Sigma Factor N, Liaison to an ntrC and rpoS Dependent Regulatory Pathway Controlling Acid Resistance and the LEE in Enterohemorrhagic Escherichia coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is dependent on acid resistance for gastric passage and low oral infectious dose, and the locus of enterocyte effacement (LEE) for intestinal colonization. Mutation of rpoN, encoding sigma factor N (σN), dramatically alters the growth-phase dependent regulation of both acid resistance and the LEE. This study reports on the determinants of σN-directed acid resistance and LEE expression, and the underlying mechanism attributable to this phenotype. Glutamate-dependent acid resistance (GDAR) in TW14359ΔrpoN correlated with increased expression of the gadX-gadW regulatory circuit during exponential growth, whereas upregulation of arginine-dependent acid resistance (ADAR) genes adiA and adiC in TW14359ΔrpoN did not confer acid resistance by the ADAR mechanism. LEE regulatory (ler), structural (espA and cesT) and effector (tir) genes were downregulated in TW14359ΔrpoN, and mutation of rpoS encoding sigma factor 38 (σS) in TW14359ΔrpoN restored acid resistance and LEE genes to WT levels. Stability, but not the absolute level, of σS was increased in TW14359ΔrpoN; however, increased stability was not solely attributable to the GDAR and LEE expression phenotype. Complementation of TW14359ΔrpoN with a σN allele that binds RNA polymerase (RNAP) but not DNA, did not restore WT levels of σS stability, gadE, ler or GDAR, indicating a dependence on transcription from a σN promoter(s) and not RNAP competition for the phenotype. Among a library of σN enhancer binding protein mutants, only TW14359ΔntrC, inactivated for nitrogen regulatory protein NtrC, phenocopied TW14359ΔrpoN for σS stability, GDAR and ler expression. The results of this study suggest that during exponential growth, NtrC-σN regulate GDAR and LEE expression through downregulation of σS at the post-translational level; likely by altering σS stability or activity. The regulatory interplay between NtrC, other EBPs, and σN–σS, represents a mechanism by which EHEC can coordinate GDAR, LEE expression and other cellular functions, with nitrogen availability and physiologic stimuli.

Coordinated regulation of acid resistance in Escherichia coli

“…Although previous studies have demonstrated that deletion of either σ 54 or ntrC leads to increased expression of GDAR and increased acid resistance [74, 75], the lack of direct interactions between NtrC and known regulators of AR2 suggests this effect is indirect. The role of Nac in AR2 suggests that part of this effect may be through the known σ 70 –dependent regulation of nac by NtrC.…”

“…However, the activation of GDAR by ΔntrC cannot be easily explained by this link alone. Previous studies have also demonstrated that NtrC, RcsB, and GadX regulate the locus of enterocyte effacement (LEE) pathogenicity island in enterohemorrhagic E. coli , indicating that the coordination of nitrogen metabolism and AR can play both non-specific (through acid resistance) and specific (through LEE) roles in E. coli pathogenesis [74–77]. …”

σN‐dependent control of acid resistance and the locus of enterocyte effacement in enterohemorrhagic Escherichia coli is activated by acetyl phosphate in a manner requiring flagellar regulator FlhDC and the σS antagonist FliZ

“…Mitra et al. () demonstrated that this effect of σ N on σ S stability/activity is indirect and dependent on transcription from a σ N promoter, and not competition of these sigma factors for core RNA polymerase (RNAP). What additional regulatory component(s) is required downstream of σ N for control of σ S , GDAR, and the LEE is not yet known.…”

“…Of the 11 EBPs encoded within the EHEC O157:H7 background, only deletion of ntrC (encoding NtrC) phenotypically reproduces the rpoN null background for control of σ S , GDAR, and the LEE (Mitra et al. ). Nitrogen regulatory protein C, NtrC (also NRI), is the response regulator of a two‐component system that activates σ N ‐dependent transcription of genes for the assimilation and utilization of nitrogen, relieving slowed growth under nitrogen‐limiting conditions (Zimmer et al.…”

Global Regulator of Virulence A (GrvA) Coordinates Expression of Discrete Pathogenic Mechanisms in Enterohemorrhagic Escherichia coli through Interactions with GadW-GadE

“…Four different gadE P -lacZ promoter fusions were created by cloning BamHI/EcoRI-digested PCR products into similarly digested pRS551 using primers GadEϪ320/EcoRI and GadEϪ1/BamHI, GadEϪ276/EcoRI and GadEϪ516/BamHI, GadEϪ773/EcoRI and GadEϪ497/BamHI, and GadEϪ773/EcoRI and GadEϪ1/BamHI. ␤-Galactosidase activity (Miller units) was measured as previously described (38,44), and the numbers of Miller units were compared using the appropriate t test or by Tukey's HSD test following a significant F test (n Ն 3, ␣ ϭ 0.05; R software, version 3.1.0).…”