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.
Highlights
Alternative sigma factor N when bound to RNA polymerase directs the transcription of genes for carbon and nitrogen metabolism, stress fitness, and regulation (Reitzer and Schneider 2001)
During growth in Dulbecco’s Modified Eagle’s Medium (DMEM) (OD600 = 0.5), both crl and fliZ expression were shown to be reduced in TW14359DntrC and TW14359DrpoN when compared to TW14359 (P < 0.05) (Fig. 1A), only TW14359DfliZ phenocopied TW14359DntrC and TW14359DrpoN for the control of glutamate-dependent acid resistance (GDAR) and locus of enterocyte effacement (LEE) genes (Fig. 1B)
Consistent with the effect of fliZ deletion on gadE and gadB expression, CFU/mL of TW14359DfliZ recovered following exposure to acidified E minimal glucose (EG) media for 1 h increased by 10- to 100-fold compared to TW14359, TW14359DfliZpRAM-8, and TW14359DfliZDrpoS, yet remained ~10-fold less than that observed for TW14359DntrC and TW14359DrpoN (Fig. 1C)
Summary
Alternative sigma factor N (rN) when bound to RNA polymerase directs the transcription of genes for carbon and nitrogen metabolism, stress fitness, and regulation (Reitzer and Schneider 2001). In B. burgdorferi, the causative agent of Lyme borreliosis, rN activates the expression of genes encoding outer surface lipoproteins (OspA and OspC) essential for transmission from the tick vector to a mammalian host, and for establishment of infection (Pal et al 2000; Hubner et al 2001; Grimm et al 2004). This Osp activation pathway requires another sigma factor, rS, the transcription of which is directly activated from a rN-promoter in what has been dubbed a rN-rS regulatory cascade (Smith et al 2007; He et al 2008). In EHEC serotype O157:H7, a food-borne pathogen attributed to outbreaks and sporadic cases of bloody diarrhea (hemorrhagic colitis)
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