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

作　　者：王梦莹 刘马峰 程安春[1,2,3] WANG Mengying;LIU Mafeng;CHENG Anchun(Institute of Preventive Veterinary Medicine,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,Sichuan,China;Avian Disease Research Center,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,Sichuan,China;Key Laboratory of Animal Disease and Human Health of Sichuan Province,Sichuan Agricultural University,Chengdu 611130,Sichuan,China)

机构地区：[1]四川农业大学动物医学院预防兽医研究所,四川成都611130 [2]四川农业大学动物医学院禽病防治中心,四川成都611130 [3]四川农业大学动物疫病与人类健康四川省重点实验室,四川成都611130

出　　处：《微生物学报》2023年第8期3083-3095,共13页Acta Microbiologica Sinica

基　　金：国家自然科学基金(32072825,32273003);四川省自然科学基金(2022NSFSC0007)

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ATCC11845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。hem H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Hem H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RAATCC11845Δhem H缺失株,并检测亲本株和hem H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hem H缺失株进行转录组测序并进行比较分析。【结果】RAATCC11845Δhem H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,hem H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(Kyotoencyclopediaofgenesandgenomes,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】Hem H参与了血红素的合成,hem H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Hem H的功能奠定基础。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,△hemH,the hemH-deleted mutant of RA ATCC 11845,was constructed,and the growth curves of the parental strain and△hemH in the GCB liquid medium and the GCB liquid medium supplemented with hemoglobin(Hb)were established.Further,the transcriptomes of the parental strain and hemH were sequenced and analyzed.[Results]RA ATCC 11845△hemH 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 AhemH 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 found

关 键 词：鸭疫里氏杆菌 血红素 hemH 

分 类 号：S852.61[农业科学—基础兽医学]