Graduate Program

Chemistry

Degree Name

Master of Science (MS)

Semester of Degree Completion

Fall 2019

Thesis Director

Gopal R. Periyannan

Thesis Committee Member

Thomas Canam

Thesis Committee Member

Michael W. Beck

Thesis Committee Member

Zhiqing Yan

Abstract

Bacteria present unique opportunities to explore the molecular mechanisms of nutrient acquisition processes at single cell level under various experimental conditions. Caulobacter crescentus is a gram-negative oligotrophic environmental bacterium with adaptive physiological responses, such as slow growth or complete arrest of cell cycle, presence of sessile and motile progenies with the sessile cells growing stalk to enhance nutrient uptake for growth in low-nutrient conditions. Details of plant polysaccharide-derived carbon compound, such as cellobiose, maltose, and xylose utilization by C. crescentus can be found but molecular details or mechanisms of polysaccharide utilization, such as xylan, has not been reported. Database analyses indicate C. crescentus has a repertoire of xylanases and carbohydrate-modifying enzymes, along with membrane transporters for efficient breakdown and uptake of xylan. Growth and metabolic properties of C. crescentus in M2 minimal medium containing xylan or xylo-oligosaccharides as the sole carbon source was investigated in this work. Growth curves of C. crescentus in xylan show typical phases as seen in other mono- and disaccharide carbon sources in similar conditions but with significantly extended lag phase. Enzyme assays using p-nitrophenol xylopyranoside and xylo-oligosaccharides substrates show high level expression of periplasmic xylosidases on the second day of growth on xylan substrate coinciding with the beginning of the log phase as well as possible membrane-bound xyloside hydrolases as well as glucuronidase. Gene expression analysis of cells grown in xylan shows expression of a select number of xylanases, suggesting induced expression of xylan deconstructing genes. Chromatography (HPLC & TLC) and 1H-NMR based metabolomic analyses of the extracellular medium combined with enzyme assay data suggest xylan binding onto the bacterial surface and xylan fragment uptake across the outer membrane. Growth in the presence of inhibitors that blocks transport across TonB-dependent receptor (TBDR) suggest that TBDR may be involve in the transport of xylo-oligosaccharides across the outer membrane. A comprehensive model emerges for xylan utilization by C. crescentus that resembles components of previously proposed gut and environmental bacterial models and further advances the molecular level understanding of xylan derived nutrient acquisition in environmental bacteria.

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