To study the underlying mechanisms for microbial cellulose biosythesis, a novel compound, CBI28, was used as an inhibitor along with classical genetics and EMS mutagenesis. An EZ-Link Biotin Hydrazide Kit was used to create a CBI28-Biotin conjugate for further studies. Gluconacetobacter xylinus cells were exposed to 10 uM CBI28 to induce cellulose biosythesis inhibition, lysed and small hydrophobic molecules were extracted using methanol and Waters Oasis HLB SPE-Paks. Samples were separated and detected using the Ultra Performance Liquid Chromatograph-Mass Spectrometer/Photo Diode Array. Putative mutants were isolated but did not survive for further study. An ion with the expected mass of a CBI28-Biotin conjugate (552 m/z) was detected but not in sufficiently high concentrations for characterization. Metabolite studies revealed putative metabolites derived from the HLB SPE and methanol extractions with no significant difference in extraction methods. Potential metabolites with masses of ~281.77 m/z and ~79 m/z were detected in CBI28 exposed cells. Further analysis needs to be performed to determine if CBI28 metabolites prevent cellulose production. / UOIT

The cell wall is a multifunctional structure which is implicated in plant growth and development as well as responding to any environmental changes including biotic and abiotic stresses. One of the practical approaches in cell wall integrity studies is the modification of the quality and quantity of particular cell wall components or destroying the specific step in cell wall synthesis pathway using Cellulose Biosynthesis Inhibitors (CBIs). In this case, chemical screen for swollen organ phenotype has proved to be an important technique to identify the genes that are directly or indirectly involved in cellulose biosynthesis. In the present research, a number of synthetic CBIs were obtained through a chemical library screen from Chembridge Company for the root swollen phenotype which is believed to be the response to a defect in cellulose biosynthesis. Therefore, a genome-wide expression profiling based on Affymetrix ATH1 GeneChip arrays (contains 22810 probe sets) were applied to investigate the altered transcriptome of four different CBIs including CBI-15, 18, 22, and 27 and isoxaben in 5 day-old Arabidopsis thaliana seedlings. The results of this project revealed overlapped up and down-regulated genes as well as discriminate responses to each CBI. The most striking modification were found in genes involve in response to the stress as well as cell wall integrity and restructuring. Thus, the identification of regulated genes under CBIs treatment suggests a robust candidate group of genes that likely to be correlated to cell wall biosynthesis. / UOIT