The role of denitrification genes in anaerobic growth and virulence ofFlavobacterium columnare

摘要

Aims Comparative genomics analyses indicated that theFlavobacterium columnaregenome has unique denitrification genes relative toFlavobacterium psychrophilumandFlavobacterium johnsoniae, includingnasA(nitrate reductase),nirS(nitrite reductase),norB(nitric oxide reductase) andnosZ(nitrous oxide reductase). The current study determines the roles ofnasA,nirS,norBandnosZin anaerobic growth, nitrate reduction, biofilm formation and virulence. Methods and Results Four in-frame deletion mutants in virulentF. columnarestrain 94-081 were constructed by allelic exchange using pCP29 plasmid. Compared with parent strain 94-081,Fc Delta nasA,Fc Delta nirSandFc Delta nosZmutants did not grow as well anaerobically, whereas the growth ofFc Delta norBstrain was similar to the parent strain (FcWT). Exogenous nitrate was not significantly consumed under anaerobic conditions inFc Delta nasA,Fc Delta nirSandFc Delta nosZcompared to parent strain 94-081. Under anaerobic conditions,Fc increment nasA,Fc increment norBandFc increment nosZformed significantly less biofilm than the wild type strain at 24 and 96 h, butFc Delta nirSwas not significantly affected. The nitrite reductase mutantFc Delta nirSwas highly attenuated in catfish, whereasFc Delta nasA,Fc Delta norBandFc Delta nosZhad similar virulence toFcWT. Conclusions These results show, for the first time, that denitrification genes enableF. columnareto grow anaerobically using nitrate as an electron acceptor, and nitrite reductase contributes toF. columnarevirulence. Significance and Impact of the Study These findings indicate potential forF. columnareto grow in nitrate-rich anaerobic zones in catfish production ponds, and they suggest that aFc increment nirSstrain could be useful as a safe live vaccine if it protects catfish against columnaris disease.