Quiescence, a transitory period of non-growth, is a ubiquitous aspect that is present in all organisms. In addition to being present in all forms of life, quiescence is a feature that has been observed in cells that are important for human health, including stem cells in mammals and antibiotic tolerant cells in bacteria. In bacteria, quiescence per se has recently been suggested to underlie the transient tolerance to a wide range of antibiotics. Furthermore, most microbial life exists in a quiescent state. Despite their prevalence and importance, relatively little is known about the physiology of quiescent bacteria. One aspect of bacterial quiescence that has been repeatedly observed is their lowered metabolic activity compared to actively growing bacteria. How do cells that grow and divide enter into a temporary state of non-growth? In particular, how are the energy-intensive processes that are required for growing cells regulated during a non-growing state? The main subject of this thesis is to investigate how protein synthesis, the most energy-intensive process in growing bacterial cells, is regulated during entry into a quiescent phenotype (stationary phase). I first investigate how protein synthesis is regulated using a single cell method that fluorescently tags nascent polypeptide chains. In chapter 3, I show that during entry into stationary phase, protein synthesis is downregulated heterogeneously with one group of cells having comparatively low protein synthesis, resulting in a population that is approximately bimodal. I further show that this bimodality is dependent on a signaling system (PrkC and its partner phosphatase PrpC) that senses cell wall metabolism. I connect signaling from this system to the expression of an enzyme (SasA) that produces a group of nucleotides that are major regulators of growth in bacteria ((pp)pGpp). Lastly, I show that the bimodality is dependent on the three enzymes that synthesize (pp)pGpp. In chapter 4, I explore in detail how the bimodality in protein synthesis is generated. This heterogeneity requires the production of (pp)pGpp by three synthases: SasA, SasB, RelA. I first show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population exhibiting low protein synthesis, whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. The RelA product (pppGpp) allosterically activates SasB, and I find that the SasA product (pGpp) competitively inhibits this activation. I provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This chapter, therefore, identifies the mechanism underlying the generation of phenotypic heterogeneity in the central physiological process of protein synthesis. In chapter 5, I next turn to understand the biochemical mechanism by which cells with comparatively low levels of protein synthesis down-regulate this process. I first show that ppGpp is sufficient to inhibit protein synthesis in vivo. I then show that ppGpp inhibits protein synthesis by inhibiting translation initiation directly by binding to the essential GTPase, Initiation Factor 2 (IF2). In collaboration with Ruben Gonzalez’s lab, we also show that ppGpp prevents the allosteric activation of IF2. Finally, I demonstrate that the observed attenuation of protein synthesis during the entry into quiescence is a consequence of the direct interaction of (pp)pGpp and IF2.

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A HP1076 null mutant has been constructed to provide a better understanding of the biological significance of HP1076 in H. pylori . The DeltaHP1076 mutant displays impaired motility and resistance to the antibiotic drug metronidazole. Using a proteomic study, an overall of 40 differentially expressing proteins involved in metabolism and pH homeostasis for bacterial survival, adhesion for colonization, virulence factor to gastric epithelial cells and antigenic proteins have been identified. The virulence factor, Cag pathogenicity island protein (Cag 26) and urease UreA and UreB are confirmed to have enhanced and reduced expression in null mutants. These findings may provide new insight into the infection of H. pylori. / FliS is an export chaperone that binds to flagellin molecules in cytosol in order to prevent pre-mature polymerization. Disruption of FliS would result in formation of shorter flagella and impaired adhesion ability to epithelial cells. Previous yeast two-hybrid study has identified various FliS associated proteins in H. pylori, but with no known implications. Here, we have demonstrated the interaction of FliS and a hypothetical protein HP1076 by biochemical and biophysical methods. Moreover, HP1076 possesses anti-aggregation ability on insoluble FliS-mutants and chaperone activity. Thus, HP1076 is proposed to be a co-chaperone that promotes the folding and chaperone activity of FliS. FliS is demonstrated to have a broad range of substrate specificity that binds to flagellin and flagellar related proteins which may play a key role in flagellar export system different from other flagellated bacteria. / Helicobacter pylori is a pathogenic bacterium and adheres to the gastric mucosal cells. Chronic infection would lead to gastritis or peptic ulceration and is one of the leading causes of gastric cancer. Formation of functional flagella is essential for infection, that it aids in motility of bacteria and colonization on gastric epithelial cells. The process is complex and involves more than 50 proteins in assembly of structural proteins, regulatory proteins, an export apparatus, a motor and a sensory system. Cytosolic chaperones are required to bind to exported proteins in order to facilitate the export or prevent the aggregation of proteins in cytosol. Divergence is found in flagellar system H. pylori that may account for survival inside gastric environment. / The crystal structures of FliS, HP1076 fragment and FliS/HP1076 complex are determined at 2.7A, 1.8A and 2.7A resolution respectively to provide better understanding of their molecular interactions. FliS consists of four helices and HP1076 consists of helical rich bundle structure with three helices and three beta strands that share similar fold to that of a flagellin homologue, hook-associated protein and FliS, suggesting HP1076 is involved in flagellar system. The FliS/HP1076 complex reveals an extensive electrostatic and hydrophobic binding interface which is distinct from the flagellin binding pocket on FliS. HP1076 stabilizes two alpha helices of FliS and therefore the overall bundle structure. Our findings provide new insights into the flagellar export chaperones and other secretion chaperones in Type III secretion system. / Lam, Wai Ling. / Adviser: An Wing-Ngor. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 223-243). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 179-198). Also issued on the Internet.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves: 138-147). Also available on the Internet.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.