Bacterial RNA polymerase is composed of a core (beta, beta', 2 alphas, and omega) that associates with the sigma specificity factor. In E. coli the primary sigma is sigma70, encoded by rpoD. There are hundreds of bacterial sigma factors within the sigma 70 family, and they all share regions of homologous sequence and function. Regions 2.4/ 3, and region 4.2 recognize specific sequences within the promoter DNA, binding to the extended -10 and to the -35 promoter elements, respectively. The spacer is thought to be important for setting the distance between these elements, with limited sequence specificity. Crohns disease (CD), an inflammatory bowel disease, arises from an immune attack of the GI tract. Although the etiology of CD is unknown, Adherent Invasive E. coli, such as the pathobiont strain LF82, is frequently found associated with CD, suggesting that it may play a role in CD pathogenesis. Genome analysis of LF82 has revealed multiple nucleotide substitutions that distinguish it from commensal and toxigenic E. coli, including a change in the rpoD gene sequence to encode a D445V variant of the sigma 70. Curiously, after introduction of LF82 into altered Schaedler Flora gnotobiotic mice, whose GI tracts are colonized with only 8 bacterial species (lacking Enterobacteriaceae), the rpoD variant (as well as others) rapidly reverts to the more highly conserved D445 residue in sigma70. After constructing isogenic strains in an E. coli K-12 genetic background, we have found that the sigma70 D445V substitution confers a strong cold sensitive phenotype for growth on plates and an extreme mucoid phenotype at low temperatures. Our modeled structure of E. coli RNA polymerase with promoter DNA indicates that sigma70 D445 is 5 angstroms from the non-template strand at position -17 relative to the transcription start site. Thus, this residue is near the spacer region between the -10 and -35 elements of a sigma70-dependent promoter, a region that does not typically contain specific sequence determinants. D445 is also within 5 angstroms of beta' E42, a residue that lies within the beta'-zipper and has been reported to interact with spacer DNA. To investigate the effect of sigma70 D445V on transcription, we have purified the variant and assayed its effect on core/sigma70 association and open complex formation at an ideal -10/-35 promoter. Our results indicate that the mutation does not simply render RNA polymerase defective at colder temperatures, suggesting that it might affect transcription from certain promoters. We have performed RNA-seq analyses of the E. coli wt and variant strain grown in culture or on plates at 37o C vs. 23o C. In either 23o C or 37o C cultures, the RNA fold-difference for the variant increases significantly for several genes, including ampC (beta-lactamase), mdtK and acrZ (multi-drug efflux pump), and csrD (destabilizes the small RNAs csrB/csrC); lowered RNA values relative to wt include those of csrC, malT (regulator of maltose operon), and putP (proline uptake). Interestingly, specifically in the 37o C culture, the relative levels of dsrA and rprA RNAs, which are involved in the activation of rpoS translation, increase, while specifically in the 23o C culture, the relative levels of multiple genes involved in chemotaxis and motility decrease. We also show that the D445V variant exhibits increased resistance to ampicillin, but not to kanamycin and rifampicin, and that the motility of the variant is impaired especially at lower temperatures. Our results suggest that the rpoD sequence variant may affect the expression of sets of genes that offer the pathobiont a selective advantage within the CD gut, and may reveal a novel strategy for environmental adaptation by bacteria.
| Hook-Barnard, India G; Hinton, Deborah M (2009) The promoter spacer influences transcription initiation via sigma70 region 1.1 of Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A 106:737-42 |
