Control of Transcription Initiation

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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 promoter spacer, located between the extended -10 and -35 elements, is important for setting the distance between these elements. However, the spacer does not typically contain crucial sequence elements for recognition. 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. Although AIEC phenotypes differ from those of “commensal” or pathogenic E. coli, previous work has failed to identify genetic features accounting for these differences. Furthermore, AIEC strains, like LF82, have multiple single nucleotide polymorphism (SNPs) that differentiate them from nonpathogenic and pathogenic E. coli, but again, previous work has not assigned any importance to these SNPs. One of these SNPs occurs within a highly conserved region of rpoD, with the substitution of the nearly universally conserved aspartic acid at residue 445 to a valine. To better understand the significance of this variation in a highly conserved housekeeping gene, we constructed the D445V mutant within a nonpathogenic K-12 strain of E. coli (MG1655) as well as the V445D substitution within LF82 and investigated how this single substitution affects gene expression and strain phenotypes. We have found that the sigma70 D445V substitution within MG1655 has little effect on growth in various liquid media and does not affect the level of sigma70 in the cell at 37o C or the activity of RNA polymerase at a strong sigma70-dependent promoter. However, it confers a strong cold sensitive phenotype for growth on plates and an extreme mucoid phenotype at low temperatures. RNA-seq analyses comparing sigma70 V445 vs. sigma70 D445 identified 24 genes upregulated by sigma70 V445 in both LF82 and MG1655. Among these genes were those whose products were involved in specific cellular processes, such as methionine biosynthesis (metE), antibiotic resistance (ampC, encoding beta-lactamase, and the multi-drug efflux pumps acrZ and bcr), degradation of L-fucose (fucI), as well as several genes with membrane-associated functions, such as transporters and pumps (shiA, mlaA, cysZ, and sppA). We also found genes within the operon lptM-dapF-yigA-xerC-yigB, which encodes products of diverse functions. Finally, yobH was identified in both datasets. Although the function of YobH has not been characterized in E. coli, in Salmonella it has been shown to be involved in the invasion of macrophages. To test whether these changes were biologically significant, we investigated antibiotic resistance patterns, growth in the absence methionine, and biofilm formation. We found that the presence of sigma70 V445 in either the MG1655 or the LF82 background increases ampicillin resistance, consistent with the increase in the ampC gene; that the overall pattern of antibiotic resistance is modified by sigma70 V445, consistent with the increase in multi-drug efflux pumps; that MG1655 sigma70 V445 grows better in the absence of exogenous methionine than MG1655 sigma70 D445, consistent with the fold change increase in metE expression; and that MG1655 sigma70 V445 makes more biofilm than MG1655 sigma70 D445, consistent with the known increase in biofilm for LF82. Our results are consistent with the idea that sigma70 V445 contributes to the lifestyle of LF82. To understand whether the D445V substitution directly affects transcription by RNA polymerase, we purified sigma70 V445 and compared its activity to sigma70 D445 in in vitro transcription assays. Using multiple promoters, we showed that a promoter spacer of 16 bp and a G:C bp at position -14 correlates with increased transcription in vitro from several promoters upstream of genes whose expression increases in the RNA-seq analyses. Based on RNAP structures, the position of sigma70 D445 posits a possible effect of RNAP interaction with promoter spacer DNA. Our results suggest that the sigma70 D445V substitution affects the RNAP-spacer interactions, resulting in a direct increase in the expression of specific genes by altering transcription initiation. Importantly, the work identifies the first distinguishing SNP for LF82. More broadly, our results further suggest that mutations within the highly conserved rpoD housekeeping gene may represent an underexplored mechanism contributing to bacterial adaptation to new environments and the emergence of new pathotypes. To understand the effect of the sigma70 D445V substitution in vivo, we are now investigating the ability of MG1655, MG1655 sigma70 D445V, WT LF82, and LF82 sigma70 V445D to invade and replicate in mouse macrophages and the elicited cytokine response of those macrophages after their interaction with these various bacteria. Our goal is to determine if and how the sigma70 mutation contributes to the association of LF82 with the CD gut.