Decoding transcriptional regulatory networks activated during stringent response in Escherichia coli

The stringent response is a response network in bacteria that is triggered by nutrient limitation and involves a global reprogramming of gene transcription, orchestrated largely by a signaling molecule guanosine tetraphosphate (ppGpp). Despite decades of studies on individual genes perturbed during the stringent response in Escherichia coli, the characteristic transcriptional profile and the role of ppGpp in affecting global transcriptional changes during the stringent response had remained to be determined systematically. We provoked the stringent response, in the strain E. coli K-12, by multiple induction pathways and identified the core transcriptional profile that is characteristic of the cellular response to ppGpp accumulation. By comparison of wild type and ppGpp-deficient E. coli mutant strains, we identified the gene expression changes that are specifically dependent upon ppGpp. We found six transcriptional regulators that are recruited in the core stringent response. We show the regulatory targets of one of these six regulators, RpoS. Over 80% of transcriptional regulation was directly or indirectly dependent upon the general stress response sigma factor, RpoS, even in presence of ppGpp. Our findings suggest that ppGpp alone is not capable of mounting a global stringent response, and it relies heavily on transcriptional regulators such as RpoS. Existing regulatory models for ppGpp-mediated regulation suggest a global redistribution of RNA polymerase (RNAP), upon binding with ppGpp, during stringent response. We have shown evidence for such redistribution of RNAP in presence of ppGpp by conducting ChIP-chip analysis of ppGpp-rich and ppGpp-deficient cells with an antibody specific to the alpha-subunit of RNAP. We also correlated transcriptional changes with a phenotype of increased viability in acidic medium, and demonstrated that it was dependent upon both ppGpp and RpoS. We have shown that ppGpp enhances long-term survival, up to two hours, in acid. Our results reveal the significance of stringent response in development of phenotypes that facilitate adaptation in acidic host environments for E. coli.