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Background   Edwardsiella ictaluri  is a Gram-negative facultative intracellular anaerobe and the etiologic agent of enteric septicemia of channel catfish (ESC). To the catfish industry, ESC is a devastating disease due to production losses and treatment costs. Identification of virulence mechanisms of  E. ictaluri  is critical to developing novel therapeutic approaches for the disease. Here, we report construction of a transposon insertion library and identification of mutated genes in growth-delayed  E. ictaluri  colonies. We also provide safety and efficacy of transposon insertion mutants in catfish.    Results  An  E. ictaluri  transposon insertion library with 45,000 transposants and saturating 30.92% of the TA locations present in the  E. ictaluri  genome was constructed. Transposon end mapping of 250 growth-delayed  E. ictaluri  colonies and bioinformatic analysis of sequences revealed 56 unique  E. ictaluri  genes interrupted by the  MAR2xT7  transposon, which are involved in metabolic and cellular processes and mostly localized in the cytoplasm or cytoplasmic membrane. Of the 56 genes, 30 were associated with bacterial virulence. Safety and vaccine efficacy testing of 19 mutants showed that mutants containing transposon insertions in hypothetical protein ( Ei s:: 004 ), and Fe-S cluster assembly protein (IscX,  Ei s:: 039 ), sulfurtransferase (TusA,  Ei s:: 158 ), and universal stress protein A (UspA,  Ei s:: 194 ) were safe and provided significant protection ( p  < 0.05) against wild-type  E. ictaluri .    Conclusions  The results indicate that random transposon mutagenesis causing growth-delayed phenotype results in identification bacterial virulence genes, and attenuated strains with transposon interrupted virulence genes could be used as vaccine to activate fish immune system.

Transposon mutagenesis and identification of mutated genes in growth-delayed Edwardsiella ictaluri