Extra-Cytoplasmic Function (ECF) σ factors are a subset of σ factors many organisms use to transcribe specific genes in response to environmental cues. In the absence of an inducing signal, ECF σ factors are inhibited by an anti-σ factor that prevents the ECF σ factor from interacting with RNA polymerase. The ECF σ factor σV from Bacillus subtilis is activated in response to lysozyme stress. Lysozyme damages bacterial peptidoglycan by cleaving at the 1,4-ß-linkage between N-acetylmuramic acid and N-acetylglucosamine. In the absence of lysozyme, the activity of σV is inhibited by the anti-σ factor RsiV, a single pass transmembrane protein. The two main components of this project have been to elucidate the mechanism of σV activation and examine how this system senses lysozyme stress. In chapter 2 we show that the activation of σV is specific to lysozyme, and the anti-σ factor RsiV is degraded by a step wise proteolytic cascade known as Regulated Intramembrane Proteolysis (RIP). In the presence of lysozyme, the extracellular domain of RsiV is removed by cleavage at site-1. Upon removal of the extracellular domain, the site-2 protease, RasP, cleaves RsiV within the membrane. The remainder of RsiV is degraded by cytosolic proteases allowing σV to interact with RNA polymerase. In response to lysozyme stress σV activates an O-actyltransferase, OatA, which modifies the peptidoglycan to prevent further lysozyme damage. Our studies in chapter 3 identifed the protease(s) responsible for site-1 cleavage of RsiV and revealed RsiV directly interacts with lysozyme. We determined the cleavage site of the site-1 protease using N-terminal sequencing, and demonstrate that disruption of site-1 cleavage blocks σV activation. Site-1 cleavage occurs at what appears to be a signal peptide cleavage site. We demonstrate that four out of the five signal peptidases from B. subtilis are able to cleave RsiV at site-1 in vitro only in the presence of lysozyme. Additionally, we show that the extracellular domain of RsiV directly binds the inducing substrate lysozyme. In chapter 4 we focus on determining if the interaction between RsiV and lysozyme is necessary for σV activation. Based on the co-crystal structure of RsiV and lysozyme we mutated sveral residues predicted to be involved in binding. One combination of RsiV mutations (S169W, P259A, Y261A) was unable to activate σV and subsequently was unable to bind lysozyme. We propose a RIP dependent mechanism of σV activation that is contingent upon the anti-σ factor (RsiV) directly binding the inducing signal (lysozyme) to present the site-1 cleavage site to signal peptidase. The co-crystal structure of RsiV and lysozyme also revealed that RsiV interacts with the active site of lysozyme. We demonstrate that purified RsiV inhibits lysozyme activity suggesting RsiV provides an additional lysozyme response mechanism. Thus, the anti-σ factor RsiV senses the presence of lysozyme, activates σV, and protects against further lysozyme damage.

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.

Clostridium perfringens is a leading cause of food poisoning annually in the United States. Ingested C. perfringens vegetative cells respond to the acidic conditions of the stomach by initiating sporulation. The process of sporulation is essential in the formation of an enterotoxin (CPE) that is responsible for the symptoms of acute food poisoning. During sporulation, the cell must differentiate into the mother cell and the forespore. Studies in Bacillus subtilis have shown that gene expression during sporulation is compartmentalized, with different genes expressed in the mother cell and the forespore. The cell-specific RNA polymerase sigma factors coordinate the development of the differentiating cell. These sigma factors are Ï F, Ï E, Ï G, and Ï K. The C. perfringens cpe gene, encoding the enterotoxin CPE, is transcribed from three promoters, P1, P2, and P3. P2 and P3 were previously proposed to be Ï E-dependent, and P1 was proposed to be Ï K-dependent based on consensus recognition sequences. In this study, mutations were introduced into the sigE and sigK genes of C. perfringens. In the sigE and sigK mutants, promoter fusion assays indicated that there was no transcription of cpe in either mutant. We also determined through transcriptional analyses that Ï E-associated RNA polymerase and Ï K-associated RNA polymerase co-regulate the transcription of each other. RT-PCR analyses indicated that Ï K is a very early acting sigma factor. The evidence provided here shows that the regulation of sporulation in C. perfringens is not the same as it is in B. subtilis, as previously proposed. / Master of Science

Mycobacterium tuberculosis has one ribosomal RNA operon. The survival of this bacillus thus depends on a transcription mechanism that can effectively couple gene expression to changes in the environment. σ factors are transcription proteins that bind to the RNA polymerase (RNAP) and dictate gene expression. Extra Cytoplasmic Function σ factors (ECF) are a subset of σ factors that coordinate environment-induced changes in transcription. The environment specific binding of ECF σ factors to the RNAP presents an effective mechanism for the bacillus to modulate gene expression. ECF σ factors, in turn, are regulated by their interaction with an anti-σ factor. The active σ factor is released from this complex upon specific cellular or environmental stimuli. The aim of this study was to understand the structural and mechanistic aspects of σ factor activation. Towards this goal, two ECF σ factors, σC and σL, were examined. Structural and biophysical studies on M. tuberculosis σC provided a novel insight into ECF σ factor regulation. Inter-domain interactions in σC were sufficient to occlude the DNA recognition regions even in the absence of an interacting protein. The structure of M. tuberculosis σL in complex with the anti-σ factor RslA provides a structural basis to rationalize the release of active σL under oxidative stress. The other chapters of this thesis include a description of the structure and biochemical features of a hypothetical protein Rv2704 that is co-transcribed with the primary σ factor σA. In an effort to understand the collaboration-competition-redundancy model of prokaryotic σ factors, we performed a computational analysis of this system compiling experimental data from the E. coli and B. subtilis model systems. These results are also presented in this thesis. Put together, the structural and biochemical characteristics of the σ factors presented in this thesis suggest substantial variations in the regulatory mechanisms of the M. tuberculosis σ factors when compared to the canonical E. coli or B. subtilis model systems. This thesis is organized as follows: Chapter 1: The introductory chapter of this thesis is organized to frame the pertinent mechanistic issues involved in the σ factor-regulatory protein interactions in the context of the underlying biology of M. tuberculosis. The first part of this chapter provides an overview of σ factors and a summary of the classification of these proteins and their roles in different prokaryotes. The latter part of this chapter is a summary of the pathogen M. tuberculosis in terms of its genetic composition, gene expression as well as aspects of virulence and pathogenecity. Chapter 2: This chapter describes the characterization of the ECF σ factor, σC. Here we report the structure of an ECF σ factor σC from M. tuberculosis. σC is essential for the lethality of M. tuberculosis in a mouse model of infection. Our studies suggest that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σC2 and σC4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σC2 and C4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of E. coli σA despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σC2 as well as surface plasmon resonance measurements reveal that the σC2 and σC4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σC2 is involved in this inter-domain interaction. Interactions between the promoter recognition domains in M. tuberculosis σC are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor. Chapter 3 provides an account of the regulatory features of the ECF σ factor, σL. ECF σ factors are often regulated by their interactions with an anti-σ factor that can sense diverse environmental stimuli. Transcriptional responses to changes in the oxidation state are particularly important for M. tuberculosis as it adapts to the environment of the host alveoli and macrophages. Here we demonstrate that the protein RslA binds Zinc and can sequester σL in a reducing environment. Our data suggests that the cytosolic domain at the N-terminus of RslA alone is involved in binding σL. Under oxidizing conditions, the σL/RslA complex undergoes substantial conformational rearrangements that coincide with the release of the Zinc cofactor. In the absence of Zinc, the affinity of RslA for σL reduces by ca 8 fold compared to the holo form. The CXXC motif of RslA acts as a redox sensor. In response to oxidative stimuli, the proximal cysteines in this motif can form a disulfide bond with the release of the bound Zn2+ ion. This observation could be rationalized based on the crystal structure of the σL4/RslA complex. Put together, RslA is a distinct variant of the Zinc binding anti-σ factor (ZAS) family. The structural and biophysical parameters that control σL/RslA interactions demonstrate how variations in the rate of Zinc release and associated conformational changes in RslA could regulate the release of free σL in a measured response to oxidative stress. Chapter 4 is based on the biochemical and structural characterization of a hypothetical protein Rv2704. The gene for M. tuberculosis Rv2704 is located in the same operon as the principal σ factor σA. The biochemical and structural features of Rv2704 were thus examined to identify its role, if any, in the regulation of σA. This protein is a trimer in solution and adopts a chorismate mutase-like fold. The crystal structure reveals that Rv2704 is a member of the functionally diverse YjgF family of proteins. The important structural differences between Rv2704 and other YjgF proteins lie in the arrangement of secondary structural elements and the putative functional clefts between the subunit interface. Although Rv2704 does not interact with σA in vitro, the structural similarities to the YjgF family suggests that this protein could interact with a variety of metabolites, potentially influencing its function. Chapter 5 of this thesis is based on a computational analysis of σ factors. Four conformational segments of σ factors, referred to as σ1, σ2, σ3 and σ4 interact with specific regions of promoter DNA. ECF σ factors are a subset of σ factors that coordinate environment-induced transcription. ECF σ factors are minimalist σ factors with two DNA binding domains viz., σ2 and σ4 that recognize the –10 and –35 promoter elements and are unable to interact with either upstream-activating regions or the extended –10 element of the promoter. There are several ECF σ factors in a typical bacterium often characterized by substantial overlap in function. Here we present an analysis of B. subtilis ECF σ factors and their cognate promoters to understand functional overlap and redundancy in this class of proteins. As expected, conserved bases in the –10 element appear more critical for promoter selectivity than the –35 element. However, we note distinct conformational features in the –35 promoter interaction with the helix-turn-helix (HTH) motif when compared to a data-set of known HTH-DNA complexes. Furthermore, we note differences in –35 element interaction between σ factors that act alone and those that overlap in function. The σ factor promoter interactions were then examined vis-à-vis the estimated cellular concentration of these proteins and their affinity to bind the core RNAP. Put together, this analysis suggests that while the cellular protein concentration dictates the choice of an ECF σ factor to form a complex with the RNAP, conformational features of the –35 element serve to select potential collaborative members, a subset of which eventually initiate transcription. Collaborative arrangements and functional redundancy in ECF σ factors are thus possible within the limits placed by these two parameters. Chapter 6 is a summary of the work reported in this thesis and the conclusions that can be drawn based on these studies. The appendix section of this thesis comprises of technical details that were not included in the main text of this thesis. Appendix I describes the initial characterization of the M. tuberculosis σD/anti-σD complex. Appendix II provides the experimental protocols as well as some of the supplementary data to the work reported in Chapters 2-5 of this thesis.

Periodontal disease affects a majority of the US population. Porphyromonas gingivalis is the major etiological agent for development and progression of the disease. P. gingivalis is a hemin-dependent, obligate anaerobe that is found predominantly in periodontal pockets in infected patients. So for, little is known regarding the mechanisms which allow P. gingivalis to survive and sustain itself in the oral cavity. To better understand the adaptive mechanisms of the bacterium to the varying conditions in the oral cavity, regulatory mechanisms must be characterized. Sigma factors (σ) are responsible for initiating transcription by guiding RNA polymerase binding to specific DNA promoter sites. There are several sigma factors present in P. gingivalis, yet their roles have to be identified. Previous unpublished data indicate that a gene coding for an extracytoplasmic function sigma factor (ECF), SigH, is differentially regulated by exposure to molecular oxygen. Different assays were conducted to assess any variation in survival and/or growth between wild-type and SigH deficient strains of P. gingivalis. The ability to grow and survive in the presence of oxidative stress was compared between the mutant deficient in SigH and that of the parental strain. In addition, transcriptional profiles of the two strains were compared. Our assays indicate that growth was slower in the presence of oxygen in the Sigh-deficient mutant with an average difference of 27% compared to the wild-type. Transcriptional profiling showed down-regulation of genes encoding key enzymes associated with oxidative stress protection and oxidative metabolism in the absence of SigH, indicating that the sigma factor is a positive regulator of transcription required for survival of the bacterium in the presence of oxygen. If oxygen sensitivity can be established for this ECF-σ factor, it will aid in better understanding of P. gingivalis’ ability to survive in the oral cavity despite the presence of oxygen.

A proteobactéria Pseudomonas aeruginosa é um patógeno oportunista em humanos, sendo associado a queimaduras e infecções pulmonares crônicas em pacientes com fibrose cística. Essas infecções são difíceis de erradicar devido à resistência intrínseca de P. aeruginosa a antibióticos e à formação de biofilmes. Essa bactéria é altamente capaz de adaptar ao ambiente, tem um metabolismo versátil e pode direcionar a expressão de genes por vários fatores sigma alternativos. Estes são subunidades para transcrição de conjuntos específicos de genes em bactérias e interagem com o cerne da RNA polimerase, levando ao reconhecimento do promotor e início da transcrição. Os fatores sigma alternativos permitem que bactérias redirecionem a sua expressão genética. Um grupo de fatores sigma alternativos é o grupo dos fatores sigma de função extracitoplasmática (ECF) que são envolvidos principalmente em funções do envelope celular. Esse trabalho teve como objetivo caracterizar dois fatores sigma ECF de função desconhecida, PA14_21550 e PA14_46810. A linhagem mutante Δ21550 foi analisada quanto a sua sobrevivência a diferentes estresses, observando-se que é mais resistente ao choque de 45°C que a linhagem selvagem. Esse fator sigma não é essencial para crescimento da bactéria em meio LB e meio mínimo M63 acrescido de glicose ou succinato. Além disso, observou-se que a superexpressão desse fator sigma aumenta a expressão da proteína hipotética PA14_30100, usando-se uma abordagem proteômica. O mutante de transposon para o fator sigma PA14_46810 apresenta melhor crescimento que a bactéria selvagem em meio M63 acrescido de glicose. Essa linhagem mostrou mesmo fenótipo para biofilme e formação de exopolissacarídeo que a bactéria selvagem. Ademais, foi realizada análise de transcritoma por RNA-Seq com a superexpressão do fator sigma PA14_46810 na linhagem selvagem. Na linhagem de superexpressão Observou-se que ocorre indução de genes envolvidos com a desnitrificação, transporte de moléculas e metabolismo de uma maneira geral, em relação à linhagem controle. Por outro lado, o excesso de PA14_46810 reprime principalmente genes envolvidos com a tradução de proteínas e síntese de espermidina. Este trabalho, portanto, trouxe novas informações sobre as funções de diferentes fatores sigma ECF de P. aeruginosa, contribuindo assim para um maior entendimento da fisiologia desta bactéria e sua adaptação a diferentes condições. / The proteobacterium Pseudomonas aeruginosa is an opportunistic pathogen in humans, and it is associated to chronic pulmonary infections in patients with cystic fibrosis and burn wounds. These infections are difficult to eradicate due to P. aeruginosa intrinsic resistance to antibiotics and formation of biofilms, which allow the bacteria to adhere to biotic and abiotic surfaces. This bacterium is highly adaptaptable to the environment has a versatile metabolism and can direct the expression of genes by several alternative sigma factors. The sigma factors bind to the RNA polymerase core, providing recognition to promoter and transcription initiation. Therefore, the alternative sigma factors can redirect bacterial genetic expression by recognizing specific promoters. One subfamily of alternative sigma factors is the extracytoplasmic function (ECF) sigma factors, involved mostly in cell envelope functions. The aim of this work was characterize two ECF sigma factors with unknown function in P. aeruginosa, PA14_21550 and PA14_46810. The strain Δ21550 was analyzed for its survival in different stress conditions and it is more resistant in heat shock conditions at 45°C than the wild type strain. It was also observed that PA14_21550 sigma factor is not essential for bacterial growth in LB and M63 minimal medium added with glucose or succinate as the carbon source. Furthermore, overexpression of this sigma factor increases the expression of hypothetical protein PA14_30100, as verified by a proteomic approach. A strain insertionally inactivated in the PA14_46810 gene has better growth than the wild type strain in M63 added with glucose and the same phenotype regarding to biofilm formation and exopolysaccharide production as the wild type strain. Moreover, transcriptome analysis was carried out by RNA-Seq with overexpression of the PA14_46810 sigma factor in the wild type strain. Induction of genes involved in denitrification, transport of molecules and energetic metabolism in relation to the control strain was observed. On the other hand, excess of PA14_46810 represses genes involved in protein translation and spermidine synthesis. This work, therefore, brought new information about the functions of two ECF sigma of P. aeruginosa, thus contributing to a greater understanding of the physiology of this bacterium and its adaptation to different conditions.

Many multicellular cyanobacteria produce specialized nitrogen-fixing heterocysts. During diazotrophic growth of the model organism Anabaena (Nostoc) sp. strain PCC 7120, a regulated developmental pattern of single heterocysts separated by about 10 to 20 photosynthetic vegetative cells is maintained along filaments. Heterocyst structure and metabolic activity function to accommodate the oxygen-sensitive process of nitrogen fixation. This dissertation focuses on my research on heterocyst development, including morphogenesis, transport of molecules between cells in a filament, differential gene expression, and pattern formation. We using microarray experiments we found that conR (all0187) gene is necessary for normal septum-formation of vegetative cells, diazotrophic grow, and heterocyst morphogenesis. In our studies we characterized the expression of sigma factors genes in Anabaena PCC 7120 during heterocyst differentiation, and we found that the expression of sigC, sigG and sigE is localized primarily in heterocysts. Expression studies using sigE mutant showed that nifH is under the control of this specific sigma factor.

Pseudomonas syringae pv. syringae B728a, an aggressive bacterial pathogen of bean, utilizes large surface populations and extracellular signaling to initiate a fundamental change from an epiphytic to a pathogenic lifestyle. Extracytoplasmic function (ECF) sigma (σ) factors serve as important regulatory factors in responding to various environmental signals. Bioinformatic analysis of the B728a genome has revealed 10 ECF sigma factors, five of which have high levels of sequence similarity to the FecI-type of ECF sigma factors and play a known role in the regulation of various iron transport systems. Because iron is essential for the induction of major virulence factors in B728a, I hypothesized that these FecI-type sigma factors may play a critical role in the bacterium’s transition between lifestyles. Deletion mutants of two FecI-type sigma factors, Psyr_1040 and Psyr_1107, in B728a have been created using homologous recombination based on the phage λ Red recombinase method. This study shows that the B728a FecI-type sigma factors, Psyr_1040 and Psyr_1107 are affected by conditions of iron stress, and influence the expression of putative outer membrane receptors and transmembrane sensors associated with these genes. Moreover, Psyr_1107 contributes to the expression of a cluster of predicted pili assembly genes downstream of it. Mutations in Psyr_1040 and Psyr_1107 affect the population levels of B728a in bean plants, since in planta growth of deletion mutants of B728a lacking Psyr_1040 and Psyr_1107 appears to be slower than wild-type B728a. In this thesis, the possible roles of Psyr_1040 and Psyr_1107 in the adaptation of B728a to a pathogenic lifestyle are addressed using a combination of phenotypic characterization and quantitative real-time PCR (qRT-PCR) analyses.