Salmonella is an important food borne pathogen. Over 100,000 cases of human Salmonella infection are reported in the European Union each year, resulting in an economic burden estimated to be around 3 billion Euros per year (EFSA, 2012). In a European Food Safety Authority (EFSA) survey between 2006 and 2007 S. Typhimurium was the most common serovar of Salmonella isolated from pig carcasses (EFSA, 2008a). Pigs can be asymptomatic carriers of S. Typhimurium (Berends et al., 1996) and contaminated pork contributes significantly to the number of human infections. It has been estimated that the porcine Salmonella reservoir contributes between 10-20% of human salmonellosis cases per year (VLA, 2010). In addition to improvements in biosecurity and husbandry practices, immune-prophylaxis is an important method to reduce the prevalence of food borne pathogens such as Salmonella in reservoir species. An understanding of the molecular basis of bacterial colonisation and persistence in the reservoir host is crucial to rational vaccine design and targeting relevant species. S. Typhimurium expresses multiple surface factors involved in adherence and colonisation of gut epithelium in several host species. The aim of this project was to identify factors involved in S. Typhimurium colonisation of the porcine gut. The work presented here specifically focuses on the role of flagella in the colonisation of porcine gut epithelium. Flagella are motility organelles possessed by many bacterial species. Flagella can also function as surface adhesins, shown in Escherichia coli O157:H7 (Mahajan et al., 2009), and Pseudomonas (De Bentzmann et al., 1996, Lillehoj et al., 2002). Flagellin is the major flagellar filament structural protein approximately 50kDa in size. Salmonella enterica has the ability to switch between two alternate, antigenic forms of its flagellin filament protein, expressing either FliC or FljB (Macnab, 1996). The biological relevance of these two types of flagella filament protein is still not understood. It has been postulated that the presence of a second phase type of flagella may offer an advantage to the bacteria by avoiding recognition by the immune system. However, studies have shown that both FliC and FljB flagella activate Toll-like receptor-5 (TLR-5) mediated by nuclear factor (NF)-κB signalling (Simon and Samuel, 2007b). One specific objective of this research was to compare the role of flagellar phase types in S. Typhimurium adherence and colonisation of porcine gut. To this end a porcine colonic primary epithelial cell culture and ex vivo tissue explants were developed as in vitro infection models. Primary colonic cell cultures were phenotypically characterised using specific markers for epithelial and M cells. In addition to primary epithelial cell culture, porcine intestinal epithelial cell line, IPEC-J2, was also used for specific flagellar interaction studies. The role of flagella in interaction of S. Typhimurium to porcine intestinal epithelium was tested using S. Typhimurium strain SL1344 and flagella mutant derivative strains. Flagella mutant strains exhibited reduced binding to porcine intestinal epithelial cells. Purified flagella proteins were also shown to bind porcine intestinal epithelial cells. Moreover, flagella specific anti-sera suppressed S. Typhimurium adherence to both porcine intestinal epithelial cells as well as porcine colonic explants. The immuno-protective role of flagella as a potential S. Typhimurium vaccine candidate was tested during vaccine efficacy studies in pigs. Parenteral immunisation of pigs with purified FliC and FljB flagella proteins induced production of both IgG and IgA antibodies. The vaccination of pigs with Salmonella flagella provided some protection against challenge as fewer ileum tissue samples from the pigs in the vaccinated group tested positive for Salmonella. The intestinal contents from the vaccinated pigs tested for Salmonella post mortem appeared to also have lower levels of Salmonella compared to un-vaccinated controls, though these were not significantly different between groups. This project has identified flagella as one potential subunit of a multivalent subunit vaccine to help control salmonellosis in the porcine reservoir.

Archaeal protein trafficking is a poorly understood process that is only beginning to unfold. Integral to this process are the various signal peptidases. Two types of archaeal signal peptidases are identified thus far: signal peptidase I (SPI) and the preflagellin peptidase. SPI is responsible for processing the majority of secreted proteins. Sequence analysis of archaeal SPI enzymes indicates a Sec-11 type enzyme with two conserved Aspartic acid and a Histidine in place of the conserved Lysine in bacteria. Site directed mutagenesis and in vitro assays identified three conserved residues (Ser52, His122 and Asp148) critical for M. voltae SPI activity, distinguishing the archaeal enzyme from its bacterial and eukaryal counterparts. The archaeal preflagellin peptidase is a type IV prepilin peptidase-like enzyme, initially characterized for its essential role in preflagellin processing prior to flagellar filament assembly. Unusual substrates have been proposed for this enzyme, including preflagellins of peculiar signal peptide lengths in certain archaeal species, as well as sugar binding proteins with extremely short signal peptides in S. solfataricus. In this thesis, in vitro comparisons of the signal peptide length requirements for the two different preflagellin peptidases, FlaK in M. voltae and PibD in S. solfataricus, are presented. While a signal peptide length cut-off of 5 amino acids was found for FlaK below which preflagellins remained unprocessed, substrates with shorter (4 and 3 aa) signal peptides were recognized and properly cleaved by PibD, suggesting a diversification of the preflagellin peptidases among the archaeal species. The ability of FlaK and PibD to complement FlaK activity in an M. maripaludis flaK mutant was evaluated. M. maripaludis flaK is a markerless, stable mutant displaying pili as the sole cell surface appendage, providing the unique opportunity to closely study this structure without the interference of flagella. Here, purification and characterization of the archaeal pili is described for the first time in any archaeon. A putative pilus gene cluster with characteristics of type IV pilus genes was identified in M. maripaludis. In-frame deletion of the putative major pilin gene, MMP0237, resulted in a nonpiliated phenotype that could be restored by complementation, providing a direct link of this gene to piliation. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2007-09-28 12:59:16.801

Thesis (M.S. in microbiology)--Washington State University, May 2010. / Title from PDF title page (viewed on June 16, 2010). "School of Molecular Biosciences." Includes bibliographical references.

This work explores bacterial motility from the mechanisms of propulsion of an individual cell to the complex behavior of collective motility. The shear modulus of bacterial flagella was measured by stretching isolated flagella with an optical trap and by measuring force extension curves of the stretched flagella shedding light onto the me-chanics involved in the motility of single micro-organisms. Experiments in concentrated suspensions of bacteria show collective behavior with large scale mixing on a time and length scale greater than can be understood from the standard model of "run and tumble" motility of a single organism are reported. To further understand the transition from individual to collective motility a novel form of motility where an individual bacterium can reverse direction without changing cell orientation is reported here. These experiments further the understanding of bacterial motility.

Microbes live in diverse, challenging and competitive environments. To survive and propagate microbes must be able to sense and respond to environmental fluctuations, such as changes in pH, nutrient status or temperature. As such, bacteria have a number of signal transduction mechanisms at their disposal that allow them to detect a range of different stimuli, integrate different signals and react to them appropriately. The work presented in this thesis aimed to understand more about the signalling cascades that the Gram positive soil-dwelling bacterium <em>Bacillus subtilis</em> uses to mediate its transition from a motile lifestyle that requires rotating helical flagella, to a sessile lifestyle called the biofilm, where cells adhere to a surface and are encased in a self-produced extracellular polymeric matrix. Bacterial tyrosine phosphorylation is required for <em>B. subtilis</em> biofilm formation and has been suggested to also play a role in regulating the putative motility protein, YvyG. This led to the hypothesis that tyrosine phosphorylation might play a role in both motility and biofilm formation. The first part of this thesis investigates this hypothesis and successfully ascribes a function to YvyG as an orthologue of a flagellar type 3 secretion system chaperone that is essential for flagellar assembly. Crucially this work provides further evidence that the <em>B. subtilis</em> flagellum is regulated by both conserved and species-specific means. These experiments led to YvyG being re-named as FlgN. Despite previous work suggesting that phosphorylation of YvyG was important for protein function and localisation, the data presented here found no evidence of this, and therefore indicate that the impact of bacterial tyrosine phosphorylation must be assessed in vivo before any significance can be drawn from the identification of such modifications by in vitro approaches. The second part of this study examines the role of the DegS-DegU two component signal transduction system in mediating the transition from motility to biofilm formation. DegS-DegU is required for both motility and biofilm formation, and previous work indicated that DegS-DegU may sense flagellar assembly. The data presented show that upon an inhibition of flagellar rotation DegU~P levels are increased, as inferred from accepted proxies. This could conceivably be the first step in biofilm formation to allow cells to sense and respond to a surface and change their gene expression profile. The <em>B. subtilis </em>flagellum therefore acts as a mechanosensor to control the DegS-DegU two component system. Collectively, the work presented here contributes to our understanding of how <em>B. subtilis</em> regulates flagellar assembly, and further enhances our knowledge of how bacteria are able to use their flagella not only as devices for propulsion, but also to sample changes in the extracellular environment.

Thesis (Ph.D.)--Drexel University, 2009. / Includes abstract and vita. Includes bibliographical references (leaves 95-103).

Thesis (Ph. D., Microbiology, Molecular Biology, and Biochemistry)--University of Idaho, January 2007. / Major professor: Douglas G. Cole. Includes bibliographical references (leaves 107-119). Also available online (PDF file) by subscription or by purchasing the individual file.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 137-143). Also available in electronic version.

No description available.

A unique undescribed gram-negative rod is extensively characterized in this study. The cells of this unusual water isolate measure 1.2 x 6.5 microns. The most distinguishing characteristic of the bacterium is a polar tuft of 35-40 flagella that aggregate to function as a single organelle which is visible under phase contrast.