Single-step genome engineering in the bee gut symbiont Snodgrassella alvi .

Autor:	Lariviere PJ; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA.; Department of Integrative Biology, The University of Texas at Austin, Austin, TX 78712, USA., Ashraf AHMZ; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA., Leonard SP; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA.; Department of Integrative Biology, The University of Texas at Austin, Austin, TX 78712, USA., Miller LG; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA., Moran NA; Department of Integrative Biology, The University of Texas at Austin, Austin, TX 78712, USA., Barrick JE; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2023 Sep 20. Date of Electronic Publication: 2023 Sep 20.
DOI:	10.1101/2023.09.19.558440
Abstrakt:	Honey bees are economically relevant pollinators experiencing population declines due to a number of threats. As in humans, the health of bees is influenced by their microbiome. The bacterium Snodgrassella alvi is a key member of the bee gut microbiome and has a role in excluding pathogens. Despite this importance, there are not currently any easy-to-use methods for modifying the S. alvi chromosome to study its genetics. To solve this problem, we developed a one-step procedure that uses electroporation and homologous recombination, which we term SnODIFY ( Sn odgrassella-specific O ne-step gene D eletion or I nsertion to alter F unctionalit Y ). We used SnODIFY to create seven single-gene knockout mutants and recovered mutants for all constructs tested. Nearly all transformants had the designed genome modifications, indicating that SnODIFY is highly accurate. Mutant phenotypes were validated through knockout of Type 4 pilus genes, which led to reduced biofilm formation. We also used SnODIFY to insert heterologous sequences into the genome by integrating fluorescent protein-coding genes. Finally, we confirmed that genome modification is dependent on S. alvi 's endogenous RecA protein. Because it does not require expression of exogenous recombination machinery, SnODIFY is a straightforward, accurate, and lightweight method for genome editing in S. alvi . This workflow can be used to study the functions of S. alvi genes and to engineer this symbiont for applications including protection of honey bee health. Competing Interests: Competing interests S.P.L., J.E.B., and N.A.M. have a pending patent (US20190015528A1) on the use of engineered symbionts to improve bee health. J.E.B. is the owner of Evolvomics LLC.
Databáze:	MEDLINE
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