Iron is an essential element for the replication of most bacteria, including Riemerella anatipestifer (R. anatipestifer, RA), a gram-negative bacterial pathogen of ducks and other birds. R. anatipestifer utilizes haemoglobin-derived haemin as an iron source; however, the mechanism by which this bacterium acquires haemin from haemoglobin is largely unknown. Here, RhuA disruption was shown to impair iron utilization from duck haemoglobin in R. anatipestifer CH-1. Moreover, the putative lipoprotein RhuA was identified as a surface-exposed, outer membrane haemin-binding protein, but it could not extract haemin from duck haemoglobin. Mutagenesis studies showed that recombinant RhuAY144A, RhuAY177A and RhuAH149A lost haemin-binding ability, suggesting that amino acid sites tyrosine 144 (Y144), Y177 and histidine 149 (H149) are crucial for haemin binding. Furthermore, RhuR, the gene adjacent to RhuA, encodes a TonB2-dependent haemin transporter. The function of RhuA in duck haemoglobin utilization was abolished in the RhuR mutant strain, and recombinant RhuA was able to bind the cell surface of R. anatipestifer CH-1ΔRhuA rather than R. anatipestifer CH-1ΔRhuRΔRhuA, indicating that RhuA associates with RhuR to function. The sequence of the RhuR-RhuA haemin utilization locus exhibits no similarity with those of characterized haemin transport systems. Thus, this locus is a novel haemin uptake locus with homologues distributed mainly in the Bacteroidetes phylum.
IMPORTANCE
In vertebrates, haemin from haemoglobin is an important iron source for infectious bacteria. Many bacteria can obtain haemin from haemoglobin, but the mechanisms of haemin acquisition from haemoglobin differ among bacteria. Moreover, most studies have focused on the mechanism of haemin acquisition from mammalian haemoglobin. In this study, we found that the RhuR-RhuA locus of R. anatipestifer CH-1, a duck pathogen, is involved in haemin acquisition from duck haemoglobin via a unique pathway. RhuA was identified as an exposed outer membrane haemin-binding protein, and RhuR was identified as a TonB2-dependent haemin transporter. Moreover, the function of RhuA in haemoglobin utilization is RhuR dependent, not vice versa. The homologues of RhuR and RhuA are widely distributed in bacteria in marine environments, animals, and plants, representing a novel haemin transportation system of gram-negative bacteria. This study not only was important for understanding haemin uptake in R. anatipestifer but also enriched the knowledge about the haemin transportation pathway in gram-negative bacteria.