Academic literature on the topic 'Σ/anti–σ factor Interactions'

The host–pathogen interactions inMycobacterium tuberculosisinfection are significantly influenced by redox stimuli and alterations in the levels of secreted antigens. The extracytoplasmic function (ECF) σ factor σKgoverns the transcription of the serodominant antigens MPT70 and MPT83. The cellular levels of σKare regulated by the membrane-associated anti-σK(RskA) that localizes σKin an inactive complex. The crystal structure ofM. tuberculosisσKin complex with the cytosolic domain of RskA (RskAcyto) revealed a disulfide bridge in the −35 promoter-interaction region of σK. Biochemical experiments reveal that the redox potential of the disulfide-forming cysteines in σKis consistent with its role as a sensor. The disulfide bond in σKinfluences the stability of the σK–RskAcytocomplex but does not interfere with σK–promoter DNA interactions. It is noted that these disulfide-forming cysteines are conserved across homologues, suggesting that this could be a general mechanism for redox-sensitive transcription regulation.

σ factors are transcriptional regulatory proteins that bind to the RNA polymerase and dictate gene expression. The extracytoplasmic function (ECF) σ factors govern the environment dependent regulation of transcription. ECF σ factors have two domains σ2 and σ4 that recognize the -10 and -35 promoter elements. However, unlike the primary σ factor σA, the ECF σ factors lack σ3, a region that helps in the recognition of the extended -10 element and σ1.1, a domain involved in the autoinhibition of σA in the absence of core RNA polymerase. Mycobacterium tuberculosis σC is an ECF σ factor that is essential for the pathogenesis and virulence of M. tuberculosis in the mouse and guinea pig models of infection. However, unlike other ECF σ factors, σC does not appear to have a regulatory anti-σ factor located in the same operon. We also note 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 σC4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of 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 interdomain interaction. Interaction 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.

In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ-factors are negatively regulated by anti–σ-factors. In Escherichia coli, Salmonella enterica, and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σS-regulon by promoting the association of σS with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σS-RNAP in an open promoter complex with a σS-regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σS (σS2), the structure, along with p-benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP β′-subunit that we call the β′-clamp-toe (β′CT). Deletion of the β′CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β′CT interaction. We conclude that Crl activates σS-dependent transcription in part through stabilizing σS-RNAP by tethering σS2 and the β′CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.

AbstractThe bacterial RNA polymerase (RNAP) holoenzyme is a multisubunit core enzyme associated with a σ factor that is required for promoter-specific transcription initiation. Besides a primary σ responsible for most of the gene expression during active growth, bacteria contain alternative σ factors that control adaptive responses. A recurring strategy in the control of σ factor activity is their sequestration by anti-sigma factors that occlude the RNAP binding determinants, reducing their activity. In contrast, the unconventional transcription factor Crl binds specifically to the alternative σ factor σS/RpoS, and favors its association with the core RNAP, thereby increasing its activity. σS is the master regulator of the general stress response that protects many Gram-negative bacteria from several harmful environmental conditions. It is also required for biofilm formation and virulence of Salmonella enterica serovar Typhimurium. In this report, we discuss current knowledge on the regulation and function of Crl in Salmonella and Escherichia coli, two bacterial species in which Crl has been studied. We review recent advances in the structural characterization of the Crl-σS interaction that have led to a better understanding of this unusual mechanism of σ regulation.

YlaD, a membrane-anchored anti-sigma (σ) factor of Bacillus subtilis, contains a HX3CXXC motif that functions as a redox-sensing domain and belongs to one of the zinc (Zn)-co-ordinated anti-σ factor families. Despite previously showing that the YlaC transcription is controlled by YlaD, experimental evidence of how the YlaC–YlaD interaction is affected by active cysteines and/or metal ions is lacking. Here, we showed that the Pyla promoter is autoregulated solely by YlaC. Moreover, reduced YlaD contained Zn and iron, while oxidized YlaD did not. Cysteine substitution in YlaD led to changes in its secondary structure; Cys3 had important structural functions in YlaD, and its mutation caused dissociation from YlaC, indicating the essential requirement of a HX3CXXC motif for regulating interactions of YlaC with YlaD. Analyses of the far-UV CD spectrum and metal content revealed that the addition of Mn ions to Zn–YlaD changed its secondary structure and that iron was substituted for manganese (Mn). The ylaC gene expression using βGlu activity from Pyla:gusA was observed at the late-exponential and early-stationary phase, and the ylaC-overexpressing mutant constitutively expressed gene transcripts of clpP and sigH, an important alternative σ factor regulated by ClpXP. Collectively, our data demonstrated that YlaD senses redox changes and elicits increase in Mn ion concentrations and that, in turn, YlaD-mediated transcriptional activity of YlaC regulates sporulation initiation under oxidative stress and Mn-substituted conditions by regulating clpP gene transcripts. This is the first report of the involvement of oxidative stress-responsive B. subtilis extracytoplasmic function σ factors during sporulation via a Mn-dependent redox-sensing molecular switch.

The alternative sigma (σ) factor E, RpoE or HrpL, has been reported to be involved in stress- and pathogenicity-related transcription initiation in Escherichia coli and many other Gram-negative bacteria, including Erwinia spp. and Pseudomonas spp. A previous study identified the hrpL/rpoE transcript as one of the significant differentially expressed genes (DEGs) during early E. mallotivora infection in papaya and those data serve as the basis of the current project. Here, the full coding DNA sequence (CDS) of hrpL from E. mallotivora (EmhrpL) was determined to be 549 bp long, and it encoded a 21.3 kDa HrpL protein that possessed two highly conserved sigma-70 (σ70) motifs—σR2 and σR4. Nucleotide sequence alignment revealed the hrpL from E. mallotivora shared high sequence similarity to rpoE/hrpL from E. tracheiphila (83%), E. pyrifoliae (81%), and E. tasmaniensis (80%). Phylogenetics analysis indicated hrpL from E. mallotivora to be monophyletic with rpoEs/hrpLs from Pantoea vagans, E. herbicola, and E. tracheiphila. Structural analysis postulated that the E. mallotivora’s alternative σ factor was non-transmembranic and was an extracytoplasmic function (ECF) protein—characteristics shared by other σ factors in different bacterial species. Notably, the protein–protein interaction (PPI) study through molecular docking suggested the σ factor could be possibly inhibited by an anti-σ. Finally, a knockout of hrpL in E. mallotivora (ΔEmhrpL) resulted in avirulence in four-month-old papaya plants. These findings have revealed that the hrpL is a necessary element in E. mallotivora pathogenicity and also predicted that the gene can be inhibited by an anti-σ.

ABSTRACT FpvR is a presumed cytoplasmic membrane-associated anti-sigma factor that controls the activities of extracytoplasmic function sigma factors PvdS and FpvI responsible for transcription of pyoverdine biosynthetic genes and the ferric pyoverdine receptor gene, fpvA, respectively. Using deletion analysis and an in vivo bacterial two-hybrid system, FpvR interaction with these σ factors was confirmed and shown to involve the cytoplasmic N-terminal 67 amino acid resides of FpvR. FpvR bound specifically to a C-terminal region of FpvI corresponding to region 4 of the σ70 family of sigma factors. FpvR and FpvI mutant proteins compromised for this interaction were generated by random and site-directed PCR mutagenesis and invariably contained secondary structure-altering proline substitution in predicted α-helices within the FpvR N terminus or FpvI region 4. PvdS was shown to bind to the same N-terminal region of FpvR, and FpvR mutations compromising FpvI binding also compromised PvdS binding, although some mutations had a markedly greater impact on PvdS binding. Apparently, these two σ factors bind to FpvR in a substantially similar but not identical fashion. Intriguingly, defects in FpvR binding correlated with a substantial drop in yields of the FpvI and to a lesser extent PvdS σ factors, suggesting that FpvR-bound FpvI and PvdS are stable while free and active sigma factor is prone to turnover.

Response to heat stress (HSR) is a key stress response for endurance in Escherichia coli mediated by transcriptional factor σ-32. Even though there has been extensive investigation on the contribution of proteins and chaperones in retaining protein stability in cells under stress conditions, limited information is available regarding the dynamic nature of mechanisms regulating the activity of the highly conserved heat shock proteins (Hsps). Several gene expression-based studies suggest the pivotal role of Hsp70 (DnaK) in the regulation of the expression of heat shock genes (Hsg). Direct interaction of Hsp70 with σ-32 may regulate this function in E. coli. Recent studies revealed that localization of σ-32 to the membrane interior by SRP-dependent pathway enables them to function appropriately in their role as regulators. The contributions of different cellular components including cell membrane remain unknown. Other cellular components or σ-32 interfere with polypeptides which could play a crucial role in cell survival. Sigma factor monitors and preserves outer membrane integrity of E. coli by stimulating the genes regulating outer membrane proteins (OMPs) and lipopolysaccharide (LPS) assemblage as well as through expression of small RNAs to down-regulate surplus unassembled OMPs. σ-E activity is regulated by the rate at which its membrane-encompassing anti-sigma factor, RseA is degraded. Mutations in rseA are reported to constitutively increase the sigma (E) activity that is validated at both genetic and biochemical levels. In this review, the basic mechanism of heat stress regulation in gram-negative bacteria has been elaborated using E. coli as a model organism.

Extra Cytoplasmic Function (ECF) σ factors are a diverse family of alternative σ factors that allow bacteria to sense and respond to changes in the environment. σV is an ECF σ factor found primarily in low GC Gram-positive bacteria and is required for lysozyme resistance in several opportunistic pathogens. In the absence of lysozyme, σV is inhibited by the anti-σ factor RsiV. In response to lysozyme, RsiV is degraded via the process of Regulated Intramembrane Proteolysis (RIP). RIP is initiated by cleavage of RsiV at site-1 which allows the intramembrane protease RasP to cleave RsiV within the transmembrane domain at site-2 and leads to activation of σV. Previous work suggested that RsiV is cleaved by signal peptidase at site-1. Here we demonstrate in vitro that signal peptidase is sufficient for cleavage of RsiV only in the presence of lysozyme and provide evidence that multiple Bacillus subtilis signal peptidases can cleave RsiV in vitro. This cleavage is dependent upon the concentration of lysozyme consistent with previous work that showed binding to RsiV was required for σV activation. We also show that signal peptidase activity is required for site-1 cleavage of RsiV in vivo. Thus, we demonstrate that signal peptidase is the site-1 protease for RsiV.

Adaptation to external environmental conditions is essential for the survival of a bacterial cell. Bacteria have thus evolved multiple mechanisms to sense environmental stimuli and to couple this information into an appropriate cellular response. The cellular response, in its simplest sense involves a change in the cellular content which is achieved by regulating gene expression. Gene expression in bacteria is primarily regulated at the first step, also referred to as transcription initiation. Extensive studies on this mechanism over the past two decades provide a molecular picture of this process. The main enzyme in this process is the DNA dependent RNA polymerase (RNAP). The RNAP enzyme however lacks a specificity factor that dictates which genes are to be selectively expressed. Selectivity is enforced by a dissociable subunit, the sigma (σ) factor. σ factors have specificity determinants that allow recognition of promoter sequences in the DNA. The sequence features on DNA include the –10 elements (Pribnow box), –35 elements, the extended -10 region, the spacing between –10 and –35 elements and the upstream sequence to –35 promoter element. σ factors recognize a few or several of these promoter sequence features thereby providing an efficient mechanism for RNAP recruitment at a specific promoter. σ factors substantially differ in sequence and structural elements. The principal σ factor of the σ70 family has several domains. This includes specific domains that interact with –10 and –35 promoter elements and the sequence between these two promoter elements. The N-terminal domain of σ factors of the σ70 family also encodes regions enabling auto-regulation of this initiation factor. A defining feature that distinguishes σ70 members from other σ factors (σ38 and σ54) is that σ70 does not require ATP for its activity. The number of σ factors in a bacterial cell varies across species. For example Streptococcus pneumonia has one σ factor, Lactococcus lactis has two, Haemophilus influenza has four while Mycobacterium tuberculosis has thirteen σ factors. The exceptions are Streptomyces coelicolor with 65and Sorangium cellulosum with 109 σ factors. The number of σ factors in a bacterial cell is suggested to be correlated with the diverse environmental conditions encountered by the bacterium and the genomic size. The focus of work reported in this thesis is on M. tuberculosis σ factors. There are large variations in the number of σ factors in different mycobacterial species. While M. leprae has two, M. tuberculosis has thirteen σ factors. This variation in the number of σ factors has widely been believed to aid rapid signal transduction of environmental stimuli into changes in gene expression. While other transcription factors enable the recruitment of RNAP to specific promoter sequences, repressors that abrogate transcription and effectors that modulate transcription also dictate transcription levels. The intra cellular levels of a σ factor is often the primary determinant of the expression profile. A specific group of σ factors referred to as Extra Cytoplasmic Function (ECF) σ factors, govern the cellular response to specific environmental stimuli. The ECF family of σ factors has been shown to be regulated by diverse mechanisms. These include transcriptional, translational and post translational control by protein – protein interactions. The focus of this thesis was to understand the regulatory mechanism that involves σ factor interacting proteins. ECF σ factors that are governed by protein – protein interactions are often co-expressed with a regulator protein, the anti-σ factor. In several cases, they are a part of the same operon. This ensures similarity in the expression levels of σ factors and their cognate anti-σ factors. The selective dissociation of an inactive σ factor from a σ/anti-σ complex is also effected by diverse mechanisms. These include structural changes in response to environmental stimuli, conformational changes brought about by the binding of metabolites or by targeted proteolysis of anti-σ factors. The cellular concentration of an activated σ factor is thus often subject to the rate at which it is released from an inactive complex. The notion of specific σ/anti σ factor pairs has recently been challenged with a suggestion that a σ factor could also make non-specific interactions with other anti-σ factors. This finding has implications for the widely accepted model for bacterial gene expression that relies on a cellular estimate of free active σ factors. In this model, referred to as the partitioning of σ factor space model of bacterial transcription, σ factors compete for a limited pool of apo-RNAP and recruit the enzyme to target promoter elements. Two aspects of the mechanism that governs σ factor activity were explored in the course of the studies reported in this thesis. The first is recognition of a particular stress by structurally similar anti-σ factors. This is described in the second chapter of this thesis. The relative sensitivity of redox sensors plays an important role in providing a calibrated response to environmental stimuli and cellular homeostasis. This cellular machinery plays a crucial role in the human pathogen M. tuberculosis as it encounters diverse microenvironments in the host. The redox sensory mechanism in M. tuberculosis is governed by two component and one component systems, alongside ECF σ factors. ECF σ factors that govern the cellular response to redox stimuli are negatively regulated by forming a complex with proteins called zinc associated anti – σ factors (ZAS). ZAS proteins release their cognate σ factor in response to oxidative stress. The relative sensitivity of the ZAS sensors to redox processes dictates the concentration of free ECF σ factors in the cell. However, factors governing the redox threshold of these sensors remain unclear. The molecular characterization of three σ factor/ZAS pairs - σL/RslA, σE/RseA and σH/RshA using a combination of biochemical, biophysical and electrochemical techniques revealed the differences in redox sensitivity in these proteins despite apparent structural similarity. This finding can potentially rationalize the hierarchy in the activation of the cognate ECF σ factors under oxidative stress. Put together, the study described in chapter 2 provides a basis for examining sequence and conformational features that modulate redox sensitivity within the confinement of a conserved structural scaffold. The other aspect that was examined in the course of this study, described in chapter 3 of this thesis, was on crosstalk between σ/anti σ factors. In this study, we evaluated the affinity and specificity for σ/anti-σ factor interaction. We then analyzed the conformational determinants that enforce fidelity in σ/anti-σ interactions. The experimental data that we obtained on interactions between cognate and non-cognate σ/anti-σ pairs provide a template to evaluate tolerance between specific and non-specific interactions. The results from this analysis suggest non-cognate interactions are feasible. These interactions are likely to govern the extent to which different σ factors are activated in response to a particular environmental stimulus. It appears likely that this mechanism could provide a route to bacterial survival, perhaps allowing phenotypic diversity that help circumvent an environmental insult. Another aspect we addressed in the course of this work was the evaluation of specific targeting of a prokaryotic transcription initiation factor. Peptide ligands of a M. tuberculosis σ factor, targeting the RNAP-σ interface were evaluated biochemically and biophysically. A sixteen residue long helical peptide from the core RNAP that could interact with σ4 region and suitably designed control peptides are validated in vitro. Consistent findings from in silico and in vitro observations validated the design strategy that can also be extended across σ factors. This study suggests that designed peptide binders can be explored as chemical tools for studying the regulation of transcription. The fifth chapter of this thesis provides a summary of the findings from the three research themes described in this thesis. This thesis has three appendices. The first two report projects that were discontinued as they were not feasible from the perspective of structural characterization. While Appendix I reports preliminary studies on hemagglutinin-antibody complex, Appendix II describes structural studies on Escherichia coli toxin–anti toxin pairs to evaluate a conformational rationale for protein–protein interactions. Appendix III is a compilation of sequence and structural data that was used for the bioinformatics analysis presented in chapter 3. Put together the studies described in this thesis reveal exquisite adaptation strategies employed by M. tuberculosis to ensure survival under diverse micro-environments in the host.

A distinctive feature of host-pathogen interactions in the case of Mycobacterium tuberculosis is the asymptomatic latent phase of infection. The ability of the bacillus to survive for extended periods of time in the host suggests an adaptive mechanism in M. tuberculosis that can cope with a variety of environmental stresses and other host stimuli. Extensive genomic studies and analysis of knock-out phenotypes revealed elaborate cellular machinery in M. tuberculosis that ensures a rapid cellular response to host stimuli. Prominent amongst these are two-component systems and σ factors that exclusively govern transcription re-engineering in response to environmental stimuli. M. tuberculosis σK is a σ factor that was demonstrated to control the expression of secreted antigenic proteins. The study reported in this thesis was geared to understand the molecular basis for σK activity as well as to explore conditions that would regulate σK activity. Transcription in bacteria is driven by the RNA polymerase enzyme that can associate with multiple σ factors. σ factors confer promoter specificity and thus directly control the expression of genes. The association of different σ factors with the RNA polymerase is essential for the temporal and conditional re-engineering of the expression profile. Environment induced changes in expression rely on a subset of σ factors. This class of σ factors (also referred to as Class IV or Extra-cytoplasmic function (ECF) σ factors) is regulated by a variety of mechanisms. The regulation of an ECF σ factor activity at the transcriptional, translational or posttranslational steps ensures fidelity in the cellular concentration of free, active ECF σ factors. In general, ECF σ factors associate with an inhibitory protein referred to as an anti-σ factor. The release of a free, active σ factor from a σ /anti-σ complex is thus a mechanism that can potentially control the cellular levels of an active σ factor in the cell. M. tuberculosis σK is associated with a membrane bound anti-σK (also referred to as RskA) (Said-Salim et al., Molecular Microbiology 62: 1251-1263: 2006). The extracellular stimulus that is recognized by RskA remains unclear. However, recent studies have suggested the possibility of a regulated proteolytic cascade that can selectively degrade RskA and other membrane associated anti-σ factors. The goal of the study was to understand this regulatory mechanism with a specific focus on the M. tuberculosis σK/RskA complex. The structure of the cytosolic σK/RskA complex and the associated biochemical and biophysical characteristics revealed several features of this /anti-σ complex that were hitherto unclear. In particular, these studies revealed a redox sensitive regulatory mechanism in addition to a regulated proteolytic cascade. These features and an analysis of the M. tuberculosis σK/RskA complex vis-à-vis the other characterized σ/anti σfactor complexes are presented in this thesis. This thesis is organized as follows- Chapter 1 provides an overview of prokaryotic transcription. A brief description of the physiology of M. tuberculosis is presented along with a summary of characterized factors that contribute to the pathogenecity and virulence of this bacillus. The pertinent mechanistic issues of σ/anti-σ factor interactions are placed in the context of environment mediated changes in M. tuberculosis transcription. A summary of studies in this area provides a background of the research leading to this thesis. Chapters 2 and 3 of this thesis describe the structural and mechanistic studies on the σK/RskA complex. The crystal structure of the σK/RskA complex revealed a disulfide bond in domain 4 (σK4). σK4 interacts with the -35 element of the promoter DNA. The disulfide forming cysteines were seen to be conserved in more than 70% of σK homologs, across both gram-positive and gram-negative bacteria. The conservation of the disulfide-forming cysteines led us to further characterize the role of this disulfide in σK/RskA interactions. These were examined by several biochemical and biophysical experiments. The redox potential of these disulfide bond forming cysteine residues were consistent with the proposed role of a sensor. The crystal structure and biochemical studies thus suggest that M. tuberculosis σK is activated under reducing conditions. Chapter 4 of this thesis describes the progress made thus far in the structural and biochemical characterization of an intra-membrane protease, M. tuberculosis Rip1 (Rv2869c). This protein is an essential component of the proteolytic cascade that selectively cleaves RskA. The proteolytic steps that govern the selective degradation of an anti-σ factor were first characterized in the case of E. coli σE (Li, X. et al. Proc. Natl. Acad. Sci. USA, 106:14837-14842, 2009). This cascade is triggered by the concerted action of a secreted protease (also referred to as a site-1 protease) and a trans-membrane protease (also referred to as a site-2 protease). M. tuberculosis Rip1 was demonstrated to be bona-fide site 2 protease that acts on three anti-σ factors viz., RskA, RslA and RsmA (Sklar et al., Molecular Microbiology 77:605-617; 2010). To further characterize the role of Rip1 in the proteolytic cascade, this intra-membrane protease was cloned, expressed and purified for structural, biochemical and biophysical analysis. The preliminary data on this membrane protein is described in this chapter. The conclusions from the studies reported in this thesis and the scope for future work in this area is described in Chapter 5. Put together, the σK/RskA complex revealed facets of σ/anti-σ factor interactions that were hitherto unrecognized. The most prominent amongst these is the finding that an ECF σfactor can respond to multiple environmental stimuli. Furthermore, as seen in the case of the σK/RskA complex, the σ factor can itself serve as a receptor for redox stimuli. Although speculative, a hypothesis that needs further study is whether these features of the σK/RskA complex contribute to the variable efficacy of the M. bovis BCG vaccine. In this context it is worth noting that σK governs the expression of the prominent secreted antigens- MPT70 and MPT83. The studies reported in this thesis thus suggest several avenues for future research to understand mycobacterial diversity, immunogenicity and features of host-pathogen interactions. The appendix section is divided into two subparts- Appendix 1 of the thesis is a review on peptidase V. This is a chapter in The Handbook of Proteolytic enzymes (Elsevier Press, ISBN:9780123822192). Appendix 2 of the thesis includes technical details and an extended materials and methods section.

A distinctive feature of host-pathogen interactions in the case of Mycobacterium tuberculosis is the asymptomatic latent phase of infection. The ability of the bacillus to survive for extended periods of time in the host suggests an adaptive mechanism in M. tuberculosis that can cope with a variety of environmental stresses and other host stimuli. Extensive genomic studies and analysis of knock-out phenotypes revealed elaborate cellular machinery in M. tuberculosis that ensures a rapid cellular response to host stimuli. Prominent amongst these are two-component systems and σ factors that exclusively govern transcription re-engineering in response to environmental stimuli. M. tuberculosis σK is a σ factor that was demonstrated to control the expression of secreted antigenic proteins. The study reported in this thesis was geared to understand the molecular basis for σK activity as well as to explore conditions that would regulate σK activity. Transcription in bacteria is driven by the RNA polymerase enzyme that can associate with multiple σ factors. σ factors confer promoter specificity and thus directly control the expression of genes. The association of different σ factors with the RNA polymerase is essential for the temporal and conditional re-engineering of the expression profile. Environment induced changes in expression rely on a subset of σ factors. This class of σ factors (also referred to as Class IV or Extra-cytoplasmic function (ECF) σ factors) is regulated by a variety of mechanisms. The regulation of an ECF σ factor activity at the transcriptional, translational or posttranslational steps ensures fidelity in the cellular concentration of free, active ECF σ factors. In general, ECF σ factors associate with an inhibitory protein referred to as an anti-σ factor. The release of a free, active σ factor from a σ /anti-σ complex is thus a mechanism that can potentially control the cellular levels of an active σ factor in the cell. M. tuberculosis σK is associated with a membrane bound anti-σK (also referred to as RskA) (Said-Salim et al., Molecular Microbiology 62: 1251-1263: 2006). The extracellular stimulus that is recognized by RskA remains unclear. However, recent studies have suggested the possibility of a regulated proteolytic cascade that can selectively degrade RskA and other membrane associated anti-σ factors. The goal of the study was to understand this regulatory mechanism with a specific focus on the M. tuberculosis σK/RskA complex. The structure of the cytosolic σK/RskA complex and the associated biochemical and biophysical characteristics revealed several features of this /anti-σ complex that were hitherto unclear. In particular, these studies revealed a redox sensitive regulatory mechanism in addition to a regulated proteolytic cascade. These features and an analysis of the M. tuberculosis σK/RskA complex vis-à-vis the other characterized σ/anti σfactor complexes are presented in this thesis. This thesis is organized as follows- Chapter 1 provides an overview of prokaryotic transcription. A brief description of the physiology of M. tuberculosis is presented along with a summary of characterized factors that contribute to the pathogenecity and virulence of this bacillus. The pertinent mechanistic issues of σ/anti-σ factor interactions are placed in the context of environment mediated changes in M. tuberculosis transcription. A summary of studies in this area provides a background of the research leading to this thesis. Chapters 2 and 3 of this thesis describe the structural and mechanistic studies on the σK/RskA complex. The crystal structure of the σK/RskA complex revealed a disulfide bond in domain 4 (σK4). σK4 interacts with the -35 element of the promoter DNA. The disulfide forming cysteines were seen to be conserved in more than 70% of σK homologs, across both gram-positive and gram-negative bacteria. The conservation of the disulfide-forming cysteines led us to further characterize the role of this disulfide in σK/RskA interactions. These were examined by several biochemical and biophysical experiments. The redox potential of these disulfide bond forming cysteine residues were consistent with the proposed role of a sensor. The crystal structure and biochemical studies thus suggest that M. tuberculosis σK is activated under reducing conditions. Chapter 4 of this thesis describes the progress made thus far in the structural and biochemical characterization of an intra-membrane protease, M. tuberculosis Rip1 (Rv2869c). This protein is an essential component of the proteolytic cascade that selectively cleaves RskA. The proteolytic steps that govern the selective degradation of an anti-σ factor were first characterized in the case of E. coli σE (Li, X. et al. Proc. Natl. Acad. Sci. USA, 106:14837-14842, 2009). This cascade is triggered by the concerted action of a secreted protease (also referred to as a site-1 protease) and a trans-membrane protease (also referred to as a site-2 protease). M. tuberculosis Rip1 was demonstrated to be bona-fide site 2 protease that acts on three anti-σ factors viz., RskA, RslA and RsmA (Sklar et al., Molecular Microbiology 77:605-617; 2010). To further characterize the role of Rip1 in the proteolytic cascade, this intra-membrane protease was cloned, expressed and purified for structural, biochemical and biophysical analysis. The preliminary data on this membrane protein is described in this chapter. The conclusions from the studies reported in this thesis and the scope for future work in this area is described in Chapter 5. Put together, the σK/RskA complex revealed facets of σ/anti-σ factor interactions that were hitherto unrecognized. The most prominent amongst these is the finding that an ECF σfactor can respond to multiple environmental stimuli. Furthermore, as seen in the case of the σK/RskA complex, the σ factor can itself serve as a receptor for redox stimuli. Although speculative, a hypothesis that needs further study is whether these features of the σK/RskA complex contribute to the variable efficacy of the M. bovis BCG vaccine. In this context it is worth noting that σK governs the expression of the prominent secreted antigens- MPT70 and MPT83. The studies reported in this thesis thus suggest several avenues for future research to understand mycobacterial diversity, immunogenicity and features of host-pathogen interactions. The appendix section is divided into two subparts- Appendix 1 of the thesis is a review on peptidase V. This is a chapter in The Handbook of Proteolytic enzymes (Elsevier Press, ISBN:9780123822192). Appendix 2 of the thesis includes technical details and an extended materials and methods section.

Abstract This study experimentally investigates the statistics of wall-pressure fluctuations and their source inside attached cavitation under different cavity regimes. Experiments were conducted in the divergent section of a convergent-divergent channel at a constant Reynolds number of Re = 7.8 × 105 based on throat height, and different cavitation numbers σ = 1.18, 0.92, 0.82 and 0.78. Four high-frequency unsteady pressure transducers were flushed-mounted in the divergent section downstream the throat where cavitation develops to sample the unsteady pressure signals induced by cavity behaviors. Flow visualization and wall-pressure measurement in high frequency on the order of MHz were employed using a synchronizing sampling technique. Results are presented for sheet/cloud cavitating flows. Specifically, sheet cavitation with both inception shear layer and fully cavitated shear layer and cloud cavitation under re-entrant jet dominated shedding and shock wave dominated shedding are studied. Compared with re-entrant jet, the interactions between shock wave and cavity could induce pressure peaks with high magnitude within cavity, which will collapse the local vapor along its propagating path and reduce local void fraction. Furthermore, statistics analysis shows that within the cavity, wall-pressure fluctuations increase with the distance to cavity leading edge increase in the first half of cavity length, and the moments of the probability density distribution skewness and kurtosis factor decrease, indicating the asymmetry and intermittency of wall-pressure fluctuation signals decrease. In shock wave dominated cavity shedding condition, the skewness and kurtosis factor increase. These results can provide data to improve the accuracy of turbulence modeling in numerical simulation of turbulent cavitating flow.