鸭疫里氏杆菌hemH基因功能初步鉴定及其缺失株的转录组学分析

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

摘要:

血红素是绝大多数细菌生长繁殖所必需的一种营养物质，其参与了细菌多种重要的生理过程。细菌可以通过自身合成和从外界摄取2种方式获得血红素。然而过多的血红素对细菌是有毒性的，细菌则会利用外排、螯合和降解等多种方式减轻血红素毒性。鸭疫里氏杆菌(Riemerella anatipestifer, RA)是一种感染禽类的革兰氏阴性菌，前期研究表明该菌可通过转运的方式从外界环境摄取血红素。然而，是否该菌也可以自身合成血红素未知。基因组分析发现鸭疫里氏杆菌ATCC 11845菌株中的基因RA0C_RS08070编码铁螯合酶(ferrochelatase) HemH，是参与将铁插入卟啉中心，形成血红素的关键酶。hemH缺失后会导致铁离子和卟啉的积累，对细菌造成毒性。【目的】为鉴定HemH在合成血红素中的功能及查找参与鸭疫里氏杆菌铁离子和卟啉解毒相关基因。【方法】本研究构建了RA ATCC 11845ΔhemH缺失株，并检测亲本株和hemH缺失株在GCB以及GCB添加血红蛋白液体培养基中的生长情况；随后对亲本株和hemH缺失株进行转录组测序并进行比较分析。【结果】RA ATCC 11845ΔhemH缺失株不能在GCB培养基中生长，而在GCB培养基补充血红蛋白后生长良好。转录组测序及比较分析发现，与亲本株相比，hemH缺失株中有354个显著差异表达基因(differentially expressed genes, DEGs)。基因本体论(gene ontology, GO)功能富集分析发现差异表达基因主要富集在催化活性、生物调节和代谢过程等途径，京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)通路富集分析发现差异表达基因主要富集在氨基酸代谢、氧化磷酸化和三羧酸循环(tricarboxylic acid cycle, TCA cycle)等途径。【结论】HemH参与了血红素的合成，hemH缺失后导致大量的基因表达改变来适应代谢的改变，为进一步研究鸭疫里氏杆菌的HemH的功能奠定基础。

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.