σ^N‐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 σ^S antagonist FliZ

Abstract

In enterohemorrhagic Escherichia coli (EHEC), sigma factor N (σN) regulates glutamate-dependent acid resistance (GDAR) and the locus of enterocyte effacement (LEE); discrete genetic systems that are 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, phenocopied rpoN (σN) and ntrC null strains for GDAR and LEE control, acid resistance, and adherence. Upregulation of fliZ by NtrC-σN was shown to be indirect and required an intact flagellar regulator flhDC. Activation of flhDC by NtrC-σN and FlhDC-dependent regulation of GDAR and the LEE was dependent on σN-promoter flhDP2, and a newly described NtrC upstream activator sequence. Addition of ammonium chloride significantly altered expression of GDAR and LEE, acid resistance, and adherence, 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, abrogated NtrC-σN-dependent control of flhDC, fliZ, GDAR, and the LEE.

Investigating the genetic regulation of the ECF sigma factor σS in Staphylococcus aureus

“…Whole genome sequencing was performed using an Ion Torrent Personal Genome Machine (PGM, Life Technologies) according to manufacturer’s protocols, as described by us previously [ 23 ]. Genomic DNA was extracted as described above and 1 μg of total DNA was fragmented using The Ion Xpress™ Plus Fragment Library Kit (Life Technologies).…”

The RNA polymerase gene specificity factor σ54 is required for homogeneous non-planktonic growth of uropathogenic Escherichia coli

“…4B, we noted that the majority (76 out of 149) of the conserved upregulated genes in the ΔrpoN CFT073 strain either belonged to or are associated with the rpoS regulon (as defined in the non-pathogenic E. coli strain MG1655 (18,19)) and transcribed by the general stress response σ factor, σ 38 (product of rpoS). The functional interconnection between the rpoN and rpoS regulons is undisputed as the absence of rpoN has been shown to lead to either increased σ 38 levels or σ 38 stability in both enterohemorrhagic and non-pathogenic E. coli strains (7,8,20). Therefore, we considered whether the biphasic non-planktonic growth property of the ΔrpoN CFT073 strain was linked to increased σ 38 activity.…”

“…Although pathogenic E. coli can cause intestinal/enteric and extra-intestinal infections -both with the potential to lead to serious systemic disease -most of our knowledge on the involvement of σ 54 in E. coli pathogenesis is limited to studies on the intestinal pathogen, enterohemorrhagic E. coli (EHEC). To establish itself in the host, EHEC bacteria must overcome the acidic gastrointestinal environment and the σ 54 is involved in the cascade of processes associated with conferring acid resistance and attachment to intestinal cells for competitive host colonization (6)(7)(8)(9). Uropathogenic E. coli (UPEC) are members of the extra-intestinal pathogenic E. coli and are the major causative agent of urinary tract infections (UTIs) worldwide.…”

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

“…Electrophoretic mobility shift assays (EMSAs) were performed as previously described (42). Briefly, the forward primers RcsBϩ4/NcoI and RcsBϩ682/XhoI, used to PCR amplify rcsB from TW14359 DNA, contained a new start codon and an N-terminal 6ϫHis epitope tag, and the PCR product was cloned into the NcoI/XhoI sites of similarly digested vector pET-24d to create pRJM-27.…”