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Myxococcus xanthus is a δ-proteobacterium that displays a complex, multicellular life cycle involving vegetative growth, coordinated motility, and a developmental program culminating in sporulation. M. xanthus utilizes two genetically distinct yet coordinated systems to regulate motility comprised of either Type IV pili (T4P) or focal adhesion complexes (FAC). Both motility systems are regulated, in part, by multiple chemosensory systems. During development, cells aggregate into foci and gradually lose motility to become quiescent fruiting bodies containing mature heat- and sonication-resistant myxospores. In this study, I have characterized a new chemosensory system, Che6, in M. xanthus for its role in motility and development. The Che6 chemosensory system is comprised by six homologs to known chemotaxis proteins, Mcp6, CheW6a, CheW6b, CheA6, CheR6, and CheB6 and includes two previously identified proteins, a potassium efflux channel (KefC) and a sensor kinase (SocD). Mutations in each of the corresponding genes were found to affect motility and to disrupt development. Notably, the motility defects were well defined in a background strain with an impaired T4P system. In this background, the che6 mutants were observed to either positively or negatively affect reversal frequencies, highlighting their role in either FAC or T4P-based motility systems. Developmental defects were also identified for the che6 mutants and affected the timing of aggregation and sporulation. Interestingly, the overproduction of the CheB6 methylesterase resulted in spore formation independent of fruiting body formation. Uncoupling of sporulation and fruiting body formation for this mutant was found to be independent of a key developmental signal known as the C-signal. Moreover, we have demonstrated for the first time the association of a potassium efflux channel (KefC) with a chemosensory system (Che6) in bacteria. In this study, we provide evidence demonstrating that KefC provides an input signal for Mcp6 which transduces a signal through CheA to the histidine kinase SocD which serves as an output to regulate both motility and development in M. xanthus.

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.

Staphylococcus aureus is a commensal organism capable of causing a diverse array of acute and chronic infections. Biofilm formation plays an important role in pathogenesis for many chronic infections by promoting antibiotic tolerance and immune evasion. Both protein and eDNA have previously been identified as major components of the biofilm matrix. Many recent studies have shown that DNA-binding proteins contribute to biofilm structure and stability in other organisms, and that these proteins represent potential targets for therapeutic development. However, little has been done to investigate the role of eDNA-binding proteins in S. aureus biofilms. To address this, we conducted a screen to identify potential eDNA-binding proteins in the S. aureus biofilm matrix. Secreted and cell surface-associated proteins displaying DNA-binding activity were detected using Southwestern blotting and were identified by mass spectrometry. From this screen, we selected Eap, IsaB, and SaeP as candidates for further study. To gain insight into the function of these proteins, in vitro biofilm formation of single and double mutants was assessed in several strain backgrounds. While no major change in overall biomass was observed in these mutants, we found that loss of both eap and isaB resulted a loss of surface-associated eDNA from the biofilm matrix. Furthermore, we observed that overexpression of SaeP resulted in a significant increase in both overall biofilm formation and an increase in surface-associated eDNA. This phenotype was still observed in both sae and nuc mutants at high levels of SaeP expression, suggesting that SaeP-based biofilm enhancement was not completely dependent on Sae-based regulation of the staphylococcal nuclease. The high pIs of these proteins suggest that the association with eDNA is primarily mediated through ionic interactions. These findings suggest that Eap, IsaB, and SaeP function as non-specific eDNA-binding proteins that are important for the maintenance of eDNA in the biofilm matrix. We suggest these proteins represent a new class of functionally redundant matrix proteins capable of significantly impacting biofilm matrix composition.

Francisella tularensis is a Gram-negative, facultative intracellular bacterium and the etiologic agent of the zoonosis tularemia. Inhalation of as few as 10 organisms can cause a severe pneumonic disease with a mortality rate reported to be around 30-60% in untreated cases. Due to its highly infectious nature, high mortality rate, and ease of aerosolization, the Centers for Disease Control considers F. tularensis a Tier 1 select agent and potential bioweapon. This organism is capable of producing a severe infection as it gains entry into a number of different host cell types and modulates numerous key innate immune responses. It is noteworthy that neutrophils contribute to tissue destruction and disease severity, as exemplified by studies demonstrating that blocking neutrophil recruitment into infected tissues leads to reduced bacterial load and an overall increase in host survival. Therefore, we hypothesized that neutrophil function is dysregulated in the context of tularemia, a result of modification of neutrophil antimicrobial mechanisms by F. tularensis. Previously, we demonstrated that F. tularensis prolongs human neutrophil lifespan by interfering with the intrinsic, extrinsic, and phagocytosis-induced apoptotic pathways. How this prolongation occurs is incompletely defined; however, our published data suggest secreted or extracellular factors function in this process. The major aims of the studies outline in this thesis were aimed at investigating the effectors produced by F. tularensis that function in the inhibition of human neutrophil apoptosis and the survival signaling within these immune cells that may lead to their prolonged lifespan. Moreover, we examined the related, less virulent strain, F. novicida, to determine the extent to which this organism shares the ability to modulate neutrophil apoptosis like F. tularensis. Herein, we report that F. tularensis activates multiple survival signaling pathways in neutrophils. In addition, we provide insight into the properties of the extracellular, antiapoptotic factors produced by F. tularensis, and furthermore, describe the identification of Francisella-derived lipoproteins as functional antiapoptotic effectors acting specifically via TLR2/1. Lastly, we report the novel finding that F. novicida manipulates apoptosis and extends neutrophil lifespan by utilizing a similar, yet distinct mechanism as F. tularensis.

Klebsiella pneumoniae is an opportunistic, Gram-negative pathogen that is responsible for a variety of infections, including those of the respiratory and urinary tracts, following insertion of medical devices. It is believed that epithelial surfaces are mechanically disrupted through insertion of these devices thereby exposing the underlying extracellular matrix components. This disruption, as well as the in situ accumulation of matrix components on the indwelling device, has been proposed to provide a unique colonization niche for K. pneumoniae as the type 3 fimbriae of this organism have been shown to adhere to, and subsequently facilitate biofilm formation on, human extracellular matrix coated surfaces. To identify regulators of type 3 fimbrial expression, a bank of transposon mutants was generated and screened using a colony immunoblotting assay, for isolates that were phenotypically non-fimbriate. One insertion identified was within a gene encoding a putative transcriptional regulator named MrkI. Mutants of mrkI were found to be non-fimbriate only under aerobic conditions, and as a result, were deficient for biofilm formation on both biotic and abiotic surfaces. The decrease in type 3 fimbrial production was at the level of fimbrial structural gene (mrk) transcription, though purified MrkI could not be shown to specifically bind the mrk promoter. The two genes that flank mrkI, mrkH and mrkJ, encode proteins that contain domains involved in the binding or degradation, respectively, of the second messenger molecule cyclic diguanylate (c-di-GMP). Deletion of mrkJ from the chromosome resulted in an increase in type 3 fimbrial surface expression, which led to a concomitant increase in biofilm formation capability. This increase in fimbrial production was found to be due to intracellular accumulation of c-di-GMP as MrkJ was shown to be a functional phosphodiesterase. Further study found that MrkH binds c-di-GMP and positively regulates type 3 fimbrial expression independently of aerobic growth conditions. The ability of MrkH to induce the production of type 3 fimbriae was also found to be dependent on binding c-di-GMP as evidenced by the inability of a site-directed mutant of MrkH to bind c-di-GMP and restore fimbrial expression in a mutant background. Using an mrk transcriptional reporter, it was found that production of MrkH and MrkI together was required for maximal expression of mrk. This observation, as well as others, alludes to an apparent synergistic effect whereby MrkH and MrkI may interact, forming a novel, c-di-GMP-dependent transcriptional activation complex.

Pseudomonas aeruginosa is a Gram-negative bacterium capable of causing infections in immunocompromised individuals. The CsrA family of RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the post-transcriptional level. P. aeruginosa has a canonical CsrA family member (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for both RsmA and RsmF. The initial target aptamer was a 57 nt RNA transcript containing a central core randomized at 15 sequential positions. Most of the selected aptamers were the expected size and shared a common consensus sequence (CAnGGAyG). Longer aptamers (80-140 nts) containing two consensus-binding sites were also identified. Representative short (single consensus site) and long (two consensus sites) aptamers were tested for RsmA and RsmF binding. Whereas RsmA bound the short aptamers with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long aptamers with high affinity. Mutation of either consensus GGA site in the long aptamers reduced or eliminated RsmF binding, suggesting a requirement for two binding sites. Based on our observations that high affinity binding by RsmF appears to require two binding sites, we used an in-silico approach to search for candidate RsmF targets in the P. aeruginosa genome. We queried a library of 5’ UTRs (untranslated regions) for potential targets of RsmF based on the number and positions of GGA motifs, and secondary structure. Experimental validation of potential targets yielded few direct targets for both RsmA and RsmF indicating that additional factors contribute to differential binding in vivo. P. aeruginosa has distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA activity is controlled by two small, non-coding regulatory RNAs (RsmY and RsmZ). In addition, we recently identified a sRNA (RsmV) that also contributes to RsmA and RsmF activity. Bioinformatic analyses suggest that these sRNAs each have 3-4 putative RsmA/RsmF bindings sites. Each site contains a GGA motif presented in the loop portion of a predicted stem-loop structure. These sRNAs regulate RsmA activity, and possibly RsmF, by sequestering RsmA and/or RsmF from target mRNAs. We characterize the contribution of each GGA site in RsmV, RsmY, and RsmZ using functional assays. We provided evidence that RsmF has more restrictive binding preferences compared to RsmA. The type III secretion system (T3SS) is an important virulence factor that contributes to P. aeruginosa pathogenesis. Production of the T3SS is activated by host-associated signals and is tightly controlled at several levels. Global regulators including cAMP-Vfr signaling and Hfq contribute to tight regulation of the T3SS. Vfr (virulence factor regulator) is a transcription factor that responds to increased intracellular levels of cAMP. Vfr directly activates exsA transcription. ExsA activates transcription of the entire T3SS regulon. Hfq is an RNA chaperone that stabilizes sRNA and/or facilitates their binding to mRNA targets. Hfq is found in many bacteria and regulates stress responses, metabolism, and virulence. P. aeruginosa Hfq regulates about 5% of the genome and has a role in post-transcriptional control of T3SS in many Gram-negative bacteria. The mechanism of Hfq control of P. aeruginosa T3SS remains to be described. To better understand how Hfq regulates the T3SS we sought to identify mRNA targets of Hfq. Utilizing several reporters to genes involved in T3SS gene expression, we found that exsA transcription is decreased by Hfq activity. ExsA translation is also decreased by Hfq in conjunction with sRNA 179. Our findings show that Hfq may indirectly and directly regulate exsA translation.

Despite their essential role in protection, T cells can be dangerous if unregulated. Dysfunctional T cell activity has been implicated in numerous diseases, including the failure of organ transplants, allergic reactions, multiple sclerosis, and coronary artery disease. Signal transduction pathways activated by the T cell receptor (TCR) are good targets for the development of therapies. However, we must first better understand the mechanisms of intracellular signaling that occur when a T cell is activated. This thesis focuses on the scaffold protein LAT and its role in T cell activation. The localization of signaling proteins into LAT-nucleated complexes and subsequent aggregation of these complexes into microclusters is vital for the activation of intracellular signaling pathways, and the effector functions of T cells. Following TCR stimulation, LAT is phosphorylated and binds SH2 domain containing molecules, such as the adaptor protein Grb2. One LAT molecule is capable of binding up to three Grb2 molecules at one time. Grb2 also binds to the proline rich regions of several proteins, including SOS1. Recent studies indicate that at physiological ratios of Grb2 and SOS1, two Grb2 molecules bind to one SOS1 proline rich region, and this 2:1 stoichiometry is essential for LAT oligomerization and cluster formation. The interaction of Grb2 and SOS1 is considered to be a model SH3 domain interaction, and has biased our understanding of these relationships for decades. Many studies have focused on the association between the Grb2 SH3 domains and the proline rich region of SOS1. This previous work identified four consensus-binding sites for Grb2 in the proline rich region of SOS1 using short 10-15 amino acid peptides, and indicated that this interaction has a low affinity. Interestingly, the interaction of full SOS1 with Grb2 appears to be at least 100-fold stronger than these peptide-based studies imply. While informative, the use of short peptides leaves the physiological relevance of the peptide-SH3 domain interaction ambiguous. In this thesis, we specifically emphasize the LAT multi-protein complex and its role in the activation of T cells. First, we compare the differences in the phosphorylation states of various LAT-proximal molecules in two T cell lines and peripheral blood T cells. We then focus on the formation of this complex by investigating the essential interaction between Grb2 and SOS1. Using biochemical and biophysical techniques, we clearly demonstrate that although the previously identified consensus binding sites are important in the context of short peptides, they do not facilitate the interaction of full length Grb2 with the full proline rich region of SOS1. We also attempt to ascertain the role of LAT microclusters in T cell signaling.

Bacillus subtilis is a Gram-positive spore-forming soil bacterium. Under environmental stress conditions such as starvation, B. subtilis enters the pathway of sporulation. Earlier work demonstrated that B. subtilis can delay sporulation by undergoing cannibalism. Sporulating cells secrete toxins that kill nearby siblings, thus allowing cells to feed on the released contents. One of these toxins, SdpC, is encoded by the sdpABC operon. To uncover the requirements for SdpC toxic activity during cannibalism, all proteins in the sdpABC operon were analyzed. We report that mutations of SdpC which block signal peptidase cleavage also block toxin production. In addition, production and secretion of SdpC do not require SdpA and SdpB. Our results indicate that SdpC secretion is indispensable for induction of the immunity operon sdpRI . Furthermore, SdpC secreted from a Δ sdpAB strain does not fully induce sdpRI expression and has decreased toxicity to cells that are sensitive to wild type SdpC. Lastly, differences in SdpC mobility are observed in the presence of SdpA and SdpB. Thus, we propose that SdpA and SdpB may function by post-translationally modifying SdpC into the active form of the toxin.

The major aim of this research was to investigate the molecular mechanisms of regulation of transcription of CIT1 and CIT2, the genes encoding citrate synthase in yeast. Specifically addressed are the questions of (1) localization of cis-acting sites required for expression or regulation, (2) the roles of HAP1 and HAP2,3,4 in expression of both genes, (3) identification of other trans-acting factors involved in expression of either gene, (4) localization of cis-acting sites involved in up regulation of CIT2 in response to disruption of CIT1 or rho° status (Liao et al., 1991; Liao and Butow, 1993). I show here that mutations in HAP2, HAP3, or HAP4 specifically prevent derepression of CIT1. Using deletions and base substitutions, derepression of CIT1 is shown to require candidate HAP2,3,4 binding sites at -290 and -310 (distance upstream from the translational start site). Attempts at demonstrating binding of HAP2,3,4 to CIT1 upstream DNA were unsuccessful. HAP1 appeared to play an important role in lactate-derepressed, but not glucose repressed expression of w. No regions were identified as being responsible for negative regulation by glucose plus glutamate. HAP2,3,4 is not required for this regulation. HAP2,3,4 also was shown to regulate CIT2 to a small degree in glucose- repressed expression and to a large degree in lactate-derepressed expression. No regions were identified as responsible for this activation. To identify additional m-acting factors involved in expression CIT1 or CIT2 (eg. activators of glucose-repressed expression), yeast containing CIT1-lacZ or CIT2-lacZ fusions were mutagenized and screened for altered expression. Seven mutants with reduced expression of a ClT1-lacZ fusion are currently under study. Expression of CIT2 (peroxisomal citrate synthase) is not regulated by glucose, but is regulated by the rho status of the strain and by the presence or absence of a functional CIT1 gene (Liao et al., 1991; Liao and Butow, 1993). We identified a region critical for expression of w in lactate-grown cells located between -300 and -370, which is the same region Butow’s laboratory has found to be important in regulation by rho.