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Biochemical Characterization of the Activity and Control of the Autolysins Involved in Flagella Assembly in Gram-Negative Bacteria

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Title: Biochemical Characterization of the Activity and Control of the Autolysins Involved in Flagella Assembly in Gram-Negative Bacteria
Author: Herlihey, Francesca
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Clarke, Anthony
Abstract: The flagellum is an important virulence factor for many pathogenic bacteria. An important step during flagellum assembly is the localized and controlled degradation of the peptidoglycan sacculus to allow for the insertion of the rod as well as to facilitate anchoring for proper motor function. The peptidoglycan lysis events necessary for the insertion of cellular machinery as well as cellular growth and division require enzymes such as lytic transglycosylases and β-N-acetylglucosaminidases. Due to their structural similarities and the use of crude assays, the nature of the peptidoglycan lytic activity of many β-glycosidases remains unknown. In this thesis, I present the development of a novel assay for glycoside lytic enzymes and its use to provide the first enzymatic characterization of the lytic domain of the dedicated flagellar enzymes FlgJ and SltF from Salmonella enterica serovar Typhimurium and Rhodobacter sphaeroides, respectively, as the model organisms. In β- and γ-proteobacteria, FlgJ functions as an endo-β-N-acetylglucosaminidase representing the first autolysin with this activity to be characterized from a Gram-negative bacterium. In contrast, in α-proteobacteria SltF functions as an endo- lytic transglycosylase and it defines a new sub-family of the family 1 lytic transglycosylases. Given their lethal potential, these enzymes have to be regulated and this is achieved at the enzyme level in many bacteria. The control of SltF’s activity was investigated and it is modulated by two flagellar rod proteins, FlgB and FlgF; FlgB stabilizes and stimulates SltF activity while FlgF inhibits it. In addition, the specificity of the Ivy proteins, inhibitors originally identified as inhibitors of vertebrate lysozyme, was also investigated. Ivyp1 and Ivyp2 from Pseudomonas aeruginosa were found to be more specific for the soluble lytic transglycosylases, SltB1 and Slt70 than for lysozymes. This work has provided important insights into the flagellum assembly of α-, β- and γ- proteobacteria as well as the specificities of the Ivy proteins from P. aeruginosa. Further insight into the modulation of the sacculus may provide new avenues for the development of novel antibacterials.
Date: 2017-01
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