Aquaculture America 2024

February 18 - 21, 2024

San Antonio, Texas

PRE-METHYLATION OF FOREIGN DNA OVERCOMES THE RESTRICTION-MODIFICATION BARRIER OF Flavobacterium psychrophilum AND ALLOWS EFFICIENT SPECIES-WIDE GENETIC MANIPULATION

 

Yongtao Zhu* , Seada Sloboda,  Daqing Jiang, Xinwei Ge, Carly A. Beveridge, Gregory D. Wiens, Mark J. McBride, Tatiana Rochat, Eric Duchaud

 

Department of Biological Sciences

Xi’an Jiaotong-Liverpool University

111 Ren’ai Road

Suzhou Industrial Park

Su zhou, Jiangsu,  China 215123

yongtao.zhu@xjtlu.edu.cn

 



Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (BCWD) in salmonids and other fish, resulting in substantial economic losses in aquaculture worldwide. The mechanism s of F. psychrophilum  in causing the disease are poorly understood. Currently t he control of BCWD mainly relies on the use of antibiotics. E fficient and universal vaccines against this disease are not commercially available. One of the major obstacles for the identification of virulence factors and  development of live attenuated vaccines in F. psychrophilum is the difficulties in genetic manipulation.  Most strains of this fish pathogen are resistant to  foreign DNA transfer, due to the presence of the protective restriction-modification (RM) “immune” systems.  In this study, we identified two critical methyltransferases, HpaIIM and ScrFIM, belonging to the type II RM systems, in F. psychrophilum CSF259-93. A helper plasmid pSS05 carrying both HpaIIM and ScrFIM encoding genes was constructed, and was shown to protect the target DNA from restriction digestion and ensure successful conjugative DNA transfer from Escherichia coli to F. psychrophilum  CSF259-93.  By using pSS05 and a previously developed sacB -mediated deletion system, we constructed the first gene deletion mutant in F. psychrophilum CSF259-93. The mutant  lacking gldN, a core component of the T9SS,  is deficient in secreted proteolytic activity, colony spreading, single cell motility, and virulence on rainbow trout. G enomic analysis of 16 F. psychrophilum strains, isolated in Chile, China, Denmark, Finland, France, and the United States,  revealed the strains that are phylogenetically close tend to carry the same RM systems , and the HpaII and ScrFI RM systems are  present in 13 of the 16  analyzed genomes. We further found the pre-methylation system developed in this study  functions in  most of the 16  strains. These newly developed genetic tools may allow the identification of key virulence factors and facilitate the development of live attenuated vaccines in  multiple F. psychrophilum  strains to prevent BCWD.