Bacteria can quickly adapt to changing environmental conditions by activating alternative sigma factors. It has been shown previously that single cell approaches can reveal hidden dynamics in sigma factor activation. Here, we investigate the single cell response dynamics of the B. subtilis extracytoplasmic function sigma factors, which are an important part of the cell envelope stress response, under their specific stresses. To do this we use transcriptional reporters of sigma factors, quantitative single cell snapshots, time-lapse microscopy, and microfluidics. By developing an improved microfluidics setup for single cell time-lapse microscopy, as well as improved single cell analysis code, we are able to observe new sigma factor dynamics. First, we observe heterogeneous entry into a higher $\sigma^{V}$ activity state in response to lysozyme, which displays a memory, as the heterogeneity is lost on removal and reapplication of the stress. Next, we observe a pulse amplitude and duration modulated sigma factor response of $\sigma^{M}$ to bacitracin. Finally, for $\sigma^{M}$ under ethanol and acidic stress, and for $\sigma^{Y}$ under ethanol stress, we observe a noisy increase in activity to a new steady state level, where the degree of variability between cells depends on the stress condition. This thesis also discusses efforts on building a single cell microfluidic device based on the ”mother machine” design, for the rod-shaped cyanobacterium, S. elongatus, which forces the cells to grow in a straight line. Growing this organism in a traditional mother machine device has, so far, proved challenging. To adapt the mother machine for cyanobacteria we modify the channel geometry using electron beam lithography, and improve the loading protocol. The research presented here reveals the range of regulatory dynamics possible for ECF sigma factors in B. subtilis, and provides improved microfluidics and analysis code that will enable easier quantification of bacterial gene circuits at the single cell level in the future.

Porphyromonas gingivalis is a major etiological agent that is responsible for the cause and progression of periodontal diseases. The bacterium is exposed to various environmental conditions and oxidative stress conditions while it is in the oral cavity. So, P. gingivalis should have an efficient regulatory system in order to adjust and survive in the oral cavity. But little is known about the regulatory mechanisms that help the bacteria to survive in the oral cavity. So, it is essential to understand and characterize these regulatory mechanisms. The response and adaptation of P. gingivalis to environmental stress conditions occur at the level of transcription which involves the alternative sigma factors. Extracytoplasmic function (ECF) sigma factors are the largest group of alternative sigma factors that play a major role in bacterial response to environmental stress conditions. Here we characterize the σ-70 factor, SigH and SigG, the extracytoplasmic function sigma factors encoded in P. gingivalis genome. Our results show that the expression of SigH is upregulated when P. gingivalis is grown in the presence of oxygen. However, there is no change in the expression of SigG when grown in the presence of oxygen. Furthermore several genes involved in oxidative stress protection such as sod, trx, tpx, ftn, feOB and the hemin uptake locus, hmu, are downregulated in the mutant deficient in SigH designated as V2948. Our RNA-seq analysis of SigG showed that there is no change in the regulation of genes involved in oxidative stress protection and metal homeostasis in SigG deficient mutant designated as V3085. Our survival studies showed that both SigH and SigG are essential for P. gingivalis to grow in host cells. Collectively our studies demonstrate that SigH is a positive regulator of gene expression required for survival of the bacterium in the presence of oxygen and oxidative stress, hemin uptake and virulence. However our studies show that SigG is essential for the bacteria to grow in host cells and hence helps in the virulence of P. gingivalis.