Functional Characterization of ?-Linked Diglucoside Metabolism in Cellvibrio japonicus

Abstract

Bacterial survival is determined by their ability to acquire and utilize nutrients from their environment. Organisms capable of exploiting diverse or niche nutrients gain a competitive advantage. The ?-diglucosides trehalose, kojibiose, nigerose, and isomaltose are comprised of two glucose moieties bound in different positions by an ?-glycosidic linkage. As the ?-diglucosides are either found in low concentrations, are produced by few biological processes, or are metabolized by few organisms, they are considered rare or underutilized nutrient sources. Additional nutrient sources take the form of insoluble particulate substrates; substrates under 1 mm2, which present challenges for high-throughput growth and enzyme assays. These challenges include difficulties in maintaining homogenous solutions, interference with optical density, and requiring processing to remove the substrate from the desired sample. The saprophytic soil bacterium Cellvibrio japonicus was shown to metabolize each of the ?-diglucosides and was used to validate a method for capturing insoluble particulates. C. japonicus Ueda107 possesses two predicted Glycoside Hydrolase family 37 genes, predicted to hydrolyze the ?-diglucoside trehalose. A combination of functional genetics and biochemical assays identified that the tre37A gene product is required for trehalose metabolism in C. japonicus and is highly biochemically active, while the tre37B gene product had no discernable phenotype or biochemical activity in the conditions tested. Further analysis utilizing cell free extract, the General Secretory Pathway deletion strain ?gsp, and bioinformatics indicate that Tre37A and Tre37B are likely periplasmic. In addition, although C. japonicus Ueda107 is not predicted to possess enzymes capable of degrading kojibiose, nigerose, or isomaltose, extended incubation with the substrates resulted in stable genomic adaptations. Genome sequencing identified a shared mutation among the Kojibiose adapted and Isomaltose adapted strains; amy13E P606S. Functional genetics in C. japonicus Ueda107 and each of the adapted strains indicated that amy13E is required for adaptation to each of the ?-diglucosides, maintenance of the adapted phenotype, and is likely periplasmic. Finally, an Agar Capture System was designed to contain insoluble particulates for high-throughput assays. The system was determined to be sufficient for microbial and enzyme assays when used in conjunction with previously designed microplate Biomass Containment Devices.