Pre-Methylation of Foreign DNA Improves Conjugation Efficiency in the Fish Pathogen, Flavobacterium psychrophilum
Location
CSU Ballroom
Start Date
12-4-2022 2:00 PM
End Date
12-4-2022 3:30 PM
Student's Major
Biological Sciences
Student's College
Science, Engineering and Technology
Mentor's Name
Yongtao Zhu
Mentor's Department
Biological Sciences
Mentor's College
Science, Engineering and Technology
Description
very year, millions of dollars are spent to combat outbreaks of fish diseases due to the presence of fish pathogens, such as Flavobacterium psychrophilum, a member of the phylum Bacteroidetes. F. psychrophilum is the causative agent of bacterial cold-water disease (BCWD) and rainbow trout fry syndrome (RTFS), predominantly in salmonids. The bacterium causes tissue damage and tail rot in young and adult fish. The issue is prevalent in fisheries in the Pacific Northwest and the treatments often entail the use of antibiotics. The virulence mechanisms of F. psychrophilum are not well understood. Genetic manipulations in F. psychrophilum CSF 259-93, the most problematic strain in rainbow trout fisheries in the U.S., are scarce, due to the strain’s ability to destroy foreign DNA via its restriction enzymes, which hampers the identification of virulence factors by using genetic tools. The goals of this project are to identify methyltransferase (MTase) genes of the DNA restriction-modification systems in F. psychrophilum CSF 259-93, and improve the efficiency of DNA transfer via conjugation by pre-methylation of foreign DNA. Individual or multiple MTase encoding genes were cloned into pACYC184, a compatible vector with the reporter plasmid pCP11. pACYC184 derived plasmids carrying the MTase genes were co-transformed with pCP11 into the conjugation donor E. coli for pre-methylation of pCP11. Two MTase genes (hpaIIM and scrF1M) were identified and shown to be effective in improving the conjugation efficiency in CSF 259-93 using pCP11. A helper plasmid (pSS05), containing both hpaIIM and scrF1M genes, was constructed and a more significant increase in conjugation efficiency of pCP11 was observed by using pSS05. We further generated a deletion construct pSS12 to delete gldN, a component of the type IX secretion system required for the virulence of F. psychrophilum. By using pSS12 and the helper plasmid pSS05, we successfully constructed the gldN deletion mutant. We intend to test this newly developed pre-methylation/gene deletion method in other virulence factors of interest. Our goal was generating avirulent or less virulent mutants. These mutants can be developed as live attenuated vaccines and used to prevent BCWD and RTFS diseases, and thus to reduce the economic losses in aquaculture.
Pre-Methylation of Foreign DNA Improves Conjugation Efficiency in the Fish Pathogen, Flavobacterium psychrophilum
CSU Ballroom
very year, millions of dollars are spent to combat outbreaks of fish diseases due to the presence of fish pathogens, such as Flavobacterium psychrophilum, a member of the phylum Bacteroidetes. F. psychrophilum is the causative agent of bacterial cold-water disease (BCWD) and rainbow trout fry syndrome (RTFS), predominantly in salmonids. The bacterium causes tissue damage and tail rot in young and adult fish. The issue is prevalent in fisheries in the Pacific Northwest and the treatments often entail the use of antibiotics. The virulence mechanisms of F. psychrophilum are not well understood. Genetic manipulations in F. psychrophilum CSF 259-93, the most problematic strain in rainbow trout fisheries in the U.S., are scarce, due to the strain’s ability to destroy foreign DNA via its restriction enzymes, which hampers the identification of virulence factors by using genetic tools. The goals of this project are to identify methyltransferase (MTase) genes of the DNA restriction-modification systems in F. psychrophilum CSF 259-93, and improve the efficiency of DNA transfer via conjugation by pre-methylation of foreign DNA. Individual or multiple MTase encoding genes were cloned into pACYC184, a compatible vector with the reporter plasmid pCP11. pACYC184 derived plasmids carrying the MTase genes were co-transformed with pCP11 into the conjugation donor E. coli for pre-methylation of pCP11. Two MTase genes (hpaIIM and scrF1M) were identified and shown to be effective in improving the conjugation efficiency in CSF 259-93 using pCP11. A helper plasmid (pSS05), containing both hpaIIM and scrF1M genes, was constructed and a more significant increase in conjugation efficiency of pCP11 was observed by using pSS05. We further generated a deletion construct pSS12 to delete gldN, a component of the type IX secretion system required for the virulence of F. psychrophilum. By using pSS12 and the helper plasmid pSS05, we successfully constructed the gldN deletion mutant. We intend to test this newly developed pre-methylation/gene deletion method in other virulence factors of interest. Our goal was generating avirulent or less virulent mutants. These mutants can be developed as live attenuated vaccines and used to prevent BCWD and RTFS diseases, and thus to reduce the economic losses in aquaculture.
Recommended Citation
Sloboda, Seada. "Pre-Methylation of Foreign DNA Improves Conjugation Efficiency in the Fish Pathogen, Flavobacterium psychrophilum." Undergraduate Research Symposium, Mankato, MN, April 12, 2022.
https://cornerstone.lib.mnsu.edu/urs/2022/poster-session-02/14
