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Edwardsiella piscicida  is a severe fish pathogen. Haem utilization systems play an important role in bacterial adversity adaptation and pathogenicity. In this study, a speculative haem utilization protein, HutZ Ep , was characterized in  E. piscicida .  hutZ   Ep   is encoded with two other genes,  hutW  and  hutX , in an operon that is similar to the haem utilization operon  hutWXZ  identified in  V. cholerae . However, protein activity analysis showed that HutZ Ep  is probably not related to hemin utilization. To explore the biological role of HutZ Ep , a markerless  hutZ   Ep   in-frame mutant strain, TX01Δ hutZ , was constructed. Deletion of  hutZ   Ep   did not significantly affect bacterial growth in normal medium, in iron-deficient conditions, or in the presence of haem but significantly retarded bacterial biofilm growth. The expression of known genes related to biofilm growth was not affected by  hutZ   Ep   deletion, which indicated that HutZ Ep  was probably a novel factor promoting biofilm formation in  E. piscicida . Compared to the wild-type TX01, TX01Δ hutZ  exhibited markedly compromised tolerance to acid stress and host serum stress. Pathogenicity analysis showed that inactivation of  hutZ   Ep   significantly impaired the ability of  E. piscicida  to invade and reproduce in host cells and to infect host tissue. In contrast to TX01, TX01Δ hutZ  was defective in blocking host macrophage activation. The expression of  hutZ   Ep   was directly regulated by the ferric uptake regulator Fur. This study is the first functional characterization of HutZ in a fish pathogen, and these findings suggested that HutZ Ep  is essential for  E. piscicida  biofilm formation and contributes to host infection.

HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida