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| 论文摘要： | 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 (GH19 domain) containing proteins, ghd-1 and ghd-2, were deleted separately and together from the F. columnare G(4) wild type strain. Surprisingly, the single-, Delta ghd-1 and Delta ghd-2, and double-gene mutants, Delta ghd-1 Delta ghd-2, all had rhizoid and non-rhizoid colony morphotypes, which we named Delta ghd-1, Delta ghd-2, Delta ghd-1 Delta ghd-2, and N Delta ghd-1, N Delta ghd-2, and N Delta ghd-1 Delta 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 Delta 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 Delta ghd -1 versus (vs) Delta ghd-1, N Delta ghd-2 vs Delta ghd-2, and N Delta ghd-1 Delta ghd-2 vs Delta ghd-1 Delta 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. |
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