副猪嗜血杆菌高自然转化效率菌株的筛选及全基因组测序
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摘要
旨在筛选具有高自然转化效率的副猪嗜血杆菌(HPS)菌株,为自然转化机制及基因功能研究提供生物材料;对所得菌株进行全基因组测序,通过比较基因组分析,挖掘HPS自然转化发生的分子背景。本研究采用自然转化方法对75株HPS田间分离株及15株标准株进行自然感受态菌株的筛选,并测定各自然感受态菌株的转化效率。采用PacBio三代测序技术对具有高自然转化效率的HPS菌株SC1401进行全基因组测序,对测序数据进行组装、基因预测、功能注释及共线性分析。将SC1401株测序结果与NCBI发布的SH0165(CP001321)、SH03(CP009158)、KL0318(CP009237)和ZJ0906(CP005384)进行全基因组比对。结果如下:1)共筛选出11株HPS自然感受态菌株,均为野生型,其中SC1401菌株自然转化效率最高;2)全基因组测序结果显示SC1401菌株整个基因组大小为2 277 540bp,GC含量为40.03%,共编码2 220个基因,占整个基因组序列的87.75%(序列已上传至GenBank数据库,登录号为CP015099);3)基因组共线性分析表明SC1401与其他四株全基因组测序菌株的共线性良好;4)比较基因组分析表明,SC1401菌株含特有基因385个,远多于其他4株菌株所含有的特有基因;5株HPS含有一系列相同的与自然转化相关的基因,且除tfox基因外,各基因氨基酸序列一致性均达90%以上。本研究首次报道了一株强自然转化能力的副猪嗜血杆菌SC1401的全基因组序列,并分析了其基因组基本特征,为后续HPS自然转化机制的研究提供一定理论基础。
This study aimed to screen highly transformable Haemophilus parasuis(HPS)strains,which in the hope of providing biological materials for the further study of natural competence mechanism and gene function research.Based on the genome sequences of the selected strain and comparative genome analysis,we attempted to investigate the molecular genetic basis of natural transformation of HPS.In this study,75 HPS field isolates and 15 standard strains were screened by natural transformation method,and the transformation efficiency of the natural competent strain was determined.The genome of the strain SC1401 with high natural transformation efficiency was sequenced using the third-generation sequencing technology on PacBio,and thenanalyzed using related software for genome assembly,gene prediction,functional annotation and synteny analysis.The sequencing result of SC1401 strain was compared with those of SH0165 strain(GenBank:CP001321),SH03 strain(GenBank:CP009158),KL0318 strain(GenBank:CP009237)and ZJ0906 strain(GenBank:CP005384).The results showed that:1)11 HPS strains were identified as natural competent strain,which all were wild type,and SC1401 had the highest transformation efficiency among them.2)The genome size is 2 277 540 bp with GC content of 40.03%.The total gene quantity is 2 220,accounting for 87.75% of the whole genome.This genome sequence had been submitted to GenBank database under the accession number CP015099.3)Synteny analysis indicated that the relationship between the 5 HPS strains were close-related.4)Comparative genomics analysis showed that SC1401 isolates contained 385 unique genes,much more than the other 4 strains.Five HPS strains contained a series of identical genes related to natural transformation,and in addition to the tfox,the homogeneity of the amino acid sequences of each gene had reached over 90%.This study is the first report of the whole genome sequence of SC1401,a highly naturally competent strain,and analyzed the basic characterization of the genome,so as to provide a theoretical basis for later research on the mechanism of natural transformation of HPS.
This study aimed to screen highly transformable Haemophilus parasuis(HPS)strains,which in the hope of providing biological materials for the further study of natural competence mechanism and gene function research.Based on the genome sequences of the selected strain and comparative genome analysis,we attempted to investigate the molecular genetic basis of natural transformation of HPS.In this study,75 HPS field isolates and 15 standard strains were screened by natural transformation method,and the transformation efficiency of the natural competent strain was determined.The genome of the strain SC1401 with high natural transformation efficiency was sequenced using the third-generation sequencing technology on PacBio,and thenanalyzed using related software for genome assembly,gene prediction,functional annotation and synteny analysis.The sequencing result of SC1401 strain was compared with those of SH0165 strain(GenBank:CP001321),SH03 strain(GenBank:CP009158),KL0318 strain(GenBank:CP009237)and ZJ0906 strain(GenBank:CP005384).The results showed that:1)11 HPS strains were identified as natural competent strain,which all were wild type,and SC1401 had the highest transformation efficiency among them.2)The genome size is 2 277 540 bp with GC content of 40.03%.The total gene quantity is 2 220,accounting for 87.75% of the whole genome.This genome sequence had been submitted to GenBank database under the accession number CP015099.3)Synteny analysis indicated that the relationship between the 5 HPS strains were close-related.4)Comparative genomics analysis showed that SC1401 isolates contained 385 unique genes,much more than the other 4 strains.Five HPS strains contained a series of identical genes related to natural transformation,and in addition to the tfox,the homogeneity of the amino acid sequences of each gene had reached over 90%.This study is the first report of the whole genome sequence of SC1401,a highly naturally competent strain,and analyzed the basic characterization of the genome,so as to provide a theoretical basis for later research on the mechanism of natural transformation of HPS.
引文
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[16]周鹏.副猪嗜血杆菌HAPS_RS00465蛋白的免疫保护性及其基因缺失株生物学特性研究[D].雅安:四川农业大学,2016.ZHOU P.Studies on the immunoprotection of H.Parasuis HAPS_RS00465protein and the biological characteristics of the gene deletion strains[D].Ya'an:Sichuan Agricultural University,2016.(in Chinese)
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[19] HOWELL K J,PETERS S E,WANG J H,et al.Development of a multiplex PCR assay for rapid molecular serotyping of Haemophilus parasuis[J].J Clin Microbiol,2015,53(12):3812-3821.
[20]谨瑾,代科,文心田,等.副猪嗜血杆菌自然转化方法的建立及条件优化[J].农业生物技术学报,2017,25(12):2058-2065.JIN J,DAI K,WEN X T,et al.Establishment and condition optimization for natural transformation method of Haemophilus parasuis[J].Journal of Agricultural Biotechnology,2017,25(12):2058-2065.(in Chinese)
[21] KRISTENSEN B M,SINHA S,BOYCE J D,et al.Natural transformation of Gallibacterium anatis[J].Appl Environ Microbiol,2012,78(14):4914-4922.
[22] LIU M F,ZHANG L,HUANG L,et al.Use of natural transformation to establish an easy knockout method in Riemerella anatipestifer[J].Appl Environ Microbiol,2017,83(9):e00127-17.
[23] BOSSEJ T,SINHA S,SCHIPPERS T,et al.Natural competence in strains of Actinobacillus pleuropneumoniae[J].FEMS Microbiol Lett,2009,298(1):124-130.
[2] BROCKMEIER S L,REGISTER K B,KUEHN J S,et al.Virulence and draft genome sequence overview of multiple strains of the swine pathogen Haemophilus parasuis[J].PLoS One,2014,9(8):e103787.
[3] ANGEN?,OLIVEIRA S,AHRENS P,et al.Development of an improved species specific PCR test for detection of Haemophilus parasuis[J].Vet Microbiol,2007,119(2-4):266-276.
[4] ZHOU Q,FENG S X,ZHANG J M,et al.Two glycosyltransferase genes of Haemophilus parasuis SC096implicated in lipooligosaccharide biosynthesis,serum resistance,adherence,and invasion[J].Front Cell Infect Microbiol,2016,6:100.
[5] ZHANG L H,LI Y,DAI K,et al.Establishment of a successive markerless mutation system in Haemophilus parasuis through natural transformation[J].PLoS One,2015,10(5):e0127393.
[6] PERRON G G,LEE A E G,WANG Y,et al.Bacterial recombination promotes the evolution of multidrug-resistance in functionally diverse populations[J].Proc R Soc B Biol Sci,2012,279(1733):1477-1484.
[7] BERTOLLA F, SIMONET P. Horizontal gene transfers in the environment:natural transformation as a putative process for gene transfers between transgenic plants and microorganisms[J].Res Microbiol,1999,150(6):375-384.
[8] CLAVERYS J P,PRUDHOMME M,MARTIN B.Induction of competence regulons as a general response to stress in gram-positive bacteria[J].Ann Rev Microbiol,2006,60:451-475.
[9] NIKAIDO H.Multidrug resistance in bacteria[J].Annu Rev Biochem,2009,78:119-146.
[10] KRUGER N J,STINGL K.Two steps away from novelty-principles of bacterial DNA uptake[J].Mol Microbiol,2011,80(4):860-867.
[11]孙东昌,张衍梅,施跃峰.细菌自然转化的分子机制研究进展[J].微生物学报,2012,52(1):6-11.SUN D C,ZHANG Y M,SHI Y F.Advances in the molecular mechanism of natural bacterial transformation-a review[J].Acta Microbiologica Sinica,2012,52(1):6-11.(in Chinese)
[12] BOSSEJ T,NASH J H E,KROLL J S,et al.Harnessing natural transformation in Actinobacillus pleuropneumoniae:a simple method for allelic replacements[J].FEMS Microbiol Lett,2004,233(2):277-281.
[13] BIGAS A,GARRIDO M E,DE ROZAS A M P,et al.Development of a genetic manipulation system for Haemophilus parasuis[J].Vet Microbiol,2005,105(3-4):223-228.
[14] SEITZ P,BLOKESCH M.Cues and regulatory pathways involved in natural competence and transformation in pathogenic and environmental Gram-negative bacteria[J].FEMS Microbiol Rev,2012,37(3):336-363.
[15] DUBNAU D.DNA uptake in bacteria[J].Annu Rev Microbiol,1999,53:217-244.
[16]周鹏.副猪嗜血杆菌HAPS_RS00465蛋白的免疫保护性及其基因缺失株生物学特性研究[D].雅安:四川农业大学,2016.ZHOU P.Studies on the immunoprotection of H.Parasuis HAPS_RS00465protein and the biological characteristics of the gene deletion strains[D].Ya'an:Sichuan Agricultural University,2016.(in Chinese)
[17] MAUGHAN H,REDFIELD R J.Tracing the evolution of competence in Haemophilus influenzae[J].PLoS One,2009,4(6):e5854.
[18] HE L Q,WEN X T,YAN X F,et al.Effect of cheY deletion on growth and colonization in a Haemophilus parasuis serovar 13clinical strain EP3[J].Gene,2016,577(1):96-100.
[19] HOWELL K J,PETERS S E,WANG J H,et al.Development of a multiplex PCR assay for rapid molecular serotyping of Haemophilus parasuis[J].J Clin Microbiol,2015,53(12):3812-3821.
[20]谨瑾,代科,文心田,等.副猪嗜血杆菌自然转化方法的建立及条件优化[J].农业生物技术学报,2017,25(12):2058-2065.JIN J,DAI K,WEN X T,et al.Establishment and condition optimization for natural transformation method of Haemophilus parasuis[J].Journal of Agricultural Biotechnology,2017,25(12):2058-2065.(in Chinese)
[21] KRISTENSEN B M,SINHA S,BOYCE J D,et al.Natural transformation of Gallibacterium anatis[J].Appl Environ Microbiol,2012,78(14):4914-4922.
[22] LIU M F,ZHANG L,HUANG L,et al.Use of natural transformation to establish an easy knockout method in Riemerella anatipestifer[J].Appl Environ Microbiol,2017,83(9):e00127-17.
[23] BOSSEJ T,SINHA S,SCHIPPERS T,et al.Natural competence in strains of Actinobacillus pleuropneumoniae[J].FEMS Microbiol Lett,2009,298(1):124-130.