Thesis (Ph. D.)--Drexel University, 2004. / Includes abstract and vita. Includes bibliographical references (leaves 210-223).

Tuberculosis is an enormous global health problem. Despite decades of research, the mechanism(s) by which Mycobacterium tuberculosis (Mtb) mediates virulence remains incompletely understood. The ESX-1 secretion system is critical for Mtb to survive and cause disease in vivo, but its primary function and mechanism of action are unclear. The many inherent challenges of working with this slow-growing pathogen often limit the experimental approaches that can be used to address these questions. Thus, we have developed a model system in the nonpathogenic bacterium Bacillus subtilis to study ESX-type secretion systems. Here, we demonstrate that the B. subtilis yuk operon encodes an ESX-type secretion system responsible for the secretion of YukE. Additionally, we demonstrate that the yuk system is active in B. subtilis during conditions of nutrient deprivation and is required for normal biofilm formation. Interestingly, this is similar to our findings that the Mtb ESX-1 system plays dual roles in protein secretion and modulating cell wall integrity. One defining feature of all ESX loci is the presence of an FtsK/SpoIIIE family ATPase. Interestingly, these ATPases have a domain structure unique to ESX-associated ATPases, where each protein contains multiple (2-3) enzymatic domains. We used our B. subtilis system to dissect the mechanism of action of this unique class of motor proteins. We find that the yuk-encoded ATPase YukBA dimerizes to form a hexamer of enzymatic subunits that are differentially required for secretion. Strikingly, we find a unique requirement for rotational symmetry in the nucleotide binding activity of the subunits. Finally, we compared the energy requirements of the Mtb ESX-1 system and the B. subtilis yuk system. We find that these systems have some overlapping ATPase requirements for protein secretion and cell wall integrity/biofilm formation, suggesting that there is a conservation of function among ESX-type systems. We also find that some ATPase domains are differentially required for function between these two systems, which we postulate is due to the split protein architecture of the ESX-1-encoded ATPases. Together, these findings highlight the power of using a B. subtilis model system to understand the function and mechanism of action of ESX-type secretion systems.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

Alternative sigma factors play a key role in the physiology of Bacillus anthracis by regulating the transcription of the appropriate genes required for adaptation and survival. Under specific conditions, alternative sigma factors activate transcription by binding to the promoter of the genes relevant to the condition and initiate synthesis of RNA. Here we report that the transcription of predicted sigI gene in B. anthracis, BAS3231, is induced by elevated temperatures and involved in the regulation of virulence gene expression. We show that BAS3231 is required for cell viability at elevated temperatures. We have also demonstrated that mutation in the BAS3231 gene results in a decrease in virulence gene expression. Our study provides new insight into the role of alternative sigma factors in B. anthracis.

The molecular mechanics of bacterial cell division remain one of the most fundamental unsolved problems in bacterial cell biology. During bacterial cytokinesis, bacteria divide symmetrically to give rise to two identical daughter cells. This tightly regulated process is orchestrated by an assembly of essential cell division proteins that form a supramolecular structure known as the divisome. The divisome, which forms at the cell centre, is responsible for the invagination and fusion of the cell’s membrane and peptidoglycan layers. The Escherichia coli divisome is comprised of at least ten essential proteins whose individual functions are mostly unknown. These divisomal proteins are recruited in a semi-hierarchical order, with the early recruits being predominantly cytoplasmic and the later recruits being predominantly extracytoplasmic or multi-pass transmembrane proteins. DivIB and its ortholog FtsQ are essential members of the divisome in Gram-positive and Gram-negative bacteria, respectively. DivIB is a bitopic membrane protein composed of an N-terminal cytoplasmic domain, a single-pass transmembrane domain, and a C-terminal extracytoplasmic region comprised of three separate protein domains. The α domain is located next to the transmembrane segment and is a polypeptide-transport-associated (POTRA) domain. The β domain comprises approximately half of the extracytoplasmic region, and has a unique three-dimensional fold. The most C-terminal domain, the γ domain, is relatively unstructured. This protein has been proposed to play a role in septal peptidoglycan cross-linking or lengthening. The primary aims of these studies were to further characterise the structure and function of the bacterial cell division protein DivIB as well as investigate the interactions this protein has with the other divisomal proteins. It was anticipated that the knowledge gained should aid in the development of antimicrobials that target this protein’s function or protein-protein interactions. A molecular dissection approach was used to determine which of DivIB’s domains are essential for its recruitment to incipient division sites and for its cell division functions. It was determined that DivIB has three molecular epitopes that mediate its localisation to division septa; two epitopes are encoded within the extracytoplasmic region while the third is located in the transmembrane domain. It is proposed that these epitopes represent sites of interaction with other divisomal proteins, and this information was used to develop a model of the way in which DivIB and FtsQ are integrated into the divisome. Remarkably, two of the three DivIB localisation epitopes are dispensable for vegetative cell division; this suggests that the divisome is assembled using a complex network of protein-protein interactions, many of which are redundant and likely to be individually nonessential. Yeast and bacterial two-hybrid studies have revealed that most of these proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate the interaction between pairs of interacting proteins. Recently, a heterologous septal targeting approach was introduced to study the interaction between Bacillus subtilis divisomal proteins in E. coli. This technique allows the interaction between pairs of divisomal proteins to be studied in vivo without the complications caused by other interacting proteins. This approach was used to perform a molecular dissection of the interaction between B. subtilis DivIB and the divisomal transpeptidase PBP 2B. Although both proteins have septal localisation determinants in their transmembrane domains, it was found that these regions do not mediate their interaction. Rather, it was shown that DivIB interacts with PBP 2B through its extracytoplasmic region. Dissection studies revealed that all three extracytoplasmic domains of DivIB are necessary for interaction with PBP 2B, suggesting that the two proteins make multiple interactions, each of which is not strong enough in isolation to mediate formation of a stable complex. Finally, it was shown that E. coli FtsQ localises to the division septum in B. subtilis but cannot complement a divIB null. Multi-angle laser light scattering (MALLS) analysis revealed that the extracytoplasmic domain of Geobacillus stearothermophilus DivIB is predominantly monomeric at high concentrations. This indicated that if DivIB does exist as a dimer in vivo, it dimerises through its cytoplasmic or transmembrane region. In vitro observations suggest the C-terminal residues of DivIB may play a role in peptidoglycan binding. Finally, attempts were made to determine the three-dimensional structure of the complete extracytoplasmic domain of DivIB. Although it proved impossible to determine the structure using NMR spectroscopy, crystals were obtained under many different crystallisation conditions. Despite diffracting to 3.5 Å, we were unable to solve the protein structure using X-ray crystallography. However, this work has laid the groundwork for future attempts at solving the structure of this protein using X-ray crystallography.

Thesis (Ph.D.) OGI School of Science & Engineering at OHSU, November 2007. / Includes bibliographical references (leaves 141-160).

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 115-120). Also available in electronic version.

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: E.D. Brown. Includes bibliographical references (leaves 95-104).

Thesis (M.S.)--Rochester Institute of Technology, 2008. / Typescript. Includes bibliographical references (leaves 57-59).

Thesis (Ph.D.). / Written for the Dept. of Natural Resource Sciences. Title from title page of PDF (viewed 2009/06/08). Includes bibliographical references.