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Bacterial type IV pili (T4P) are filamentous surface appendages with a variety of functions including motility, surface attachment, and biofilm formation. In many species of bacteria a clear understanding of how the functions of T4P in lifestyle switching are regulated remains to be elucidated. Here, we focus on understanding the regulation of the T4P assembly ATPase PilB. We examined its interactions with the secondary messenger cyclic-di-GMP (cdG). Specifically we investigated how cdG binding regulates PilB functions not only as the assembly ATPase, but also as an EPS signaling molecule in Myxococcus xanthus biofilm regulation. Chapter 2 focuses on the development of a microplate-based biofilm assay for M. xanthus. This new assay allows for the analysis of the M. xanthus submerged biofilms under vegetative conditions in a high throughput format which has been absent in the published literature. M. xanthus biofilm formation tightly correlates with EPS production, suggesting that the assay can be used as a convenient method of examining EPS production. Chapter 3 examines the regulation of M. xanthus PilB (MxPilB) by cdG binding in vivo. We carried out a mutational analysis of the MshEN cdG binding domain in MxPilB. Mutations were created that either diverge with or converge from the MshEN consensus sequence. These two classes of MxPilB variants are expected to either decrease or increase cdG binding affinity, respectively. We examined the motility, EPS production, and piliation phenotypes of these mutants. Our results were consistent with a model where the function of MxPilB is altered in response to cdG binding, and suggesting that PilB responds to different thresholds of cdG concentration. In Chapter 4, we examine the ligand binding to the N-terminal cdG binding domain and C-terminal ATPase domain of Chloracidobacterium thermophilum PilB (CtPilB) in vitro. Our results confirm that these two domains bind to their respective ligands specifically, and demonstrate these domains communicate with each other in response to ligand binding. The results from all of the studies help us to establish a model where cdG binding fine tunes the functions of PilB to regulate the switch of bacteria between the motile and planktonic states. In addition to their roles in motility and biofilm formation, T4P are key virulence factors in many significant human pathogens. Antivirulence chemotherapeutics are considered to be a promising alternative to antibiotics, as they target disease processes rather than bacterial viability. Because PilB is essential for T4P biogenesis, we sought to identify PilB inhibitors for the development of antivirulence therapies. In Chapter 5, we describe the development of the first high throughput screen (HTS), for PilB inhibitors. This assay is uses the reduction of the binding of a fluorescent ATP analog to CtPilB in vitro, leading to the discovery of the plant flavonoid quercetin as a PilB inhibitor. Using M. xanthus as a model a bacterium, quercetin was found to inhibit T4P-dependent motility and T4P assembly in vivo. Builds on this initial success with CtPilB, Chapter 6 describes the development and implementation of a second HTS based on the inhibition of CtPilB as an ATPase. Screening a large chemical library led to the identification of benserazide and levodopa as CtPilB inhibitors. We show that both compounds inhibit T4P assembly in M. xanthus without any detrimental effects on bacterial growth. Furthermore we demonstrate that both levodopa and benserazide inhibit T4P-mediated motility in Acinetobacter nosocomialis, a human pathogen, providing the first evidence that the compounds identified with CtPilB can be effective against a pathogenic bacterium. Both of these studies validate the effectiveness not only of our HTSs, with of CtPilB as a model protein for the identification of PilB inhibitors. Doctor of Philosophy Bacteria can be motile or sessile. Motile bacteria can use hair like structures on their surface, called pili, to move in their natural environment, whereas sessile bacteria produce intricate structures attached to solid surfaces known as biofilms. Bacteria are able to switch between being motile and sessile states depending on their environment conditions. However, it isn't clear how this switch is controlled in bacteria that use pili to move. To answer this question, we studied how PilB the protein that assembles pili, might control this switching process. We specifically investigated PilB because it has two known roles. The first is that it can assemble pili, to enable pili-mediated motility. The second is that it can stimulate or promote biofilm formation. This places PilB at the intersection of these two lifestyles, suggesting that this protein may play a key role in deciding whether a bacterium is to be motile or sessile. Another important reason to understand how PilB functions is because pili are used by some antibiotic resistant pathogenic bacteria. Since PilB is essential for the formation of pili, if the actions of PilB could be blocked, bacteria would be unable to make pili. This could stop bacteria from causing disease. By searching for new chemicals which stop PilB from creating pili, we can potentially identify new drugs to treat bacterial infections.

Positive-strand RNA viruses associate with specific organelle membranes of host cells to establish viral replication complexes. The replication protein 1a of brome mosaic virus associates strongly with the nuclear endoplasmic reticulum (ER) membranes, invaginates membranes into the lumen, and recruits various host proteins to establish replication complexes termed spherules. 1a has a strong affinity towards the perinuclear ER membrane, however, the structural features in 1a that dictate its membrane associations and thereby membrane remodeling activities are unclear. This study examined the possible role of an amphipathic α-helix, helix B, in BMV 1a's membrane association. Deletion or single substitution of multiple amino acids of helix B abolished BMV 1a's localization to nuclear ER membranes. Additional reporter-based, gain-of-function assays showed that helix B is sufficient in targeting several soluble proteins to the nuclear ER membranes. Furthermore, we found that the helix B-mediated organelle targeting is a functionally conserved feature among positive-strand RNA viruses of the alphavirus-like superfamily that includes notable human viruses such as Hepatitis E virus and Rubella virus as well as plant viruses such as cucumber mosaic virus and cowpea chlorotic mottle virus. Our results demonstrate a critical role for helix B across members of the alphavirus-like superfamily in anchoring viral replication complexes to the organelle membranes. We anticipate our findings to be a starting point for the development of sophisticated models to use helix B as a novel target for the development of antivirals for positive-strand RNA viruses that belong to the alphavirus-like superfamily. Doctor of Philosophy Among the seven classes of viruses, the positive-strand RNA viruses dominate the domain of viral diseases of the world. Brome mosaic virus (BMV) is a positive-strand RNA virus that infects cereal crops such as wheat, barley, and rice. BMV has a simple genome organization and serves as a suitable model virus to study and characterize positive-strand RNA viruses. The replication of all positive-strand RNA viruses occurs at the organelle membranes of the host. Membrane association of the replication is one of the early steps and a crucial event in the life cycle of positive-strand RNA viruses. One of the proteins produced early on during BMV infection is the replication protein 1a, which is also the master regulator of viral replication; 1a recruits viral factors in addition to hijacking the necessary host factors at the membranous sites to initiate replication. Upon reaching the organelle membranes, 1a induces membrane rearrangements to form viral replication complexes that safeguard the recruited factors from the deleterious effects of the host cell. The structural determinants within 1a that are responsible for such membrane association are unknown. This study explored the potential roles of a short helical motif within the 1a protein for its ability to dictate such site-specific membrane associations. We show here that this helical region is necessary and sufficient for 1a's membrane-binding activity. We also discovered it to be a functionally conserved feature that is responsible for membrane associations in various viruses of the alphavirus-like superfamily that includes some of the notable human viruses such as Hepatitis E virus and Rubella virus in addition to plant viruses such as cucumber mosaic virus and cowpea chlorotic mottle virus.