarrays representing 6,088 ORF from EDL933, Sakai, K12 and pO157. Significance analysis of microarrays (SAM) was used to analyze expression measurements, correcting for multiple comparisons using an FDR=0.05, and for biological significance using a 2-fold cutoff. qRT-PCR was used to validate select datasets. Inactivation of rpoS significantly down-regulated 323 genes, and up-regulated 46 genes, whereas, rpoN inactivation downregulated 92 genes and upregulated 127 genes. Most of the annotated genes functioned in carbohydrate utilization, nitrogen assimilation and stress-fitness. rpoS and rpoN similarly co-regulated the expression of 64 genes (48 up- and 16 down-regulated), and dissimilarly co-regulated 73 genes. Thirty-five genes, absent in MG1655 and W3110, were modulated by rpoS and rpoN, including: pOSAK1 (2 and 1), prophage (8 and 6), backbone (8 and 2), and locus of enterocyte effacement (LEE) genes (6 and 7), respectively. Stress-fitness genes modulated by rpoS and rpoN included: acid fitness island genes (12 and 8), osmotic- (10 and 4) and general stress genes (29 and 14), respectively. These results support the hypothesis that rpoS and rpoN contribute to O157:H7 pathogenesis through the expression of LEE virulence genes, and through stress-fitness gene regulation. Redundancy in the regulation of carbon- and nitrogen-utilization pathways may contribute to survival in the host and environment.