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ARS Home » Northeast Area » Leetown, West Virginia » Cool and Cold Water Aquaculture Research » Research » Publications at this Location » Publication #416040
Research Project: Improving Salmonid Health through Breeding, Vaccination and Microbiome Modulation

Location: Cool and Cold Water Aquaculture Research

Title: Methylation of foreign DNA overcomes the restriction barrier of Flavobacterium psychrophilum and allows efficient genetic manipulation

Author
item SLOBODA, SEADA - Minnesota State University
item GE, XINWEI - Xi'An Jiaotong-Liverpool University
item JIANG, DAQING - University Of Wisconsin
item SU, LIN - Xi'An Jiaotong-Liverpool University
item Wiens, Gregory
item BEVERIDGE, CARLY - Minnesota State University
item DUCHAUD, ERIC - Inrae
item MCBRIDE, MARK - University Of Wisconsin
item ROCHAT, TATIANA - Inrae
item ZHU, YONGTAO - Xi'An Jiaotong-Liverpool University

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2024
Publication Date: 1/10/2025
Citation: Sloboda, S., Ge, X., Jiang, D., Su, L., Wiens, G.D., Beveridge, C.A., Duchaud, E., Mcbride, M.J., Rochat, T., Zhu, Y. 2025. Methylation of foreign DNA overcomes the restriction barrier of Flavobacterium psychrophilum and allows efficient genetic manipulation. Applied and Environmental Microbiology. 0:e01448-24. https://doi.org/10.1128/aem.01448-24.
DOI: https://doi.org/10.1128/aem.01448-24

Interpretive Summary: Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum is a problem for aquaculture worldwide and current control measures are inadequate. An obstacle in understanding and controlling BCWD is that most F. psychrophilum strains resist DNA transfer, thus limiting genetic studies of its virulence mechanism. F. psychrophilum restriction enzymes that destroy foreign DNA were suspected to contribute to this problem. Here we used F. psychrophilum DNA methyltransferases to modify and protect foreign DNA from digestion. This allowed efficient conjugative DNA transfer into 9 diverse strains of F. psychrophilum that had previously resisted gene transfer. Using this approach, we constructed a targeted gene deletion that eliminated the ability to cause disease. Further genetic studies could help determine the mechanisms responsible for the disease, and may aid development of vaccines and other control measures.

Technical Abstract: Flavobacterium psychrophilum causes bacterial cold-water disease (BCWD) in salmonids and other fish, resulting in substantial economic losses in aquaculture worldwide. The mechanisms F. psychrophilum uses to cause disease are poorly understood. Despite considerable effort, most strains of F. psychrophilum have resisted attempts at genetic manipulation. The reasons for this were not known but F. psychrophilum restriction endonucleases (REases) may contribute to the problem by destroying foreign DNA. REases are often paired with modification enzymes, such as methyltransferases (MTases), that modify and protect the DNA of organisms that produce them. Together, these constitute restriction-modification (R-M) defense systems. Here, we identified two critical R-M systems, HpaII-M.FpsJI and ScrFI-M.FpsJVI, in F. psychrophilum CSF259-93. Protection of foreign DNA against HpaII and ScrFI by a methylation plasmid expressing M.FpsJI and M.FpsJVI, or by deletion of the two REase genes, resulted in efficient conjugative DNA transfer from Escherichia coli to CSF259-93. This allowed us to use a sacB-mediated deletion system to construct a CSF259-93 mutant lacking gldN, a core component of the type IX protein secretion system. The mutant was deficient in secreted proteolytic activity, gliding motility, and virulence on rainbow trout. Genomic analyses revealed HpaII-M.FpsJI and ScrFI-M.FpsJVI systems in 14 of 17 F. psychrophilum genomes. The pre-methylation system developed in this study functions in at least 9 diverse F. psychrophilum strains. These newly developed genetic tools may allow the identification of key virulence factors and facilitate the development of live attenuated vaccines or other measures to control BCWD.