Construction Of UvrC Gene Deletion Mutant Of Rimerella Anatipestifer And Analysis Of Its Biological Characteristics

Riemerella anatipestifer（RA）mainly causes infectious serositis,high infection rate and mortality,which brings great economic losses to the duck industry.The previous results in our study indicated that the mutant strain of the Riean＿1413（uvrC）gene encoding the ABC subunit of RA endonuclease has significantly reduced pathogenicity in ducks.But the molecular mechanism remains unclear.In this study,a mutant of RA Yb2strain with uvrC gene deletion was constructed,and the effect of deletion uvrC gene on virulence of Yb2 strain was analyzed by comparing with the Yb2 wild-type strain.Our results showed that the uvrC gene was associated with the formation capacity of the RA biofilm.Deletion of the uvrC gene results in weakening of RA biofilm production.1.Construction of uvrC gene deletion mutant strain and its complemented strain.The left and right homologous arm fragments of uvrC gene were amplified by PCR and ligated into the 313 suicide plasmid.The suicide plasmid p313-uvrC-L-R were constructed and transferred into the E.coli S17-1 competent cells.Then the recombinant plasmid was transferred into Yb2 by conjugation using S17-1（p313-uvrC-L-R）as the donor and the wild type Yb2 as the recipient,and results in generating deletion mutant Yb2ΔuvrC.Furthermore,the uvrC ORF was amplified by PCR and ligated into the E.coli-RA shuttle expression plasmid p RES1 to obtain the complemented plasmid p RES1-uvrC.The plasmids were transferred into the E.coli S17-1 competent cells using S17-1（p RES1-uvrC）as the donor and the recombinant plasmid p RES1-uvrC was introduced into the deletion mutant Yb2ΔuvrC by conjugation to obtain the complemented strain c Yb2ΔuvrC.2.Biological characteristics of the deletion mutant Yb2ΔuvrC.The wild-type Yb2 strain,the deletion strain Yb2ΔuvrC and complemented strains c Yb2ΔuvrC were grown respectively.The protease hydrolytic activity was detected on the defatted plates.Analysis of the biofilm formation capacity.Adadhesion and invasion assay against Vero cells and analysis of bacterial invasion.The 7-day-old ducklings were inoculated with different dose of bacteria with gradient dilution and the LD₅₀and bacterial load in tissues were analyzed.There was no significant difference between the wild type strain and the deleted strain Yb2ΔuvrC in the growth curve,protease hydrolysis,LD₅₀and bacterial load.But the deletion strain Yb2ΔuvrC significantly reduced the ability of adhesion and invasion to Vero cells（P<0.05）and a very significant reduction in the biofilm-formation ability（P<0.001）.In conclusion,we constructed the uvrC gene mutant and complemented strain and compared the biological characteristics of wild strains Yb2 and Yb2ΔuvrC.The uvrC deletion strain had significantly reduced in biofilm formation and ability to adhere to and invade Vero cells,which indicates Uvr C is associated with biofilm-producing ability and invasiveness of RA Yb2 strain.