莠去津降解菌泛基因组测序及比较基因组分析
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摘要
Arthrobacter aurescens TC1和Pseudomonas sp. ADP是目前莠去津降解菌的模式菌株,筛选出Microbacterium sp.HBT4,旨在挖掘这3株不同种属细菌基因组间生物学信息的异同,并预测重要基因。通过Illumina Hiseq 4000测序平台采用DNA小文库制备和测序技术,进行了泛基因组测序,使用相关软件进行基因组组分分析、基因功能注释、基因间变异检测和比较基因组学分析,将分离得到的微杆菌HBT4与模式菌株进行核苷酸组成、共线性及菌株间变异差异分析。得到该菌株基因组大小约为3.53Mb,预测到菌株HBT4编码基因3 397个、重复序列含量为1.33%、非编码RNA 63个,通用数据库基因功能注释共3 324个,专用数据库基因功能注释共1 149个,通过菌株间差异变异分析发现SNP、Small InDel和水平转移基因,未发现结构变异基因,获得该菌株特有基因中GO注释到的基因在细胞组分、分子功能和生物学进程中的数量和比例,从KEGG代谢通路富集图中发现特有基因编码的二氢硫基赖氨酸残基琥珀酰转移酶位于三羧酸循环中α-酮戊二酸和琥珀酰辅酶A的代谢通路之间。获得3个菌株核心基因组与非必需基因组比例分布、系统进化树和共线性关系,发现三者之间共有基因家族986个、菌株HBT4特有基因家族1 171个。得到的菌株HBT4与两株模式菌株相比,其基因家族之间既有相同之处,又有较大差异。
Arthrobacter aurescens TC1 and Pseudomonas sp. ADP are currently the model strains of atrazine-degrading bacteria.Microbacterium sp. HBT4 was screened in this experiment for discovering the similarities and differences of biological information among the 3 bacterial genomes and predicting important genes. In this study,pan-genome sequencing was carried out by using small DNA library preparation and sequencing technology on Illumina Hiseq 4000 sequencing platform. Genome composition analysis,gene function annotation,gene mutation detection and comparative genomics analysis were carried out by using related software. The nucleotide composition,collinearity and variation differences between the isolated Microbacterium HBT4 and model strains were analyzed. The genome size of the strain was about3.53 Mb. It was predicted that there were 3 397 coding genes,1.33% repetitive sequences and 63 non-coding RNA in the strain HBT4. There were 3 324 annotations of gene function in general database and 1 149 annotations of gene function in special database. SNP,Small InDel and horizontal transfer genes were found through analyzing variance among strains and no structural variation genes were found. The number and proportion of GO-annotated genes in the specific genes of the strain in cell components,molecular functions and biological processes were obtained. From KEGG metabolic pathway enrichment map,it was found that the dihydrothiosyllysine residue succinyltransferase encoded by the specific gene was located between the metabolic pathway of α-ketoglutaric acid and succinyl coenzyme A in the tricarboxylic acid cycle. The proportional distribution,phylogenetic tree and collinearity of the core genome and non-essential genome of the three strains were obtained. It was found that there were 986 common gene families among the three strains,and 1171 specific gene families strain HBT4. Compared with the two model strains,the strain HBT4 obtained in this study had both similarities and differences in gene family.
Arthrobacter aurescens TC1 and Pseudomonas sp. ADP are currently the model strains of atrazine-degrading bacteria.Microbacterium sp. HBT4 was screened in this experiment for discovering the similarities and differences of biological information among the 3 bacterial genomes and predicting important genes. In this study,pan-genome sequencing was carried out by using small DNA library preparation and sequencing technology on Illumina Hiseq 4000 sequencing platform. Genome composition analysis,gene function annotation,gene mutation detection and comparative genomics analysis were carried out by using related software. The nucleotide composition,collinearity and variation differences between the isolated Microbacterium HBT4 and model strains were analyzed. The genome size of the strain was about3.53 Mb. It was predicted that there were 3 397 coding genes,1.33% repetitive sequences and 63 non-coding RNA in the strain HBT4. There were 3 324 annotations of gene function in general database and 1 149 annotations of gene function in special database. SNP,Small InDel and horizontal transfer genes were found through analyzing variance among strains and no structural variation genes were found. The number and proportion of GO-annotated genes in the specific genes of the strain in cell components,molecular functions and biological processes were obtained. From KEGG metabolic pathway enrichment map,it was found that the dihydrothiosyllysine residue succinyltransferase encoded by the specific gene was located between the metabolic pathway of α-ketoglutaric acid and succinyl coenzyme A in the tricarboxylic acid cycle. The proportional distribution,phylogenetic tree and collinearity of the core genome and non-essential genome of the three strains were obtained. It was found that there were 986 common gene families among the three strains,and 1171 specific gene families strain HBT4. Compared with the two model strains,the strain HBT4 obtained in this study had both similarities and differences in gene family.
引文
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[13]Mandelbaum RT, Allan DL, Wackett LP. Isolation and characterization of a Pseudomonas sp. that mineralizes the s-Triazine herbicide atrazine[J]. Appl Environ Microbiol, 1995, 61(4):1451-1457.
[14]Pooja B, Abhinav S, Sneha S, et al. Mapping atrazine and phenol degradationgenesinPseudomonassp.EGD-AKN5[J].Biochemical Engineering Journal, 2015, 102:125-134.
[15]Ana FTF, Vania SB, Anelize B, et al. Degradation of atrazine byPseudomonassp.andAchromobactersp.isolatedfrom Brazilian agricultural soil[J]. International Biodeterioration&Biodegradation, 2018, 130:17-22.
[16]Ye J, Zhang J, Gao J, et al. Isolation and characterization of atrazine-degrading strain Shewanella sp. YJY4 from cornfield soil[J]. Letters in Applied Microbiology, 2016, 63(1):45-52.
[17]Wang J, Zhu L, Wang Q, et al. Isolation and characterization of atrazine mineralizing bacillus subtilis strain HB-6[J]. PLoS One, 2014, 9(9):e107270.
[18]Sagarkar S, Bhardwaj P, Storck V, et al. s-triazine degrading bacterial isolate Arthrobacter sp. AK-YN10, a candidate for bioaugmentation of atrazine contaminated soil[J]. Applied Microbiology and Biotechnology, 2016, 100(2):903-913.
[19]Ojha S, Rana N, Mishra S. Fructo-oligosaccharide synthesis by whole cells of Microbacterium paraoxydans[J]. Tetrahedron:Asymmetry, 2016, 27(24):1245-1252.
[20]许尤厚,周洪波.产絮凝剂微杆菌的絮凝特性及印染废水处理应用[J].工业水处理, 2016(12):59-63.
[21]程仕伟,李坦坦,梁会会,等.响应面优化金橙黄微杆菌YT9的发酵条件生产纤维素酶[J].中国酿造, 2013, 32(4):48-51.
[22]徐天宇,胡苏莹,周峻岗,等.微杆菌Microbacterium sp.FY1538降解赤霉烯酮的活性研究[J].复旦学报:自然科学版, 2016, 55(2):223-231.
[23]Corretto E, Antonielli L, Sessitsch A, et al. Draft genome sequences of 10 Microbacterium spp., with emphasis on heavy metalcontaminated environments[J]. Genome Announc, 2015, 3(3):e00432.
[24]BankevichA,NurkS,AntipovD,etal.SPAdes:Anew genome assembly algorithm and its applications to single-cell sequencing[J]. Journal of Computational Biology, 2012, 19(5):455-477.
[25]Hyatt D, Chen GL, Locascio PF, et al. Prodigal:prokaryotic gene recognition and translation initiation site identification[J]. BMC Bioinformatics, 2010, 11(1):119-129.
[26]Chen N. Using repeat masker to identify repetitive elements in genomic sequences[J]. Current Protocols in Bioinformatics,2004, Chapter 4:Unit 4. 10.
[27]Lowe TM, Eddy SR. tRNAscan-SE:A program for improved detection of transfer RNA genes in genomic sequence[J].Nucleic Acids Research, 1997, 25(5):955-964.
[28]Nawrocki EP, Eddy SR. Infernal 1. 1:100-fold faster RNA homology searches[J]. Bioinformatics, 2013, 29(22):2933-2935.
[29]Nawrocki EP, Burge SW, Bateman A, et al. Rfam 12. 0:updates to the RNA families database[J]. Nucleic Acids Research, 2015,43(Database issue):D130-137.
[30]Tatusov RL. The COG database:a tool for genome-scale analysis of protein functions and evolution[J]. Nucleic Acids Research,2000, 28(1):33-36.
[31]Kanehisa M. The KEGG resource for deciphering the genome[J].Nucleic Acids Research, 2004, 32:D277-D280.
[32]Boeckmann B. The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003[J]. Nucleic Acids Research, 2003,31(1):365-370.
[33]邓泱泱,荔建琦,吴松锋,等. nr数据库分析及其本地化[J].计算机工程, 2006(5):71-73, 76.
[34]Altschul SF. Gapped BLAST and PSI-BLAST:a new generation of protein detabase search programs[J]. Nucleic Acids Res, 1997,25(17):3389-3402.
[35]Conesa A, Gotz S, Garcia-Gomez JM, et al. Blast2GO:a universal tool for annotation, visualization and analysis in functional genomics research[J]. Bioinformatics, 2005, 21(18):3674-3676.
[36]Ashburner M, Ball CA, Blake JA, et al. Gene ontology:tool for the unification of biology. The Gene Ontology Consortium[J]. Nature Genetics, 2000, 25(1):25-29.
[37]Finn RD, Coggill P, Eberhardt RY, et al. The Pfam protein families database:towards a more sustainable future.[J]. Nucleic Acids Research, 2016, 44(D1):D279-D285.
[38]Saier MH, Tran CV, Barabote RD. TCDB:The transporter classification database for membrane transport protein analyses and information[J]. Nucleic Acids Research, 2006, 34(Database issue):D181-D186.
[39]Rainer W, Thomas KB, Martin U, et al. PHI-base:a new database for pathogen host interactions[J]. Nucleic Acids Research,2006, 34(Database issue):D459-D464.
[40]Liu B, Pop M. ARDB--antibiotic resistance genes database[J].Nucleic Acids Research, 2009, 37(Database):D443-D447.
[41]Chen L. VFDB:A reference database for bacterial virulence factors[J]. Nucleic Acids Research, 2004, 33(Database issue):D325-D328.
[42]Cantarel BL, Coutinho PM, Rancurel C, et al. The carbohydrateactive enZymes database(CAZy):an expert resource for Glycogenomics[J]. Nucleic Acids Research, 2009, 37(Database):D233-D238.
[43]Delcher AL. Fast algorithms for large-scale genome alignment and comparison[J]. Nucleic Acids Research, 2002, 30(11):2478-2483.
[44]Vernikos GS, Parkhill J. Interpolated variable order motifs for identification of horizontally acquired DNA:Revisiting the Salmonella pathogenicity islands[J]. Bioinformatics, 2006, 22(18):2196-2203.
[45]Nguyen M, Ekstrom A, Li XQ, et al. HGT-Finder:A new tool for horizontal gene transfer finding and application to Aspergillus genomes[J]. Toxins, 2015, 7(10):4035-4053.
[46]Angiuoli SV, Salzberg SL. Mugsy:Fast multiple alignment of closely related whole genomes[J]. Bioinformatics, 2011, 27(3):334-342.
[47]Li L. OrthoMCL:Identification of ortholog groups for eukaryotic genomes[J]. Genome Research, 2003, 13(9):2178-2189.
[48]Guindon S, Dufayard JF, Lefort V, et al. New algorithms and methods to estimate maximum-Likelihood phylogenies:assessing the performance of PhyML 3. 0[J]. Systematic Biology, 2010, 59(3):307-321.
[49]史延华,任磊,贾阳,等.施氏假单胞菌YC-YH1的萘降解特性及产物分析[J].微生物学通报, 2015, 42(10):1866-1876.
[50]张丹,李兆格,包新光,等.细菌降解萘、菲的代谢途径及相关基因的研究进展[J].生物工程学报, 2010, 26(6):726-734.
[51]Gao YP, Fang JG, Du MR, et al. Response of the eelgrass(Zostera marina L.)to the combined effects of high temperatures and the herbicide, atrazine[J]. Aquatic Botany, 2017, 142:41-47.
[2]吴奇,宋福强.土壤中阿特拉津生物降解的研究进展[J].土壤与作物, 2017, 6(2):153-160.
[3]Jablonowski ND, Andreas S, Burauel P. Still present after all these years:persistence plus potential toxicity raise questions about the use of atrazine[J]. Environmental Science and Pollution Research, 2011, 18(2):328-331.
[4]Zhang Y, Meng DG, Wang ZG, et al. Oxidative stress response in atrazine-degrading bacteria exposed to atrazine.[J]. Journal of Hazardous Materials, 2012, 334(2):95-101.
[5]Gorito AM, Ribeiro AR, Almeida CMR, et al. A review on the application of constructed wetlands for the removal of priority substances and contaminants of emerging concern listed in recently launched EU legislation[J]. Environmental Pollution, 2017, 227:428-443.
[6]Tao Y, Hu SB, Han SY, et al. Efficient removal of atrazine by ironmodified biochar loaded Acinetobacter lwoffii DNS32.[J]. The Science of the Total Environment, 2019, 682:59-69.
[7]Renee MZ, Zakariya A, Ashley S. Whitaker, et al. Atrazine exposure affects growth, body condition and liver health in Xenopus laevis tadpoles[J]. Aquatic Toxicology, 2011, 104(3):243-253.
[8]齐文启,孙宗光,汪志国,等.环境荷尔蒙研究的现状及其监测分析[J].现代科学仪器, 2000(4):32-38.
[9]Helena P, Lucija ZK. Evaluation of photolysis and hydrolysis of atrazine and its first degradation products in the presence of humic acids[J]. Environmental Pollution, 2004, 133(3):517-529.
[10]蔺中,张倩,李文清,等.土壤阿特拉津的生物修复机制的研究[J].科技资讯, 2018, 16(11):116-117, 119.
[11]Morillo E, Villaverde J. Advanced technologies for the remediation of pesticide-contaminated soils[J]. Science of the Total Environment, 2017, 586:576-597.
[12]Zhong L, Zhen ZH, Lei R, et al. Effects of two ecological earthworm species on atrazine degradation performance and bacterial community structure in red soil[J]. Chemosphere, 2018, 196:467-475.
[13]Mandelbaum RT, Allan DL, Wackett LP. Isolation and characterization of a Pseudomonas sp. that mineralizes the s-Triazine herbicide atrazine[J]. Appl Environ Microbiol, 1995, 61(4):1451-1457.
[14]Pooja B, Abhinav S, Sneha S, et al. Mapping atrazine and phenol degradationgenesinPseudomonassp.EGD-AKN5[J].Biochemical Engineering Journal, 2015, 102:125-134.
[15]Ana FTF, Vania SB, Anelize B, et al. Degradation of atrazine byPseudomonassp.andAchromobactersp.isolatedfrom Brazilian agricultural soil[J]. International Biodeterioration&Biodegradation, 2018, 130:17-22.
[16]Ye J, Zhang J, Gao J, et al. Isolation and characterization of atrazine-degrading strain Shewanella sp. YJY4 from cornfield soil[J]. Letters in Applied Microbiology, 2016, 63(1):45-52.
[17]Wang J, Zhu L, Wang Q, et al. Isolation and characterization of atrazine mineralizing bacillus subtilis strain HB-6[J]. PLoS One, 2014, 9(9):e107270.
[18]Sagarkar S, Bhardwaj P, Storck V, et al. s-triazine degrading bacterial isolate Arthrobacter sp. AK-YN10, a candidate for bioaugmentation of atrazine contaminated soil[J]. Applied Microbiology and Biotechnology, 2016, 100(2):903-913.
[19]Ojha S, Rana N, Mishra S. Fructo-oligosaccharide synthesis by whole cells of Microbacterium paraoxydans[J]. Tetrahedron:Asymmetry, 2016, 27(24):1245-1252.
[20]许尤厚,周洪波.产絮凝剂微杆菌的絮凝特性及印染废水处理应用[J].工业水处理, 2016(12):59-63.
[21]程仕伟,李坦坦,梁会会,等.响应面优化金橙黄微杆菌YT9的发酵条件生产纤维素酶[J].中国酿造, 2013, 32(4):48-51.
[22]徐天宇,胡苏莹,周峻岗,等.微杆菌Microbacterium sp.FY1538降解赤霉烯酮的活性研究[J].复旦学报:自然科学版, 2016, 55(2):223-231.
[23]Corretto E, Antonielli L, Sessitsch A, et al. Draft genome sequences of 10 Microbacterium spp., with emphasis on heavy metalcontaminated environments[J]. Genome Announc, 2015, 3(3):e00432.
[24]BankevichA,NurkS,AntipovD,etal.SPAdes:Anew genome assembly algorithm and its applications to single-cell sequencing[J]. Journal of Computational Biology, 2012, 19(5):455-477.
[25]Hyatt D, Chen GL, Locascio PF, et al. Prodigal:prokaryotic gene recognition and translation initiation site identification[J]. BMC Bioinformatics, 2010, 11(1):119-129.
[26]Chen N. Using repeat masker to identify repetitive elements in genomic sequences[J]. Current Protocols in Bioinformatics,2004, Chapter 4:Unit 4. 10.
[27]Lowe TM, Eddy SR. tRNAscan-SE:A program for improved detection of transfer RNA genes in genomic sequence[J].Nucleic Acids Research, 1997, 25(5):955-964.
[28]Nawrocki EP, Eddy SR. Infernal 1. 1:100-fold faster RNA homology searches[J]. Bioinformatics, 2013, 29(22):2933-2935.
[29]Nawrocki EP, Burge SW, Bateman A, et al. Rfam 12. 0:updates to the RNA families database[J]. Nucleic Acids Research, 2015,43(Database issue):D130-137.
[30]Tatusov RL. The COG database:a tool for genome-scale analysis of protein functions and evolution[J]. Nucleic Acids Research,2000, 28(1):33-36.
[31]Kanehisa M. The KEGG resource for deciphering the genome[J].Nucleic Acids Research, 2004, 32:D277-D280.
[32]Boeckmann B. The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003[J]. Nucleic Acids Research, 2003,31(1):365-370.
[33]邓泱泱,荔建琦,吴松锋,等. nr数据库分析及其本地化[J].计算机工程, 2006(5):71-73, 76.
[34]Altschul SF. Gapped BLAST and PSI-BLAST:a new generation of protein detabase search programs[J]. Nucleic Acids Res, 1997,25(17):3389-3402.
[35]Conesa A, Gotz S, Garcia-Gomez JM, et al. Blast2GO:a universal tool for annotation, visualization and analysis in functional genomics research[J]. Bioinformatics, 2005, 21(18):3674-3676.
[36]Ashburner M, Ball CA, Blake JA, et al. Gene ontology:tool for the unification of biology. The Gene Ontology Consortium[J]. Nature Genetics, 2000, 25(1):25-29.
[37]Finn RD, Coggill P, Eberhardt RY, et al. The Pfam protein families database:towards a more sustainable future.[J]. Nucleic Acids Research, 2016, 44(D1):D279-D285.
[38]Saier MH, Tran CV, Barabote RD. TCDB:The transporter classification database for membrane transport protein analyses and information[J]. Nucleic Acids Research, 2006, 34(Database issue):D181-D186.
[39]Rainer W, Thomas KB, Martin U, et al. PHI-base:a new database for pathogen host interactions[J]. Nucleic Acids Research,2006, 34(Database issue):D459-D464.
[40]Liu B, Pop M. ARDB--antibiotic resistance genes database[J].Nucleic Acids Research, 2009, 37(Database):D443-D447.
[41]Chen L. VFDB:A reference database for bacterial virulence factors[J]. Nucleic Acids Research, 2004, 33(Database issue):D325-D328.
[42]Cantarel BL, Coutinho PM, Rancurel C, et al. The carbohydrateactive enZymes database(CAZy):an expert resource for Glycogenomics[J]. Nucleic Acids Research, 2009, 37(Database):D233-D238.
[43]Delcher AL. Fast algorithms for large-scale genome alignment and comparison[J]. Nucleic Acids Research, 2002, 30(11):2478-2483.
[44]Vernikos GS, Parkhill J. Interpolated variable order motifs for identification of horizontally acquired DNA:Revisiting the Salmonella pathogenicity islands[J]. Bioinformatics, 2006, 22(18):2196-2203.
[45]Nguyen M, Ekstrom A, Li XQ, et al. HGT-Finder:A new tool for horizontal gene transfer finding and application to Aspergillus genomes[J]. Toxins, 2015, 7(10):4035-4053.
[46]Angiuoli SV, Salzberg SL. Mugsy:Fast multiple alignment of closely related whole genomes[J]. Bioinformatics, 2011, 27(3):334-342.
[47]Li L. OrthoMCL:Identification of ortholog groups for eukaryotic genomes[J]. Genome Research, 2003, 13(9):2178-2189.
[48]Guindon S, Dufayard JF, Lefort V, et al. New algorithms and methods to estimate maximum-Likelihood phylogenies:assessing the performance of PhyML 3. 0[J]. Systematic Biology, 2010, 59(3):307-321.
[49]史延华,任磊,贾阳,等.施氏假单胞菌YC-YH1的萘降解特性及产物分析[J].微生物学通报, 2015, 42(10):1866-1876.
[50]张丹,李兆格,包新光,等.细菌降解萘、菲的代谢途径及相关基因的研究进展[J].生物工程学报, 2010, 26(6):726-734.
[51]Gao YP, Fang JG, Du MR, et al. Response of the eelgrass(Zostera marina L.)to the combined effects of high temperatures and the herbicide, atrazine[J]. Aquatic Botany, 2017, 142:41-47.
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