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

Abstract

ObjectivePhocaeicolavulgatus (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 methodsThe 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. ResultsIn 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. ConclusionThe 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.