Functional characterization of the chitin utilization system in the saprophytic bacterium Cellvibrio japonicus
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Date
2020-01-01
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Biological Sciences
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Biological Sciences
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This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu
This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu
Abstract
Chitin is a linear polymer of ? (1-4)-linked N-acetylglucosamine and is the second most abundant polysaccharide on earth. It is a major source of fixed carbon and nitrogen to microorganisms, and the strategies used by microbes to degrade chitin are of interest to the biotechnology industry as current chemical methods used are inefficient and wasteful. Due to the environmental and industrial relevance of chitin conversion the strategies used by microbes able to degrade this polysaccharide are of interest. Our current understanding of mechanistic and biochemical aspects of chitin degradation is fairly strong, however the physiological and genetic aspects of chitin degradation are not well characterized. The saprophytic Gram-negative bacterium Cellvibrio japonicus is a potent chitin degrader and has a suite of nine Carbohydrate Active enZymes (CAZymes) with predicted functions for chitin degradation: four chitinases from the Glycoside Hydrolase (GH) family 18, one GH19 chitinase, two GH20 hexosaminidases, one GH46 chitosanase, and one chitin-specific lytic polysaccharide mono-oxygenase (LPMO; Auxiliary Activity, AA10). My dissertations work had the goal to characterize the physiological roles of the chitinolytic machinery of Cellvibrio japonicus. During my dissertations work, I characterized the physiological roles of the C. japonicus GH18 chitinases during the degradation of chitin-rich substrates. I determined that the four GH18 chitinases (Chi18A, Chi18B, Chi18C, and Chi18D) have specific roles during the degradation of chitin and using a systems biology approach, and I determined that Chi18D is essential for chitin degradation. I also characterizated the mechanisms that C. japonicus employs to transport and metabolize chitin degradation products. Specifically, I functionally characterized the role of the two GH20 hexosaminidades in chito-oligosaccharide (CHOS) catabolism and I concluded that the gene product of hex20B has a major role in CHOS catabolism. Using transcriptomics and functional characterization I identified two TonB-dependent receptor genes cttA (CJA_0353) and cttB (CJA_1157). These two genes encode proteins essential for the transport of the products of chitin degradation. Furthermore, I characterized an operon for GlcNAc and GlcN transport and utilization by C. japonicus that has a glucosamine transporter and GlcNAc transporter. Finally, I developed a method to perform high-throughput screening of mutants using a custom designed 3D printed biocontainment devices, which allowed for the elucidation of subtle and complex phenotypes. I have not only characterized the physiological roles of the GH18 and the GH20 families of the saprophyte Cellvibrio japonicus but also proposed a model for chitin degradation in a soil bacterium. My dissertations expands our understanding of chitin degradation from an organismal perspective and validates the usage of multidisciplinary approaches to characterize the physiological roles of carbohydrate active enzymes.