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.

No description available.

A citricultura é de grande importância para as atividades agrícolas brasileiras, uma vez que o Brasil é o principal produtor e exportador de suco de laranja. O cancro cítrico, causado pela bactéria Xanthomonas axonopodis pv. citri (Xac) é um grave problema nesse setor, causando um elevado prejuízo na produção de frutos e seus derivados. O fator sigma é a subunidade da RNA polimerase que tem a função de direcionar o núcleo da RNA polimerase a uma classe específica de sequências promotoras. Como a maioria das bactérias sintetiza diversos fatores sigma, essa característica proporciona à bactéria a oportunidade de manutenção basal da sua expressão gênica, assim como, a regulação em resposta a alterações ambientais e a sinais durante o desenvolvimento bacteriano. O genoma de Xac codifica para 14 fatores sigma. Nesse presente trabalho, detectamos interações dos fatores σECF (Xac2814. Xac3989, Xac0922, Xac1319, Xac1380, Xac1682, Xac4129 e Xac2191) e seus fatores anti-σ cognatos (Xac2815. Xac3988, Xac0921, Xac1320, Xac1379, Xac1681, Xac4130 e Xac2192). Além disso, observamos interações entre o fator σFliA (Xac1933) e o anti-σFlgM (Xac1989), seu fator anti-σ cognato. A caracterização das cepas nocautes para alguns fatores σ apontaram o envolvimento do fator σ54Xac1969 no mecanismo de formação de flagelo, a contribuição do fator σECFXac1682 na resposta ao choque térmico e a participação do fator σECFXac2191 no crescimento bacteriano em condições de carência de ferro. / Citriculture is an important sector of the economy of the State of São Paulo. Citrus canker, caused by Xanthomonas axonopodis pv. citri (Xac), is a devastating disease responsible for large agribusiness losses every year. several sigma factors. The sigma factor is the subunit of RNA polymerase that serves to direct the RNA polymerase core to a specific class of promoter sequences. Most bacteria code for more than one sigma factor, which provides the cell with the means by which to maintain basal gene expression while at the same time modulate the expression of specific genes in response in environmental changes and signals during bacterial growth. The Xac genome codes for 14 sigma factors which are the objects of study in this thesis. We demonstrate that many of the sigma factors of the σECF family (Xac2814, Xac3989, Xac0922, Xac1319, Xac1380, Xac1682, Xac4129 e Xac2191) interact with cognate anti- factors (Xac2815, Xac3988, Xac0921, Xac1320, Xac1379, Xac1681, Xac4130 e Xac2192). These sigma-anti-sigma pairs are all coded by neighboring genes. Interactions between the sigma factor σFliA (Xac1933) and anti-σFlgM (Xac1989) were also observed. Xac strains with gene knockouts for several sigma factors were produced. The characterization some these knockout strains point to the involvement of σ54Xac1969 in the biosynthesis of flagella, participation of σECFXac1682 in the ability to survive heat shock and involvement of σECFXac2191 in the response to iron deficiency.

A citricultura é de grande importância para as atividades agrícolas brasileiras, uma vez que o Brasil é o principal produtor e exportador de suco de laranja. O cancro cítrico, causado pela bactéria Xanthomonas axonopodis pv. citri (Xac) é um grave problema nesse setor, causando um elevado prejuízo na produção de frutos e seus derivados. O fator sigma é a subunidade da RNA polimerase que tem a função de direcionar o núcleo da RNA polimerase a uma classe específica de sequências promotoras. Como a maioria das bactérias sintetiza diversos fatores sigma, essa característica proporciona à bactéria a oportunidade de manutenção basal da sua expressão gênica, assim como, a regulação em resposta a alterações ambientais e a sinais durante o desenvolvimento bacteriano. O genoma de Xac codifica para 14 fatores sigma. Nesse presente trabalho, detectamos interações dos fatores σECF (Xac2814. Xac3989, Xac0922, Xac1319, Xac1380, Xac1682, Xac4129 e Xac2191) e seus fatores anti-σ cognatos (Xac2815. Xac3988, Xac0921, Xac1320, Xac1379, Xac1681, Xac4130 e Xac2192). Além disso, observamos interações entre o fator σFliA (Xac1933) e o anti-σFlgM (Xac1989), seu fator anti-σ cognato. A caracterização das cepas nocautes para alguns fatores σ apontaram o envolvimento do fator σ54Xac1969 no mecanismo de formação de flagelo, a contribuição do fator σECFXac1682 na resposta ao choque térmico e a participação do fator σECFXac2191 no crescimento bacteriano em condições de carência de ferro. / Citriculture is an important sector of the economy of the State of São Paulo. Citrus canker, caused by Xanthomonas axonopodis pv. citri (Xac), is a devastating disease responsible for large agribusiness losses every year. several sigma factors. The sigma factor is the subunit of RNA polymerase that serves to direct the RNA polymerase core to a specific class of promoter sequences. Most bacteria code for more than one sigma factor, which provides the cell with the means by which to maintain basal gene expression while at the same time modulate the expression of specific genes in response in environmental changes and signals during bacterial growth. The Xac genome codes for 14 sigma factors which are the objects of study in this thesis. We demonstrate that many of the sigma factors of the σECF family (Xac2814, Xac3989, Xac0922, Xac1319, Xac1380, Xac1682, Xac4129 e Xac2191) interact with cognate anti- factors (Xac2815, Xac3988, Xac0921, Xac1320, Xac1379, Xac1681, Xac4130 e Xac2192). These sigma-anti-sigma pairs are all coded by neighboring genes. Interactions between the sigma factor σFliA (Xac1933) and anti-σFlgM (Xac1989) were also observed. Xac strains with gene knockouts for several sigma factors were produced. The characterization some these knockout strains point to the involvement of σ54Xac1969 in the biosynthesis of flagella, participation of σECFXac1682 in the ability to survive heat shock and involvement of σECFXac2191 in the response to iron deficiency.