The Discovery Of Natural Transformation And An Easy Knockout Method In Riemerella Anatipestifer

Riemerella anatipestifer(RA)is a Gram-negative of phylum bacteroidetes,the family Flavobacteriaceae and a major causative agent of duck serositis.Several bacteria are competent for natural transformation in nature environment;however,whether RA is also competent for natural transformation has not been investigated.For the first time,we showed that natural transformation occurred in RA ATCC 11845 and RA CH-1,subsequently,a new knockout method was established for RA ATCC 11845.The main results as follows:1.The discovery of natural competence in R.anatipestiferWe cultured RA ATCC 11845(sensitive to erythromycin)in GCB medium to exponential phase.A 300?L sample of the culture(OD600 was adjusted to 1)was incubated with 1?g of RA CH-1 erythromycin resistance(erm~R)chromosomal DNA.the bacteria could grow on the tablet containing erythromycin resistance.The PCR results showed that the erythromycin resistance gene was inserted into the RA ATCC 11845 genome successfully.This result indicated that RA ATCC11845 had natural competence.2.Establishment of an easy knockout method in R.anatipestiferWe selected the gene of RA0C?1193 and amplified its upstream,downstream respectively.Then fusion of erm R in sequence via the over-lap method(RA0C?1193 up-erm R-RA0C?1193 down).After RA ATCC 11845 incubated with the fusion fragements,and cultured on the tablet containing erythromycin resistance.The PCR results showed that erm R gene could replace RA0C?1193 target gene and express erythromycin resistance,natural transformation could also be used to efficiently knockout targeted gene in RA CH-1;when DNA fragments between 0.1 ng and 1?g,the transformation efficiency increased as the donor DNA concentration increased and the highest transformation efficiency was obtained at saturating DNA fragment concentrations(1?g);transformation efficiency was decreased when the flanking fragments was shorter;Erm~R transformants were obtained after one minute of exposure to DNA within 30 min,and a saturated level of transformants was obtained after approximately 20 min.3.R.anatipestifer preferentially take up its own DNAExponential phase competent RA ATCC 11845 cells were incubated with E.coli XL1-blue genomic DNA and RA0C?1193 mutagenic PCR fragments,or RA ATCC 11845genomic DNA and RA0C?1193 mutagenic PCR fragments.The result showed that a significant reduction in the transformation efficiency for the sample to which RA ATCC11845 genomic DNA was added but not the sample to which E.coli genomic DNA was added.the results indicated that the RA ATCC 11845 genomic DNA can compete with RA0C?1193 mutagenic PCR fragments,it was speculated that RA ATCC 11845preferentially takes up its own genome DNA compared to E.coli genome DNA.Moreover,apart from genome DNA and fragements,R.anatipestifer can also uptake the shuttle plasmid DNA.