In enterohemorrhagic E. coli (EHEC) sigma factor N (σN) regulates glutamate-dependent acid resistance (GDAR) and the locus of enterocyte effacement (LEE), discrete genetic systems required for transmission and virulence of this intestinal pathogen. Regulation of these systems requires nitrogen regulatory protein C, NtrC, and is a consequence of NtrC/σN-dependent reduction in the activity of sigma factor S (σS). This study elucidates pathway components and stimuli for σN-directed regulation of GDAR and the LEE in EHEC. Deletion of fliZ, the product of which reduces σS activity, phenocopies rpoN (σN) and ntrC null strains for GDAR and LEE control, acid resistance and adherence. Upregulation of fliZ by NtrC/σN is indirect, requiring an intact flagellar regulator flhDC. Activation of flhDC by NtrC/σN and FlhDC-dependent regulation of GDAR and the LEE is dependent on σN-promoter flhDP2, and a newly described NtrC upstream activator sequence. While the addition of ammonium significantly alters GDAR and LEE expression, acid resistance and adherence, it does so independently of rpoN, ntrC and the NtrC sensor kinase ntrB. Altering the availability of NtrC phosphodonor acetyl phosphate by growth without glucose, with acetate addition, or by deletion of acetate kinase, ackA, abrogates NtrC/σN-dependent control of flhDC, fliZ, GDAR and LEE genes.

Extreme acid resistance contributes to the successful transmission of enterohemorrhagic E. coli (EHEC) through acidic food matrices and the stomach, allowing it to gain access to the intestine and elicit disease in humans. Alternative sigma factor N (σN, encoded by rpoN) was previously identified as a novel regulator of extreme acid resistance in EHEC. This study investigated the role for σN and co-expressed products of the rpoN operon in the acid resistance phenotype of EHEC. The results revealed that σN primarily controls acid resistance through repression of the glutamate-dependent acid resistance (GDAR) system through control of the σS-directed GadXW pathway. σN was also determined to repress additional acid resistance systems, including arginine-dependent acid resistance, and an anaerobic acid resistance mechanism. Two gene products of the rpoN operon, hpf and ptsN, were also determined to negatively affect GDAR, as well as expression of the σN dependent genes glnA, astA, and pspA. Mutation of hpf and ptsN did not however alter the transcription of rpoN. Transcript levels of rpoN operon genes were observed to be differential, and inconsistent with the hypothesis of expression as a single transcriptional unit. Together this data signifies the importance of rpoN operon genes in the negative regulation of extreme acid resistance systems, and suggests that the products of hpf and ptsN control the activity of σN at its promoters.