Functional characterization of RhuB as a second TonB2-dependent hemin receptor inCH-1.

Abstract

In the previous study, it was shown that(, RA), a pathogen in ducks and some other birds, encodes a hemin uptake system. Thehemin uptake receptor RhuR is a TonB2-dependent hemin transporter. However, it remains unclear whetherencodes additional TonB-dependent hemin transporters. Herein, we demonstrated thathemin uptake receptor B (RhuB) ofCH-1 (RA CH-1) was negatively regulated by iron and mediated by the Fur protein, and knocking outdamaged the ability of RA CH-1 to utilize iron from duck hemoglobin (Hb) but not that from duck serum. Moreover, the ability to use iron from Hb was restored by the expression. Furthermore, the RhuB of RA CH-1 is a membrane protein, and recombinant RhuB could bind hemin at a 1:1 molar ratio. Compared to that of ΔΔ, the ability of ΔΔΔto utilize hemin was impaired; meanwhile, compared to that of ΔΔ, the hemin utilization ability of ΔΔΔwas not affected, indicating that RhuB is a TonB2-dependent receptor. Compared to Δ,did not affect hemin utilization. However, compared to Δ,had reduced ability to utilize hemin, suggesting that RhuA relies on RhuB for its activity. Finally, the deletion ofdid not affect the virulence of RA CH-1. These results suggested that RhuB encodes a TonB2-dependent hemin receptor. The characterization of the second TonB-dependent receptor inenriches our understanding of the hemin uptake system of this bacterium.IMPORTANCEIron is essential for the survival of most bacteria, and hemin of hemoglobin can serve as an important iron source. In our previous studies, we showed thatCH-1 encodes a TonB2-dependent hemin receptor RhuR, which is involved in hemin uptake. The deletion ofdid not abolish hemin utilization by RA CH-1. We hypothesized that additional hemin uptake systems exist in this bacterium. In this study, we identified the second TonB2-dependent hemin receptor RhuB in RA CH-1 through hemin utilization, protein localization, and hemin-binding experiments. The duck infection model showed that the deletion ofdid not affect the virulence of RA CH-1. This study is not only important for further understanding the hemin utilization mechanism of, but also for enriching the hemin uptake transporters of gram-negative bacteria.