Add like
Add dislike
Add to saved papers

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

Anaerobe 2023 May 26
OBJECTIVE: Phocaeicola (P.) vulgatus (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 Bacillus subtilis 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 endofructanase (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.

Full text links

We have located links that may give you full text access.
Can't access the paper?
Try logging in through your university/institutional subscription. For a smoother one-click institutional access experience, please use our mobile app.

For the best experience, use the Read mobile app

Mobile app image

Get seemless 1-tap access through your institution/university

For the best experience, use the Read mobile app

All material on this website is protected by copyright, Copyright © 1994-2024 by WebMD LLC.
This website also contains material copyrighted by 3rd parties.

By using this service, you agree to our terms of use and privacy policy.

Your Privacy Choices Toggle icon

You can now claim free CME credits for this literature searchClaim now

Get seemless 1-tap access through your institution/university

For the best experience, use the Read mobile app