Expanding the repertoire of counterselection markers for markerless gene deletion in the human gut bacterium Phocaeicola vulgatus.

OBJECTIVE: Phocaeicolavulgatus (formerly Bacteroides vulgatus) is a highly abundant and ubiquitous member of the human gut microbiota, associated with human health and disease, and therefore represents an important target for further investigations. In this study a novel gene deletion method was developed for P. vulgatus, expanding the tools available for genetic manipulation of members of the microbial order Bacteroidales. MATERIAL AND METHODS: The study used a combination of bioinformatics and growth experiments in interaction with molecular cloning to validate the applicability of SacB as a counterselection marker in P. vulgatus. RESULTS: In this study, the levansucrase gene sacB from Bacillussubtilis was verified as a functional counterselection marker for P. vulgatus, conferring a lethal sensitivity towards sucrose. Markerless gene deletion based on SacB was applied to delete a gene encoding a putative endofructosidase (BVU1663). The P. vulgatus Δbvu1663 deletion mutant displayed no biomass formation when grown on levan, inulin or their corresponding fructooligosaccharides. This system was also applied for the deletion of the two genes bvu0984 and bvu3649, which are involved in the pyrimidine metabolism. The resulting P. vulgatus Δ0984 Δ3649 deletion mutant no longer showed sensitivity for the toxic pyrimidine analogon 5-fluorouracil, allowing a counterselection with this compound in the double knockout strain. CONCLUSION: The genetic toolbox for P. vulgatus was expanded by a markerless gene deletion system based on SacB as an efficient counterselection marker. The system was employed to successfully delete three genes in P. vulgatus which all resulted in expected phenotypes as confirmed by subsequent growth experiments.

Rapid, real-time sucrase characterization: Showcasing the feasibility of a one-pot activity assay.

Sucrases can modify numerous carbohydrates, and short-chain oligosaccharides produced by the unique transfructosylation activity of levansucrases are promising candidates for the growing sugar substitute market. These compounds could counteract the increasing number of diseases associated with the consumption of high-calorie sugars. Thus, there is great interest in the characterization of novel levansucrases. The commonly used method for sucrase activity determination is to quantify d-glucose released in the sucrose-splitting reaction. This is usually done in a discontinuous mode, i.e., several samples taken from the sucrase reaction are applied to a separately performed d-glucose determination (e.g., GOPOD assay). Employing the newly isolated levansucrase LevSKK21 from Pseudomonas sp. KK21, the feasibility of a one-pot sucrase characterization was investigated by combining sucrase reaction and GOPOD-based d-glucose determination into a single, continuous assay (Real-time GOPOD). The enzyme was characterized with respect to kinetic parameters, ion dependency, pH value, and reaction temperature in a comparative approach employing Real-time GOPOD and HPLC. High data consistency for all investigated enzyme parameters demonstrated that current processes for sucrase characterization can be considerably accelerated by the continuous assay while maintaining data validity. However, the assay was not applicable at acidic pH, as decolorization of the quinoneimine dye formed during the GOPOD reaction was observed. Overall, the study presents valuable data on the potentials of real-time sucrase activity assessment for an accelerated discovery and characterization of interesting enzymes such as the hereby introduced levansucrase LevSKK21. Progress in sucrase discovery will finally foster the development of health-promoting sucrose substitutes.