Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 102, Issue 17, Pages 7475-7488Publisher
SPRINGER
DOI: 10.1007/s00253-018-9181-4
Keywords
Riemerella anatipestifer; Markerless mutant; Knock-in; Point mutant
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Funding
- National Natural Science Foundation of China [31572521]
- International S&T Cooperation Program of Sichuan Province [2016HH0052]
- China Agricultural Research System [CARS-42-17]
- Sichuan Veterinary Medicine and Drug Innovation Group of China Agricultural Research System (CARS-SVDIP)
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Riemerella anatipestifer (R. anatipestifer, RA) is an important bacterial pathogen of ducks and other birds; infection with RA causes high poultry mortality and heavy economic losses in the poultry industry. However, the pathogenesis of this bacterium is poorly understood, in part due to the lack of a suitable array of methods for genetic manipulation. In this study, we first examined the efficacy of the mutated pheS gene (pheS*) as a counterselectable marker in R. anatipestifer. A suicide vector carrying pheS*, pOES, was constructed and used for markerless deletion of the gene RA0C_2053 which encode a putative TonB-dependent receptor in RA ATCC11845. The suicide plasmid pOES was also used to introduce a knock-in Myc-tag into the C-terminus of RA0C_1912 which encode a putative Fur protein. Using pheS* as a counterselectable marker, markerless mutagenesis and knock-in genetic manipulation techniques were also developed based on natural transformation. Furthermore, this marker was used to generate a point mutation in the RA0C_1912 gene of the RA ATCC11845 genome. The genetic methods developed in this study provide new and useful tools required to investigate the physiology and pathogenic mechanisms of this bacterium. These techniques may also have wider application in many other members of the Flavobacteria.
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