Involvement of two glycoside hydrolase family 19 members in colony morphotype and virulence in Flavobacterium columnare

摘要

Flavobacterium columnare is the pathogenic agent of columnaris disease in aquaculture.Using a recently developed gene deletion strategy,two genes that encode the Glyco_hydro_19 domain (GH 19 domain) containing proteins,ghd-1 and ghd-2,were deleted separately and together from the F.columnare G4 wild type strain.Surprisingly,the single-,△ghd-1 and △ghd-2,and double-gene mutants,△ghd-1 △ghd-2,all had rhizoid and non-rhizoid colony morphotypes,which we named △ghd-1,△ghd-2,△ghd-1 △ghd-2,and N△ghd-1,N△ghd-2,and N△ghd-1 △ghd-2.However,chitin utilization was not detected in either these mutants or in the wild type.Instead,skimmed milk degradation was observed for the mutants and the wild type;the non-rhizoid strain N△ghd-2 exhibited higher degradation activity as revealed by the larger transparent circle on the skimmed milk plate.Using zebrafish as the model organism,we found that non-rhizoid mutants had higher LD50 values and were less virulent because zebrafish infected with these survived longer.Transcriptome analysis between the non-rhizoid and rhizoid colony morphotypes of each mutant,i.e.,N△ghd-1 versus (vs) △ghd-1,N△ghd-2 vs △ghd-2,and N△ghd-1 △ghd-2 vs △ghd-1 △ghd-2,revealed a large number of differentially expressed genes,among which 39 genes were common in three of the pairs compared.Although most of these genes encode hypothetical proteins,a few molecules such as phage tail protein,rhs element Vgr protein,thiol-activated cytolysin,and TonB-dependent outer membrane receptor precursor,expression of which was down-regulated in non-rhizoid mutants but up-regulated in rhizoid mutants,may play a role F.columnare virulence.