Functional identification of hemH and transcriptomic analysis of hemH mutant of Riemerella anatipestifer

Abstract:

Heme is an essential nutrient for the growth and proliferation of most bacteria since it is involved in a variety of physiological processes. Bacteria can obtain heme through biosynthesis and acquisition from the host. However, excessive heme is toxic, and bacteria can alleviate heme toxicity by efflux, sequestration, and degradation. Riemerella anatipestifer (RA), a Gram-negative bacterium that infects birds, can transport heme from hemoglobin. However, whether RA can synthesize heme remains unknown. The genome analysis revealed that the gene RA0C_RS08070 of RA ATCC 11845 strain encodes the ferrochelatase HemH, which is a key enzyme that participates in the insertion of iron into porphyrin center to form heme. The loss of hemH leads to the accumulation of iron and porphyrin, causing toxicity to bacteria. [Objective] To identify the role of HemH in the synthesis of heme and identify the genes involved in the detoxification of iron and porphyrin in RA. [Methods] In this study, ΔhemH, the hemH-deleted mutant of RA ATCC 11845, was constructed, and the growth curves of the parental strain and ΔhemH in the GCB liquid medium and the GCB liquid medium supplemented with hemoglobin (Hb) were established. Further, the transcriptomes of the parental strain and ΔhemH were sequenced and analyzed. [Results] RA ATCC 11845ΔhemH did not grow in the GCB medium, while it grew well in the GCB medium supplemented with Hb. Transcriptome analysis revealed 354 differentially expressed genes (DEGs) between ΔhemH and the parental strain. Gene ontology (GO) functional annotation showed that the DEGs were mainly involved in catalytic activity, biological regulation, and metabolic processes. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis showed that the DEGs were mainly enriched in amino acid metabolism, oxidative phosphorylation, and tricarboxylic acid cycle (TCA cycle). [Conclusion] HemH is involved in heme synthesis, and the mutant with the deletion of hemH changed the expression of the genes to adapt to the disorder of metabolism. This study lays a foundation for further studying the role of HemH in RA.