Hemerythrin-like proteins are non-heme, di-iron and O₂-binding proteins that are ubiquitous from bacteria to mammals and have been shown to function in oxygen storage and transport. Although hundreds of hemerythrin-like proteins have been predicted in bacteria, studies on the biological functions of hemerythrin-like proteins are scarce. Recently, a study showed that the multi-domain protein VcBhr-DGC (with a hemerythrin domain and a diguanylate cyclase GGDEF domain) functions as a regulatory oxygen sensor for switching between reducing or anaerobic environments in Vibrio cholerae. This is the first one to demonstrate a regulatory function of the hemerythrin domain and hints that hemerythrin-like proteins might have other unannotated functions. As a follow-up of that study, members of the lab of Dr. Mi from the Institute of Microbiology have analyzed the function and relationship between multiple hemerythrin-like proteins withinMycobacterium smegmatis, the model for Mycobacterium tuberculosis.

    M. smegmatis contains three hemerythrin-like proteins, MSMEG_3312, MSMEG_2415 and MSMEG_6212. Using knock-out, complementation and overexpression strains of these genes, researchers from the Mi lab found that MSMEG_2415 was involved in the response to H₂O₂. In addition, using promoter regulation experiments, it was shown that MSMEG_2415 regulates sigF expression via promoter P_rbsw and is essential for inhibition of the SigF-mediated H₂O₂ response (Figure 1). To our knowledge, MSMEG_2415 is the first characterized hemerythrin-like protein in mycobacteria.

   Next, the genes encoding the two other hemerythrin-like proteins in M. smegmatis were investigated in a similar manner. Experiments showed that MSMEG_6212 and MSMEG_3312 both modulate erythromycin susceptibility and the msmeg_3312 and msmeg_6212 double-knockout strain, mc²155:Δ3312-6212, has a similar resistance as the individual single-knockout strains mc²155:Δ3312 and mc²155:Δ6212. Moreover, MSMEG_3312 and MSMEG_6212 were found to inhibit MtrA-mediated erythromycin resistance, but had no effect on WhiB7-mediated erythromycin resistance. These results are the first to show that MSMEG_3312 and MSMEG_6212 normally lowers the mRNA level of MtrA,thus affecting its regulon, and thereby causing drug resistance (Figure 1).

   Interestingly, phylogenetic analysis of bacterial hemerythrin-like proteins showed that the three mycobacterial hemerythrin-like proteins are likely derived from different lineages, as MSMEG_3312, MSMEG_2415 and MSMEG_6212 are present in three different clades (Figure 2). All the proteins in the MSMEG_2415 cluster belong to the Mycobacterium genus, while those in the MSMEG_3312 cluster were derived from Mycobacterium, Rhodococcus and Sciscionellas. A large portion of the hemerythrin-like proteins that were present in the MSMEG_6212 cluster belong to the Streptomyces, Saccharomonospora and Amycolatopsis genera, suggesting that msmeg_6212 has an independent origin. Differences in origins may explain the differences in their physiological functions.

   This study is the first to analyze the function and relationship between multiple hemerythrin-like proteins within one organism. The functional and phylogenetic analyses of hemerythrin-like proteins in M. smegmatis has provided insight into the evolutionary selection of antimicrobial resistant traits.