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Riemerella anatipestifer  is a duck pathogen that has caused serious economic losses to the duck industry worldwide. Despite this, there are few reported studies of the physiological and pathogenic mechanisms of  Riemerella anatipestifer  infection. In previous study, we have shown that TonB1 and TonB2 were involved in hemin uptake. TonB family protein (TbfA) was not investigated, since knockout of this gene was not successful at that time. Here, we used a plasmid based gene over-expression and knockdown to investigate its function. First, we constructed three  Escherichia - Riemerella anatipestifer  shuttle vectors containing three different native  Riemerella anatipestifer  promoters. The shuttle plasmids were introduced into  Riemerella anatipestifer  ATCC11845 by conjugation at an efficiency of 5 × 10 −5  antibiotic-resistant transconjugants per recipient cell. Based on the high-expression shuttle vector pLMF03, a method for gene knockdown was established. Knockdown of TbfA in  Riemerella anatipestifer  ATCC11845 decreased the organism’s growth ability in TSB medium but did not affect its hemin utilization. In contrast, over-expression of TbfA in  Riemerella anatipestifer  ATCC11845 ΔtonB1ΔtonB2 . Significantly promoted the organism’s growth in TSB medium but significantly inhibited its hemin utilization. Collectively, these findings suggest that TbfA is not involved in hemin utilization by  Riemerella anatipestifer .

Investigation of TbfA in Riemerella anatipestifer using plasmid-based methods for gene over-expression and knockdown