Global Regulation Mechanics of E. Coli Stress Response

Abstract

Bacteria have evolved sophisticated global transcriptional programs to coordinate the expression of hundreds to thousands of genes in response to stresses, enhancing their chances of survival in unfavourable environments. In the bacterium <i>Escherichia coli (E. coli)</i>, several of those stress responses are coordinated by global regulators (GRs), such as σ factors. These σ factors, for instance, favour recognition of specific promoter cohorts by RNA polymerases (RNAPs), enhancing their transcription initiation in response to specific stimuli.

This thesis describes the results of a study of transcriptional programs triggered by genome-wide stresses targeting RNAP and DNA gyrase, which are two principal components of the transcription machinery of <i>E. coli</i>. Meanwhile, the stresses included temperature shifts, which the bacterium commonly encounters along the oral-faecal route. Since temperature shifts influence the activities of several hundred genes in a quasi-synchronous manner, to study them, we used RNA-seq and flow- cytometry, supported by existing large-scale strain libraries. Moreover, we used microscopy to verify cells health, validate flow-cytometry results, and investigate biophysical features. Finally, we used synthetic gene engineering to engineer a library to track the GR numbers.

We showed that, first, genome-wide responses to cold shock (CS) are influenced by temperature-driven alterations in DNA supercoiling. Moreover, we showed evidence that this can be explained by reduced efficiency of DNA Gyrase, a GR responsible for maintaining genome-wide supercoiling levels, particularly, in the resolution of positive supercoils. This is due to a depletion in cellular energy levels following CS. In agreement, many genes that exhibited higher susceptibility to CS, were also more vulnerable to the antibiotic novobiocin, which hampers DNA Gyrase’s ability to resolve positive supercoils.

Next, we conducted a comparative analysis of the stress response dynamics of neighbouring genes within <i>E. coli</i> operons, which allow transcribing them as single units. Our findings indicated that global structural features such as the internal promoters in operons, highly influence the operon’s stress response dynamics. Namely, we found that the relative positioning of internal promoters, is consistent with a need to compensate for premature terminations of RNAP elongation, and thus, ensure similar response strengths for distal genes in the same operon. Our findings further suggested that positive supercoiling buildup and collisions between elongating RNAPs with RNAPs at internal promoters are major contributors to RNAP fall-off rates.

Finally, during the studies above, we observed that existing strain libraries did not allow us to determine which genes (Transcription Factor (TF) inputs or outputs of the known transcription factor network (TFN)) were first triggered during stress responses. We thus developed a library of fluorescent reporters to measure the transcriptional dynamics of the most influential GRs in <i>E. coli</i>. These constructs, coding for the native promoter sequences of GR genes, enable sensing temporal changes in the RNA levels of the native GR genes. This makes the library a valuable experimental tool for future studies on the GR-mediated regulation in <i>E. coli</i>, which plays key roles in phenotypic adaptation to stresses.

Overall, this thesis offers mechanistic insights into the global regulatory processes that modulate large prokaryotic gene sets in response to stresses. As such, it contributes to knowledge on bacterial internal processes, which, in turn, could contribute to the development of new biotechnological and therapeutic applications.