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1 微生物学通报 Microbiology China 研究报告 Aug. 20, 2015, 42(8): by Institute of Microbiology, CAS DOI: /j.microbiol.china 分枝杆菌噬菌体 Guo1 环状基因组特征及生物学特性分析 * 姚义勇江莉莎张莉郭述良柳岩 ( ) 摘要目的初步研究分枝杆菌噬菌体 Guo1 环状基因组特征及生物学特性方法采用鸟枪法和重叠群组装策略完成 Guo1 全基因组测序 ; 使用 DNAStar trnascan-se Promoter predictions TRF Glimmer BLAST 软件分别对噬菌体的一般基因组学特征 trna 启动子串联重复序列和编码基因进行分析 ; 运用 BLAST 软件检索与 Guo1 基因组相似的噬菌体并构建系统进化树, 推断 Guo1 的分群 ; 测定最佳感染复数一步生长曲线以及对紫外线氯仿酒精温度 ph 的敏感性结果噬菌体 Guo1 的基因组为全长 nt 的环状双链 DNA, 属于 G 群分枝杆菌噬菌体 ; 含有 59 个推定基因,7 个启动子序列和 3 个串联重复序列 ;Guo1 对紫外线有较强抵抗性, 对酒精氯仿和高温敏感, 最适温度为 37 C, 最适 ph 为 7.0, 最佳感染复数为 0.01, 潜伏期 120 min, 裂解期 120 min, 裂解量为 47 个结论 Guo1 是首次发现的具有环状双链 DNA 结构的 G 群分枝杆菌噬菌体, 完成了 Guo1 的全基因组测序, 并初步分析了 Guo1 的基因组特征和生物学特性关键词 Guo1, 分枝杆菌噬菌体 G 群, 环状基因组, 生物学特性 Circular genetic information and biologic characteristics of mycobacteriophage Guo1 YAO Yi-Yong JIANG Li-Sha ZHANG Li GUO Shu-Liang * LIU Yan (Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing , China) Abstract: [Objective] To investigate the genetic and biologic characteristics of mycobacteriophage Guo1 with circular genome. [Methods] Sequenced the complete genome of Guo1 with Shot-gun library and config package strategy. Performed DNAStar, trnascan-se, Promoter predictions, TRF, Glimmer, BLAST software for general genome characteristics, trna, promoter regions, tandem repeat sequences and putative function of predicted coding sequences, respectively. Utilized BLAST software to search for mycobacteriophages with genome similar as Guo1. Constructed phylogenetic tree to identify the cluster into which Guo1 should be grouped. Then measured the optimal 基金项目 : (No. 2012ZX ) (No. [2012] 649 ) * 通讯作者 : guosl999@sina.com 收稿日期 : 接受日期 : 优先数字出版日期 (

组测序 ; 使用 DNAStar trnascan-se Promoter predictions TRF Glimmer BLAST 软件分别对噬菌体的一般基因组学特征 trna 启动子串联重复序列和编码基因进行分析 ; 运用 BLAST 软件检索与 Guo1 基因组相似的噬菌体并构建系

2 1530 微生物学通报 Microbiol. China 2015, Vol.42, No.8 multiplicity of infection and one-step growth curve, and observed the sensibility to ultraviolet, chloroform, alcohol, temperature, ph. [Results] Guo1 contains a double strand circular DNA with a full length of nt, which belongs to the cluster G mycobacteriophage and which contains 59 putative genes, 7 promoter regions and three tandem repeat sequences. Guo1 is resistant to ultraviolet but sensitive to alcohol, chloroform and high temperature. The optimal temperature is 37 C while the optimal ph is 7.0. The optimal MOI of Guo1 is about One step growth of it showed that both latent period and burst period are 120 min, and the average burst size is about 47. [Conclusion] Guo1 is a cluster G mycobacteriophage with double strand circular DNA found for the first time. We sequenced the complete genome of Guo1 and identified the circular genetic information and biologic characteristics. Keywords: Guo1, Cluster G mycobacteriophage, Circular genome, Biologic characteristics 2012 WHO % [1] [2] % 8 9 [3] ( ) (Resuscitation promoting factor Rpf) [4] Rpf [5-6] [7-9] TM4 Motif3 Rpf DNA [10-11] TM4 [12] Guo1 TM4 Motif3 Guo1 Guo1 1 材料与方法 1.1 材料宿主菌及噬菌体来源 : (CMCC93202) Guo 主要试剂及仪器 :λ DNA Bgl II Fermentas DNaseⅠ RNase A Sigma 7H10 7H9 ( 0.4% ) BD (Phage buffer) Tris-base g MgSO g NaCl 4 g ddh 2 O 980 ml ph 7.5 CO 2 Thermo Forma Sigma Bio-Rad ph Mettler Toledo 1.2 方法纯化噬菌体颗粒 : 24 h 4 C g 10 min 0.22 μm DNaseⅠ( 40 mg/l) RNase A ( 10 mg/l) 37 C 30 min NaCl ( 58.5 g/l) 1 h g 10 min 10%

three tandem repeat sequences. Guo1 is resistant to ultraviolet but sensitive to alcohol, chloroform and high temperature. The optimal temperature is 37 C while the optimal ph is 7.0.

3 : Guo h g 10 min 1 ml Phage buffer 4 C g 10 min 0.22 μm 提取分枝杆菌噬菌体 Guo1 核酸 : λ DNA Guo1 10 ml 2 ml 1 h g 4 C 10 min 500 μl 100 μl 20% SDS 70 C 10 min 100 μl g 4 C 10 min 350 μl LB r/min 30 s 700 μl r/min 1 min r/min 2 min 1.5 ml 100 μl 50 C 1 min r/min 1 min DNA (A 260 ) (A 260 /A 280 ) DNA Guo1 全基因组序列测定 : Guo1 ( ) Phred/PhraD/Consedh PCR GenBank KJ 噬菌体基因组一般特性及编码基因预测分析 : DNAStar EditSeq G+C LOW LAB trnascan-se trna Promoter predictions TRF DNA (National Institutes of Health NIH) Glimmer Guo 系统进化树构建 : BLAST (Nucleotide blast) Tree Method Neighbor-Joining Max Seq Difference 0.05 Database nr [13] 1.3 噬菌体的生物学特性最佳感染复数的测定 :(Multiplicity of infection MOI) Lu [14] MOI MOI 37 C 24 h MOI 一步生长曲线的绘制 : [15] MOI=10 37 C 15 min g 1 min 7H9 2 5 ml 7H9 37 C (160 r/min) 300 min 30 min 100 μl g 1 min 3 ( 10 4 PFU/mL) =/

2 提取分枝杆菌噬菌体 Guo1 核酸 : λ DNA Guo1 10 ml 2 ml 1 h 10 000 g 4 C 10 min 500 μl 100 μl 20% SDS 70 C 10 min 100 μl 13 000 g 4 C 10 min 350 μl LB 12 000 r/min 30 s 700 μl 12 000 r/min 1 min 13 000

4 1532 微生物学通报 Microbiol. China 2015, Vol.42, No 噬菌体对紫外线照射的抵抗力 : 3 ml 6 cm (18 W 36 cm) 90 min 15 min 100 µl 30 min 3 0 min 100% 噬菌体对氯仿的敏感性 :750 µl 37 C 10 min g 15 min ddh 2 O 3 ddh 2 O 100% 噬菌体对酒精的抵抗力 :320 µl 95% µl ( 75%) 37 C 10 min g 15 min ddh 2 O 3 ddh 2 O 100% 温度对噬菌体的影响 : ( C) 30 min 60 min ( 10 5 PFU/mL) ph 对噬菌体的影响 : ph ( ) 7H9 37 C 1 h ph 2 结果与分析 2.1 基因组一般特性 Guo1 DNA nt A=5 789 C= G= T=7 502 G+C 66.84% G G+C trnascan-se trna 2.2 启动子序列和串联重复序列预测结果 Promoter predictions Guo1 7 1 TRF 噬菌体 Guo1 的编码基因预测分析 Glimmer 59 ( 3) 表 1 噬菌体 Guo1 启动子区的预测 Table 1 Putative promoter regions of mycobacteriophage Guo1 Start site End site Score Promoter sequence AACGGGTTGAGTCCGAGCTCGGATCCGTAGATCATCGCCGCGGCAGCCTC GTCTTGAGCAGAATCGGTTTCCAGCCGGCGATGATGGTCGCGGCGATGTT TCGGGTTGTCGAGCATCTGGTCGATGAGATAGATCGCTTCCTGCGCCGGC GAATGACTTGTGGGTGTGGACGGTGCCGATGTAGTTCATCCGCTTGTCCC TCGGTTTGAGTGCGACCGTCCAGAAGACGGAAATTGCTTGGCCGAACAGT GCCAGGTTCGAAGTCGAACGGGCCCAGGTTGTAATCCCTCACGGGCCCGT GAGGTCAGTGACGGGTCGATTCCGACGGCGATCATGCGCGGTGTCCCTTG Note: The 35 region and 10 region shown in underlined type; The transcription start shown in bold type. 表 2 噬菌体 Guo1 串联重复序列的预测 Table 2 Putative tandem repeat sequences of mycobacteriophage Guo1 Start site End site Length (nt) Tandem repeat sequences Score GGTTCATCGCCGCCGCCTGCT CCAGCGTCGGCCGGC CCGCAGTTCGGGTGCTGCTA 50

0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0) 7H9 37 C 1 h ph 2 结果与分析 2.1 基因组一般特性 Guo1 DNA 40 086 nt A=5 789 C=13 375 G=13 420 T=7 502 G+C 66.84% G G+C trnascan-se trna 2.

5 : Guo 表 3 噬菌体 Guo1 的基因注释 Table 3 Gene annotation of mycobacteriophage Guo1 Number Start site End site Length Strand Start codon NCBI NCBI annotation Putative function ATG Leo 0043 Hypothetical protein ATG Leo 0042 Hypothetical protein ATG Leo 0041 PDDEXK-like domain ATG Leo 0040 Hypothetical protein ATG Leo 0039 Hypothetical protein ATG Halo gp ATG Leo ASC-1-like protein GTG Halo gp37 Hypothetical protein ATG Leo 0035 Hypothetical protein ATG Sedge repressor ATG Angel gp34 Helix-turn-helix domain TTG Leo Integrase GTG Legendre 0028 Hypothetical protein GTG DNAIII 0030 Hypothetical protein ATG Halo gp ATG Halo gp28 Lysin B ATG Halo gp27 Lysin A ATG Halo gp ATG Halo gp ATG Halo gp ATG Leo Minor tail protein ATG BPs ATG Leo Minor tail protein ATG Leo Minor tail protein ATG Halo Minor tail protein ATG Halo Minor tail protein ATG Halo Minor tail protein ATG Halo Tape measure protein GTG Leo 0015 Hypothetical protein ATG Bps Potenial virion protein ATG Bps Major tail subunit ATG BPs 112 Hypothetical protein ATG BPs 111 Hypothetical protein ATG BPs 110 Hypothetical protein ATG BPs 19 Hypothetical protein ATG BPs 18 Hypothetical protein ATG Chy2 006 Capsid protein ATG BPs 16 Hypothetical protein ATG Halo gp5 Hypothetical protein ATG BPs 14 Major surface protein1b ( )

351 ATG Leo 0039 Hypothetical protein 6 3 802 3 951 150 ATG Halo gp39 7 3 948 4 298 351 ATG Leo ASC-1-like protein 8 4 352 4 468 117 + GTG Halo gp37 Hypothetical protein 9 4 477 5 013 537 ATG Leo

6 1534 微生物学通报 Microbiol. China 2015, Vol.42, No.8 ( 3) ATG Angel gp3 Portal protein GTG Sedge 003 Portal protein ATG Leo Terminase ATG Halo gp ATG Chy Hypothetical protein ATG Halo gp ATG Leo 0056 Pfam ATG Halo gp ATG DNAIII 0054 Hypothetical protein GTG DNAIII 0055 Hypothetical protein ATG Leo 0053 Hypothetical protein ATG Legendre 006 Hypothetical protein ATG Halo gp GTG Legendre 007 Hypothetical protein ATG Leo RuvC ATG Halo gp ATG Leo 0047 Hypothetical protein ATG Leo 0046 Hypothetical protein ATG Leo 0044 Hypothetical protein 3 51 ATG 7 GTG 1 TTG Guo1 28 (4 014 nt) 4 (153 nt) B A 系统进化树构建 Guo1 BLAST Guo1 G 95% 98% E ( 1) Guo1 Leo BO4 DNAⅢ 3 G [16-18] Liefie Angel Halo Hope BPs Avrafan G Guo1 G 2.5 生物学特性分析最佳感染复数测定 : h 4 MOI= 一步生长曲线的绘制 : 2 Guo1 120 min 120 min 240 min 噬菌体对紫外线照射的抵抗力 : 3 30 min 70% 30 min 60 min 75 min 90 min 62%

672 822 + ATG Chy2 0058 Hypothetical protein 46 34 651 34 893 243 ATG Halo gp63 47 34 992 35 327 336 ATG Leo 0056 Pfam05305 48 35 359 35 541 183 ATG Halo gp61 49 35 541 35 756 216 ATG DNAIII 0054

7 : Guo 图 1 噬菌体 Guo1 的系统进化树 Figure 1 Phylogenetic tree of mycobacteriophage Guo1 BLAST (Tree method Neighbor-Joining Max Seq Difference 0.05 Database nr) GenBank. Note: Phylogenetic tree was constructed by BLAST (Tree method: Neighbor-Joining, Max Seq Difference: 0.05, Database: nr); The numbers in the bracket denote GenBank accession number, the scale plate denote evolutionary distance. MOI Table 4 Number of bacterium phage (PFU/mL) 表 4 感染复数的测定 Determination of multiplicity of infection Number of bacteria (CFU/mL) 24 h Titer at 24 h (PFU/mL) 噬菌体对氯仿的敏感性 : 10 min ddh 2 O 56% 噬菌体对酒精的抵抗力 : 10 min ddh 2 O min 75% 温度对噬菌体的影响 : 4 37 C 60 C 30 min 37 C 30 min 60 min ph 对噬菌体的影响 : 5 ph 7.0 ph>11.0 ph<5.0 (ph= PFU/mL) ph<4.0

05, Database: nr); The numbers in the bracket denote GenBank accession number, the scale plate denote evolutionary distance.

8 1536 微生物学通报 Microbiol. China 2015, Vol.42, No.8 图 5 ph 对噬菌体 Guo1 活性的影响 Figure 5 Influence of ph on the activity of Guo1 图 2 噬菌体 Guo1 一步生长曲线 Figure 2 One step growth curve of Guo1 图 3 噬菌体 Guo1 对紫外线的耐受性 Figure 3 Ultraviolet resistibility of Guo1 图 4 温度对噬菌体 Guo1 的影响 Figure 4 Influence of temperature on Guo1 3 讨论 DNA DNA RNA Guo1 G [19-20] G 50% 1% [21] Guo1 3 [22-23] trna trna trna Guo1 trna Guo1 12 Leo [18] 10 Sedge [24] Guo1

2 One step growth curve of Guo1 图 3 噬菌体 Guo1 对紫外线的耐受性 Figure 3 Ultraviolet resistibility of Guo1 图 4 温度对

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