分枝杆菌噬菌体的生物学特性及基因组学研究
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摘要
目的
结核病疫情严峻，耐药率高，新型抗结核药物研发越来越困难，分枝杆菌噬菌体逐渐成为新型抗结核药物的研究热点之一。目前还无文献报道在国内发现新分枝杆菌噬菌体，本研究在国内首次分离鉴定出5株分枝杆菌噬菌体(Mycobacterium Phage)，命名为Chy1、Chy2、Chy3、Chy4和Chy5，通过对其生物学特性和基因组学进行研究，探寻这5株分枝杆菌噬菌体抗结核菌的潜力，明确其遗传背景，为噬菌体的进一步开发利用提供理论基础和方法指导。
方法
1采用直接分离法和富集法从泥土标本中分离、纯化分枝杆菌噬菌体。
2比较噬菌体滴度测定、噬菌体大量扩增和收获的不同方法，优化实验条件。
3分枝杆菌噬菌体生物学特性研究
3.1采用PEG8000沉淀法纯化分枝杆菌噬菌体，电镜观察噬菌体形态特征。
3.2以不同MOI扩增分枝杆菌噬菌体，根据扩增后的产量找出每株噬菌体的最佳MOI。
3.3通过一步生长实验测定分枝杆菌噬菌体潜伏期、裂解期和裂解量。
3.4分离临床病原菌，用罗氏药敏培养基进行初步菌种鉴定和药敏实验，再采用单斑法测定分枝杆菌噬菌体的宿主谱。
3.5比较分枝杆菌噬菌体在pH值为5.0和7.4的7H9固体培养基中裂解耻垢分枝杆菌mc2155的能力。
3.6比较Chy1、Chy2、Chy3、D294种噬菌体组成鸡尾酒制剂与这4种噬菌体分别单独裂解耻垢分枝杆菌mc2155的能力，实验数据采用SPSS17.0统计软件中的单因素方差分析进行统计分析，以P <0.05为差异有统计学意义。
3.7培养原代巨噬细胞，巨噬细胞吞噬结核菌后分为3组，1组加入Chy3，2组加入D29，3组为不加噬菌体的空白对照组，培养过夜后，收集细胞，电镜观察噬菌体对巨噬细胞胞内结核菌的影响。
4分枝杆菌噬菌体全基因组测序
4.1用λ噬菌体DNA提取试剂盒提取分枝杆菌噬菌体基因组DNA，限制性内切酶酶切后进行琼脂糖凝胶电泳，观察电泳结果。
4.2基于鸟枪法随机测序和重叠群组装的方法进行测序：构建文库，随机挑取克隆片段测序，拼接成大小不一的重叠群，最后通过PCR法填补各重叠群之间的缺口，得到完整的全基因组序列。
4.3组装成环形的噬菌体采用DNAStar程序包中的GeneQuest软件对其基因组列进行电子酶切分析，并与实际酶切图谱进行比较，以确定噬菌体基因组是否为环形。
5分枝杆菌噬菌体基因组生物信息学分析
5.1分枝杆菌噬菌体基因预测及基因组注释
⑴分析5株分枝杆菌噬菌体基因组的一般特性：①采用DNAStar软件包中Editseq软件分析基因组大小，碱基组成，基因平均长度、基因密度以及5株噬菌体遗传密码子的使用频率及其偏嗜性。②采用TRF软件预测基因组中的串联重复序列。③通过tRNAscan软件预测基因组中的tRNA区域。
⑵采用Glimmer3.0和GeneMark基因预测软件对基因组进行基因de novo预测，推定5株分枝杆菌噬菌体基因组编码序列。
⑶用Blast和Clustal软件将推定基因与nr，swissport，tremble，cog数据库中的序列进行同源比对，E value为1e-5，对每个预测出来的基因选取同源比对上分值最高的序列，预测基因功能。
6分枝杆菌噬菌体比较基因组学
6.1将5个分枝杆菌噬菌体的基因与公共数据库数据进行Blast m8比对，e-value <=1e-5，选取每个蛋白的最好比对结果，将两两比对均是最好的结果保留，绘制共线性图。
6.2先将所有的基因序列用Clustal W软件进行两两比对，计算得到包含每对序列分歧程度的距离矩阵，再根据距离矩阵计算得到引导树，最后根据前面得到的引导树分支顺序，逐级比对，得到全部序列的全局比对结果，用tree view软件生成进化树。
结果
1采用富集法分离纯化得到5株分枝杆菌噬菌体,根据分离地点分别命名为Chy1、Chy2、Chy3、Chy4和Chy5。这5株噬菌体的噬菌斑均为圆形、透明、清晰，呈裂解性噬菌体噬菌斑特点，但Chy2、Chy4和Chy5培养超过48h后噬菌斑逐渐变混浊。
2优化实验方法，后续实验均采取小平板法测定噬菌体滴度，双层固体培养基培养的方法大量扩增噬菌体以及刮取上层培养基离心的方法收集噬菌体。
3分枝杆菌噬菌体生物学特性研究
3.1Chy1、Chy3、Chy4和Chy5噬菌体头部均为多面立体对称，而Chy2噬菌体头部呈椭圆形。5株噬菌体均具有长尾，可弯曲，但不可收缩，可见尾板和尾丝，依据ICTV第八次报告的最新病毒分类系统的标准属长尾噬菌体科(Siphoviridae)。
3.2Chy1最佳MOI为1.4×10~-6，Chy~2最佳MOI为9.58×10~-5，Chy3最佳MOI为1.1×10~-4，Chy4最佳MOI为4.5×10~-5，Chy5最佳MOI为1.8×10~-5，后续实验大量扩增这些噬菌体时，均根据最佳MOI扩增，以达到最大产量。
3.3Chy~2感染耻垢分枝杆菌mc~2155的潜伏时间约为210min，裂解期为60min，270min后进入平台期，噬菌体滴度不再增加，裂解量为23；Chy3感染耻垢分枝杆菌mc~2155的潜伏时间约为150min，裂解期为90min，240min后进入平台期，噬菌体滴度不再增加，裂解量为40；Chy1、Chy4和Chy5潜伏期超过420min。
3.4共收集临床病原菌36株，经菌种鉴定，有32株为结核分枝杆菌(结核菌)，2株为牛结核分枝杆菌，2株为非结核分枝杆菌。这5株分枝杆菌噬菌体的宿主谱较广，能裂解MS mc2155、结核菌标准株H37Rv、大部分临床分离耐药结核菌株、耐药牛结核分枝杆菌和非结核分枝杆菌，其中Chy1对广泛耐药结核菌裂解率达100％。
3.5抗菌能力最强的噬菌体是D29(P <0.05)，48h内几乎裂解所有的耻垢分枝杆菌mc~2155，48h后细菌数量逐渐回升，但仍比初始加入的细菌数少，维持在较低水平，其次是鸡尾酒制剂，24h内能裂解大多数耻垢分枝杆菌mc2155，但24h后细菌数逐渐增多，到96h时细菌数与Chy2和Chy3组已无差别(P>0.05)。Chy1、Chy2和Chy33个新分离的噬菌体中，Chy3早期(48h)杀菌能力最强(P <0.05)，但到96h时细菌数与Chy2相当(P>0.05)，Chy1杀菌能力最弱，在48h内不能抑制细菌生长，但48h后MS不再增加，维持在109CFU左右，细菌数比空白对照组少(P <0.05)。
3.6当7H9固体培养基pH值为5.0和7.4时，Chy1、Chy2和Chy3均能裂解耻垢分枝杆菌mc~2155。
3.7电镜观察发现Chy3组和D29组结核菌数量少，菌体结构明显被破坏，而空白对照组结核菌数量较多，细菌结构仍完整。
4分枝杆菌噬菌体全基因组测序
4.15株噬菌体基因组DNA均可被EcoRⅠ、Hind Ⅲ和BamH I切开，这3种核酸内切酶均能识别并切割双链DNA分子特定的核苷酸序列，表明这5株噬菌体基因组均为双链DNA。分析酶切图谱发现这5株噬菌体基因组大小均在40-50kb之间。4.25株噬菌体测序质量达到完成图标准。Chy1、Chy2、Chy4和Chy5最终均组装为线状重叠群。虽然Chy3组装为环状重叠群，但实际酶切图谱中，Hind Ⅲ将Chy3基因组切为2个条带，与线性电子酶切图谱相同，所以Chy3基因组很可能是线性。5分枝杆菌噬菌体基因预测及基因组注释5.1Chy1⑴Chy1基因组全长为47198bp，G＋C含量为63.68%，基因平均长度为562bp，基因密度为0.92。Chy1有明显的遗传密码子偏嗜性，未发现串联重复序列，共预测出5个tRNA。⑵采用glimmer软件分析，确定了77个推定基因(GL)，对这些基因进行同源检索分析，得到29个已知功能的基因。GL05、GL10、GL12、GL13、GL20、GL23、GL24和GL25为噬菌体的结构基因；GL06、GL07和GL08分别编码LysA、holin和LysB；GL31为整合酶基因，GL70推定为阻遏蛋白类似基因，与D29p72基因序列100％相似，D29p72在进化过程中丢失了N-末端的一个片段，缺乏完整的HTH模体，不能结合到宿主菌DNA上，因而不能形成溶原性反应，即Chy1为裂解性噬菌体。GeneMark软件分析，共预测出71个推定基因(GM)，其中有55个基因与glimmer软件分析结果相同，有16个不同的基因，通过比对，发现GM22与GL23， GM08与GL09， GM51与GL55， GM52与GL56具有相同功能，其余基因未能匹配上已知功能的基因。5.2Chy2
⑴Chy2基因组全长为40017bp， G+C=66.73%，基因平均长度为656bp，基因密度为0.951。Chy2有明显的遗传密码子偏嗜性，共发现3个串联重复序列，未发现tRNA。
⑵采用glimmer在线分析软件分析，确定了58个推定基因，对这些基因进行同源检索分析，得到16个已知功能的基因。GL02、GL05、GL06、GL12、GL13、GL15、GL17和GL19为噬菌体的结构基因；GL26、GL27分别编码LysA和LysB，未发现holin基因；GL31为整合酶基因，GL32推定为阻遏蛋白基因，与分枝杆菌噬菌体Halo的gp33基因序列100％相似，Halo是溶原性噬菌体，gp33表达的产物是阻遏蛋白，即Chy2为溶原性噬菌体。GeneMark软件分析，共预测出54个推定基因，其中有44个基因与glimmer软件分析结果相同，有10个不同的基因，通过比对，发现GM15与GL12具有相同功能，其余基因未能匹配上已知功能的基因。
5.3Chy3
⑴Chy3基因组全长为40070bp， G+C=66.86%，基因平均长度为667bp，基因密度为0.97。Chy3有明显的遗传密码子偏嗜性，共发现3个串联重复序列，未发现tRNA。
⑵采用glimmer在线分析软件分析，确定了58个推定基因，对这些基因进行逐个同源检索分析，得到20个已知功能的基因。GL01、GL41、GL43、GL45、GL47、GL48、GL54、GL55和GL58为噬菌体的结构基因，位于整合酶基因(GL29)的两侧；GL33、GL34分别编码LysB和LysA，未发现holin基因和编码阻遏蛋白的基因，即Chy3为裂解性噬菌体。GeneMark软件分析，共预测出56个推定基因，其中有46个基因与glimmer软件分析结果相同，有10个不同的基因，通过比对，发现GM43与GL43具有相同功能，其余基因未能匹配上已知功能的基因。
5.4Chy4
⑴Chy4基因组全长为46639bp， G+C=63.68%，基因平均长度为586bp，基因密度为0.92。Chy4有明显的遗传密码子偏嗜性，未发现串联重复序列，共发现5个tRNA。
⑵采用glimmer在线分析软件分析，确定了73个推定基因，对这些基因进行同源检索分析，得到28个已知功能的基因。GL05、GL10、GL12、GL13、GL20、GL23、GL24和GL25为噬菌体的结构基因；GL06、GL07和GL08分别编码LysA、holin和LysB；GL31为整合酶基因，GL71与编码噬菌体L5阻遏蛋白的gp71相似性为86.3％，L5gp71具有183aa，而Chy4GL71仅含155个aa，是否能编码有活性的阻遏蛋白还未知，即Chy4很可能为溶原性噬菌体。GeneMark软件分析，共预测出70个推定基因，其中有55个基因与glimmer软件分析结果相同，有15个不同的基因，通过比对，发现GM09与GL09，GM23与GL23，GM46与GL47，GM54与GL57具有相同功能，其余基因未能匹配上已知功能的基因。
5.5Chy5
⑴Chy5基因组全长为51214bp，G+C=66.74%，基因平均长度为530bp，基因密度为0.91。Chy5有明显的遗传密码子偏嗜性，未发现串联重复序列，共发现5个tRNA。
⑵采用glimmer在线分析软件分析，确定了88个推定基因，对这些基因进行逐个同源检索分析，得到29个已知功能的基因。GL05、GL10、GL12、GL13、GL20、GL23、GL24和GL25为噬菌体的结构基因；GL06、GL07、GL08分别编码LysA、holin和LysB；GL31为整合酶基因，gene70与分枝杆菌噬菌体Pukovnik编码阻遏蛋白的基因gp72相似性为70.81％，推定为编码阻遏蛋白的基因，是否能编码有活性的阻遏蛋白还未知，即Chy5很可能为溶原性噬菌体。GeneMark软件分析，共预测出86个基因，其中有68个基因与Glimmer3.0软件分析结果相同，有18个不同的基因，通过比对，发现GM09与GL09，GM12与GL12，GM23与GL23，GM55与GL54，GM63与GL62具有相同功能，其余基因未能匹配上已知功能的基因。
6分枝杆菌噬菌体比较基因组学
6.1共线性分析
⑴Chy1同源性最高的两株噬菌体为D29和Che12，可归入ClusterA，同源性基因分布在整个基因组，具有清晰的共线性。Chy1基因组77个基因中有75个基因与D29的基因具有同源性，有60个基因与Che12的基因具有同源性。
⑵Chy2同源性最高的两株噬菌体为BPs和Angel，可归入ClusterG，同源性基因分布在整个基因组，具有清晰的共线性。Chy2基因组58个基因中有57个基因均与这两个噬菌体具有同源性。
⑶Chy3同源性最高的两株噬菌体为BPs和Angel，可归入ClusterG，同源性基因分布在整个基因组，Chy3基因组58个基因组中有56个基因均与这两个噬菌体具有同源性。Chy3基因组前3个基因与BPs和Angel前3个基因匹配，但从第4个基因开始与BPs和Angel的同源基因排列顺序相反。
⑷Chy4同源性最高的两株噬菌体为D29和Che12，可归入ClusterA，同源性基因分布在整个基因组，具有清晰的共线性。Chy4基因组73个基因组中有70个基因与D29具有同源性，有54个基因与Che12具有同源性。
⑸Chy5同源性最高的两株噬菌体为D29和Pukovnik，可归入Cluster A，同源性基因分布在整个基因组，具有清晰的共线性。Chy5基因组88个基因组中有70个基因与D29具有同源性，有57个基因与Pukovnik具有同源性。
6.2进化树分析
对14株分枝杆菌噬菌体基因组进行系统发育分析构建进化树，距离越近，噬菌体间亲缘关系越近。Chy1与D29亲缘关系最近，Chy4与Chy5亲缘关系最近， Chy2与BPs、Angel亲缘关系最近，Chy3与Legendre亲缘关系最近。
结论
5株分枝杆菌噬菌体均属于双链DNA病毒群(群Ⅰ)，尾病毒目，长尾病毒科。Chy1和Chy3为裂解性噬菌体，Chy2为溶原性噬菌体，Chy4和Chy5为可疑溶原性噬菌体，基因组学研究未在噬菌体基因组中发现毒力基因。5株噬菌体均能裂解耐药结核分枝杆菌，Chy3潜伏期短，体外裂菌能力强且能裂解巨噬细胞内结核分枝杆菌，最具有抗结核潜力。本研究初步探明5株新分枝杆菌噬菌体抗结核菌的潜力和遗传背景，为下一步开发利用噬菌体提供理论基础和方法指导。
Objective
Mycobacterium Phage can be used as a tool in the diagnosis, treatmentof tuberculosis and a genetic tool for analysis of mycobacterium genetics.The study of Mycobacterium Phage biological characteristics and genomicshelps us to identify its role in the physiology, genetics, and evolution ofmycobacterium. Here I report the isolation, sequencing and comparativegenomic analysis of five new Mycobacterium Phages for the first time inchina, which are named Chy1, Chy2, Chy3, Chy4and Chy5, respectively.The purpose of this study is to explore the potential of Mycobacterium Phagein TB diagnosis and treatment and identify the genetic background of thefive new Phages in order to provide a theoretical basis for the furtherdevelopment and utilization of them.
Methods
1Mycobacterium Phages were Isolated and Purified from soil.
2Different ways of plaque assay, Phage amplification and harvest werecompared to find the optimized experimental conditions.
3The study of biological characteristics of Mycobacterium Phage
3.1Mycobacterium Phages were purified by PEG8000precipitationand the morphological characteristics were observed by electronmicroscopy.
3.2Mycobacterium Phages were amplified by double-layer agar platemethod in different multiplicity of infection (MOI) to find the optimal MOI.
3.3One step growth experiment was done to find the latent period,burst period and burst size of Mycobacterium Phages.
3.4The host range of Mycobacterium Phages was examined bysingle-spot examination.
3.5The effect of pH values on lysis ability of Mycobacterium Phageswas surveyed.
3.6The lysis ability of Phage cocktail, Chy1, Chy2, Chy3and D29wascompared and the one-way analysis of variance (ANOVA) is used todetermine whether there are any significant differences between the meansof five independent (unrelated) groups.
3.7Murine macroPhages were isolated and cultured, which wereinfected by mycobacterium tuberculosis and divided in to three groups.Group1was added in Chy3, group2was added in D29and group3wasblank control group. Three groups were cultured overnight and the cellswere collected and observed by electron microscopy.
4Mycobacterium Phage genome sequencing
4.1The genome of Phages was extracted and the type of nucleic acidwas identified by restriction enzyme analysis to check the DNA for quantity,quality and a first estimate of the genome size.
4.2DNA was broken up randomly into small segments, which were sequenced using the chain termination method to obtain reads. Multipleoverlapping reads for the target DNA were obtained by performing severalrounds of this fragmentation and sequencing. Computer programs then usedthe overlapping ends of different reads to assemble them into a continuoussequence.
4.3The electron restriction map, which were analyzed by GeneQuestsoftware in DNAStar package, and the actual restriction map were comparedto determine whether the Chy3genome is circular.
5Bioinformatics of Mycobacterium Phage genome
5.1Gene prediction and genome annotation of Mycobacterium Phages
(1) The genome size, base composition, the average gene length and thegene density of five Phages were analyzed using Editseq software inDNAStar package. TRF (Tandem Repeat Finder) software was used tosearch for tandem repeat and tRNAscan software was used to search fortRNA region.
(2) Glimmer and GeneMark were used to predict where theprotein-coding genes of five Mycobacterium Phages are and identified theputative genes.
(3) Sequence similarity analyses were performed with gapped BLASTpalgorithm against the non-redundant database provided by NCBI.
6Comparative genomics of Mycobacterium Phages
6.1A comparative analysis of five Mycobacterium Phages with their two closest relatives respectively was carried out Clustal W and created SVGimages using Perl.
6.2A phylogenetic supertree of14Phage genomes was constructedusing ClustalW and tree view.
Results
1Five new Mycobacterium Phages were successfully isolated andpurified, which were named Chy1, Chy2, Chy3, Chy4and Chy5,respectively. The plaques of five Phages all are transparent, but the plaquesof Chy2, Chy4and Chy5become turbid cultured after48h.
2The optimal method to measure the titer is small plate plaque assay.The best way to amplify Phages abundantly is that Phage and MS in optimalMOI were cultured in double-solid plat and the upper medium was scrapeddown after48h and centrifuged to collect the Phages.
3The biological characteristics of Mycobacterium Phages
3.1Chy1, Chy3, Chy4and Chy5all have an isometric head, and Chy2has a oval head. All the five Phages have long tail, base plate, tail fiber, andall belong to Siphoviridae.
3.2The optimal MOI of Chy1, Chy2, Chy3, Chy4and Chy5are1.4×10~(-6),9.58×10~(-5),1.1×10~(-4),4.5×10~(-5)and1.8×10~(-5), respectively.
3.3The one-step growth cycle of Chy2is270min, in which the latentperiod is210min, the burst period is60min and the burst size is23. Theone-step growth cycle of Chy3is240min, in which the latent period is150 min, the burst period is90min and the burst size is44. The latent period ofChy1, Chy4and Chy5are all more than420min.
3.4The Phages can lyse MS mc~2155, Mycobacterium TuberculosisH37Rv and the majority of clinical drug-resistant strains.
3.5D29has the most efficient sterilization and can kill almost all of MSwithin48hours, the number of MS roses gradually after48h, but still lowerthan the initial bacterial number. The second is the Phage cocktail, which cankill most MS within24hours, and then the number of bacteria graduallyincreased and equal to the number of bacteria with Chy2and Chy3group at96h. Chy3has the most efficient sterilization in the three newly isolatedPhages within48h, but the number of MS in the three groups has nodifference at96h. Chy1has the weakest bactericidal capacity, and can notinhibit bacterial growth within48hours, but the number of MS maintainsaround in109CFU after48h and no longer increases.
3.6Chy1、Chy2and Chy3can lyse Mycobacterium smegmatis mc~2155in solid medium with pH5.0and7.4.
3.7Electron microscopy shows that the number of Mycobacteriumtuberculosis is lower in the Chy3group and D29group, of which thebacterial structure is destroyed more seriously.
4Genome sequencing of Mycobacterium Phages
4.1The genome of five Phages can be digested by restrictionendonuclease EcoRⅠ, Hind Ⅲand BamH I, and the size of the genomes all are between40kb and50kb.
4.2Multiple overlapping reads for the target DNA were obtained andComputer programs then used the overlapping ends of different reads toassemble them into a continuous sequence. Chy1, Chy2, Chy4and Chy5areassembled into linear genome, and Chy3is circular.
5Gene prediction and genome annotation of five MycobacteriumPhages
5.1Chy1
(1) The length of Chy1genome is47198bp, the%GC content is63.68%, the average length of gene is562bp, and the gene density of thegenome is0.92. Chy1has a significant codon usage bias and has five tRNAgenes and no tandem repeats.
(2)77genes (GL) are identified in the Chy1Phage by glimmersoftware, each of which was submitted to BLAST analysis. Of the77genes,29have similarity to proteins with known functions and the rest arehomologous to those with unknown function. GL05, GL10, GL12, GL13,GL20, GL23, GL24and GL25are involved in the virion structure. GL06,GL07, GL08are assigned to be the lyses cassette. GL31is assigned to beintegrase. GL70shared100%homology with repressor gene (gp72) of D29.However, gp72of D29has lost a segment of its N-terminal during evolutionhence lacking HTH motif, which prevent it from binding to DNA, so Chy1islytic Phage.
5.2Chy2
(1) The length of Chy2genome is40017bp, the%GC content is66.73%, the average length of gene is656bp, and the gene density of thegenome is0.95. Chy2has a significant codon usage bias and has none tRNAgenes and three tandem repeats.
(2)58genes are identified in the Chy2Phage, each of which wassubmitted to BLAST analysis. Of the58genes,16have similarity to proteinswith known functions and the rest are homologous to those with unknownfunction. GL02, GL05, GL06, GL12, GL13, GL15, GL17and GL19areinvolved in the virion structure. GL26and GL27are assigned to be the lysescassette. GL31is assigned to be integrase. Gene32shared100%homologywith repressor gene (gp33) of Halo. The product of gp33is an activerepressor protein, so Chy2is lysogenic Phage.
5.3Chy3
(1) The length of Chy3genome is40070bp, the%GC content is66.86%, the average length of gene is667bp, and the gene density of thegenome is0.97. Chy3has a significant codon usage bias and has none tRNAgenes and three tandem repeats. Restriction analysis shows that the genomeof Chy3is digested into two bands by Hind Ⅲ, which is the same to thelinear electron restriction map, but there are two bands in the actualrestriction map digested by EcoRⅠ, and three in the linear electronrestriction map. So Chy3may have a linear genome.
(2)58genes are identified in the Chy3Phage, each of which wassubmitted to BLAST analysis. Of the58genes,20have similarity to proteinswith known functions and the rest are homologous to those with unknownfunction. GL01, GL41, GL43, GL45, GL47, GL48, GL54, GL55andGL58are involved in the virion structure. GL33and GL34are assigned tobe the lyses cassette. GL29is assigned to be integrase, but there is no gene isassigned to be repressor, so Chy3is lysogenic Phage.
5.4Chy4
(1) The length of Chy4genome is46639bp, the%GC content is63.68%, the average length of gene is586bp, and the gene density of thegenome is0.92. Chy4has a significant codon usage bias and has five tRNAgenes and none tandem repeats.
(2)73genes are identified in the Chy4Phage, each of which wassubmitted to BLAST analysis. Of the73genes,28have similarity to proteinswith known functions and the rest are homologous to those with unknownfunction. GL05, GL10, GL12, GL13, GL20, GL23, GL24and GL25areinvolved in the virion structure. GL06, GL07, GL08are assigned to be thelyses cassette. GL31is assigned to be integrase. GL71shared86.3％homology with repressor gene (gp71) of lysogenic Phage L5. However, gp71has188aa, and Chy4gene71has155aa, whether the repressor protein ofChy4has active function is also unknown, so Chy4may be a lysogenicPhage.
5.5Chy5
(1) The length of Chy4genome is51214bp, the%GC content is66.74%, the average length of gene is530bp, and the gene density of thegenome is0.91. Chy5has a significant codon usage bias and has five tRNAgenes and none tandem repeats.
(2)88genes are identified in the Chy5Phage, each of which wassubmitted to BLAST analysis. Of the88genes,29have similarity to proteinswith known functions and the rest are homologous to those with unknownfunction. GL05, GL10, GL12, GL13, GL20, GL23, GL24and GL25areinvolved in the virion structure. GL06, GL07, GL08are assigned to be thelyses cassette. GL31is assigned to be integrase. GL70shared70.81％homology with repressor gene (gp72) of Phage Pukovnik, whether therepressor protein of Chy5has active function is also unknown, so Chy5maybe a lysogenic Phage.
6Comparative genomics of Mycobacterium Phages
6.1Collinearity analysis
(1) The two closest relatives of Chy1are found to be D29and Che12.The homology genes distribute throughout the genome. There is clearsynteny among genes encoding the virion structure and assembly functions.
(2) The two closest relatives of Chy2are found to be BPs and Angel.The homology genes distribute throughout the genome. There is clearsynteny among genes encoding the virion structure and assembly functions.
(3) The two closest relatives of Chy3are found to be BPs and Angel.The homology genes distribute throughout the genome. The genome hasmost genes (56/58) in reverse order, comparing with BPs and Angel.
(4) The two closest relatives of Chy4are found to be D29and Che12.The homology genes distribute throughout the genome. There is clearsynteny among genes encoding the virion structure and assembly functions.
(5) The two closest relatives of Chy5are found to be D29and Pukovnik.The homology genes distribute throughout the genome. There is clearsynteny among genes encoding the virion structure and assembly functions.
6.2The Phage phylogenetic tree is constructed from14completelysequenced Mycobacterium Phage genomes. Chy1and D29have closestrelationship; Chy4and Chy5have closest relationship; Chy2and Chy3havecloser relationship than Chy1, Chy4and Chy5.
Conclusions
Five Mycobacterium Phages all belong to Siphoviridae, containingdouble-stranded DNA. Chy1and Chy3are lytic Phages, Chy2is lysogenicPhage, Chy4and Chy5may be lysogenic Phages. Chy3has a broad hostrange, shorter latent period, the most efficient sterilization in the newlyisolated Phages and can lyse Mycobacterium tuberculosis in macrophages，which makes Chy3have the potential of anti-TB treatment.
结核病疫情严峻，耐药率高，新型抗结核药物研发越来越困难，分枝杆菌噬菌体逐渐成为新型抗结核药物的研究热点之一。目前还无文献报道在国内发现新分枝杆菌噬菌体，本研究在国内首次分离鉴定出5株分枝杆菌噬菌体(Mycobacterium Phage)，命名为Chy1、Chy2、Chy3、Chy4和Chy5，通过对其生物学特性和基因组学进行研究，探寻这5株分枝杆菌噬菌体抗结核菌的潜力，明确其遗传背景，为噬菌体的进一步开发利用提供理论基础和方法指导。
方法
1采用直接分离法和富集法从泥土标本中分离、纯化分枝杆菌噬菌体。
2比较噬菌体滴度测定、噬菌体大量扩增和收获的不同方法，优化实验条件。
3分枝杆菌噬菌体生物学特性研究
3.1采用PEG8000沉淀法纯化分枝杆菌噬菌体，电镜观察噬菌体形态特征。
3.2以不同MOI扩增分枝杆菌噬菌体，根据扩增后的产量找出每株噬菌体的最佳MOI。
3.3通过一步生长实验测定分枝杆菌噬菌体潜伏期、裂解期和裂解量。
3.4分离临床病原菌，用罗氏药敏培养基进行初步菌种鉴定和药敏实验，再采用单斑法测定分枝杆菌噬菌体的宿主谱。
3.5比较分枝杆菌噬菌体在pH值为5.0和7.4的7H9固体培养基中裂解耻垢分枝杆菌mc2155的能力。
3.6比较Chy1、Chy2、Chy3、D294种噬菌体组成鸡尾酒制剂与这4种噬菌体分别单独裂解耻垢分枝杆菌mc2155的能力，实验数据采用SPSS17.0统计软件中的单因素方差分析进行统计分析，以P <0.05为差异有统计学意义。
3.7培养原代巨噬细胞，巨噬细胞吞噬结核菌后分为3组，1组加入Chy3，2组加入D29，3组为不加噬菌体的空白对照组，培养过夜后，收集细胞，电镜观察噬菌体对巨噬细胞胞内结核菌的影响。
4分枝杆菌噬菌体全基因组测序
4.1用λ噬菌体DNA提取试剂盒提取分枝杆菌噬菌体基因组DNA，限制性内切酶酶切后进行琼脂糖凝胶电泳，观察电泳结果。
4.2基于鸟枪法随机测序和重叠群组装的方法进行测序：构建文库，随机挑取克隆片段测序，拼接成大小不一的重叠群，最后通过PCR法填补各重叠群之间的缺口，得到完整的全基因组序列。
4.3组装成环形的噬菌体采用DNAStar程序包中的GeneQuest软件对其基因组列进行电子酶切分析，并与实际酶切图谱进行比较，以确定噬菌体基因组是否为环形。
5分枝杆菌噬菌体基因组生物信息学分析
5.1分枝杆菌噬菌体基因预测及基因组注释
⑴分析5株分枝杆菌噬菌体基因组的一般特性：①采用DNAStar软件包中Editseq软件分析基因组大小，碱基组成，基因平均长度、基因密度以及5株噬菌体遗传密码子的使用频率及其偏嗜性。②采用TRF软件预测基因组中的串联重复序列。③通过tRNAscan软件预测基因组中的tRNA区域。
⑵采用Glimmer3.0和GeneMark基因预测软件对基因组进行基因de novo预测，推定5株分枝杆菌噬菌体基因组编码序列。
⑶用Blast和Clustal软件将推定基因与nr，swissport，tremble，cog数据库中的序列进行同源比对，E value为1e-5，对每个预测出来的基因选取同源比对上分值最高的序列，预测基因功能。
6分枝杆菌噬菌体比较基因组学
6.1将5个分枝杆菌噬菌体的基因与公共数据库数据进行Blast m8比对，e-value <=1e-5，选取每个蛋白的最好比对结果，将两两比对均是最好的结果保留，绘制共线性图。
6.2先将所有的基因序列用Clustal W软件进行两两比对，计算得到包含每对序列分歧程度的距离矩阵，再根据距离矩阵计算得到引导树，最后根据前面得到的引导树分支顺序，逐级比对，得到全部序列的全局比对结果，用tree view软件生成进化树。
结果
1采用富集法分离纯化得到5株分枝杆菌噬菌体,根据分离地点分别命名为Chy1、Chy2、Chy3、Chy4和Chy5。这5株噬菌体的噬菌斑均为圆形、透明、清晰，呈裂解性噬菌体噬菌斑特点，但Chy2、Chy4和Chy5培养超过48h后噬菌斑逐渐变混浊。
2优化实验方法，后续实验均采取小平板法测定噬菌体滴度，双层固体培养基培养的方法大量扩增噬菌体以及刮取上层培养基离心的方法收集噬菌体。
3分枝杆菌噬菌体生物学特性研究
3.1Chy1、Chy3、Chy4和Chy5噬菌体头部均为多面立体对称，而Chy2噬菌体头部呈椭圆形。5株噬菌体均具有长尾，可弯曲，但不可收缩，可见尾板和尾丝，依据ICTV第八次报告的最新病毒分类系统的标准属长尾噬菌体科(Siphoviridae)。
3.2Chy1最佳MOI为1.4×10~-6，Chy~2最佳MOI为9.58×10~-5，Chy3最佳MOI为1.1×10~-4，Chy4最佳MOI为4.5×10~-5，Chy5最佳MOI为1.8×10~-5，后续实验大量扩增这些噬菌体时，均根据最佳MOI扩增，以达到最大产量。
3.3Chy~2感染耻垢分枝杆菌mc~2155的潜伏时间约为210min，裂解期为60min，270min后进入平台期，噬菌体滴度不再增加，裂解量为23；Chy3感染耻垢分枝杆菌mc~2155的潜伏时间约为150min，裂解期为90min，240min后进入平台期，噬菌体滴度不再增加，裂解量为40；Chy1、Chy4和Chy5潜伏期超过420min。
3.4共收集临床病原菌36株，经菌种鉴定，有32株为结核分枝杆菌(结核菌)，2株为牛结核分枝杆菌，2株为非结核分枝杆菌。这5株分枝杆菌噬菌体的宿主谱较广，能裂解MS mc2155、结核菌标准株H37Rv、大部分临床分离耐药结核菌株、耐药牛结核分枝杆菌和非结核分枝杆菌，其中Chy1对广泛耐药结核菌裂解率达100％。
3.5抗菌能力最强的噬菌体是D29(P <0.05)，48h内几乎裂解所有的耻垢分枝杆菌mc~2155，48h后细菌数量逐渐回升，但仍比初始加入的细菌数少，维持在较低水平，其次是鸡尾酒制剂，24h内能裂解大多数耻垢分枝杆菌mc2155，但24h后细菌数逐渐增多，到96h时细菌数与Chy2和Chy3组已无差别(P>0.05)。Chy1、Chy2和Chy33个新分离的噬菌体中，Chy3早期(48h)杀菌能力最强(P <0.05)，但到96h时细菌数与Chy2相当(P>0.05)，Chy1杀菌能力最弱，在48h内不能抑制细菌生长，但48h后MS不再增加，维持在109CFU左右，细菌数比空白对照组少(P <0.05)。
3.6当7H9固体培养基pH值为5.0和7.4时，Chy1、Chy2和Chy3均能裂解耻垢分枝杆菌mc~2155。
3.7电镜观察发现Chy3组和D29组结核菌数量少，菌体结构明显被破坏，而空白对照组结核菌数量较多，细菌结构仍完整。
4分枝杆菌噬菌体全基因组测序
4.15株噬菌体基因组DNA均可被EcoRⅠ、Hind Ⅲ和BamH I切开，这3种核酸内切酶均能识别并切割双链DNA分子特定的核苷酸序列，表明这5株噬菌体基因组均为双链DNA。分析酶切图谱发现这5株噬菌体基因组大小均在40-50kb之间。4.25株噬菌体测序质量达到完成图标准。Chy1、Chy2、Chy4和Chy5最终均组装为线状重叠群。虽然Chy3组装为环状重叠群，但实际酶切图谱中，Hind Ⅲ将Chy3基因组切为2个条带，与线性电子酶切图谱相同，所以Chy3基因组很可能是线性。5分枝杆菌噬菌体基因预测及基因组注释5.1Chy1⑴Chy1基因组全长为47198bp，G＋C含量为63.68%，基因平均长度为562bp，基因密度为0.92。Chy1有明显的遗传密码子偏嗜性，未发现串联重复序列，共预测出5个tRNA。⑵采用glimmer软件分析，确定了77个推定基因(GL)，对这些基因进行同源检索分析，得到29个已知功能的基因。GL05、GL10、GL12、GL13、GL20、GL23、GL24和GL25为噬菌体的结构基因；GL06、GL07和GL08分别编码LysA、holin和LysB；GL31为整合酶基因，GL70推定为阻遏蛋白类似基因，与D29p72基因序列100％相似，D29p72在进化过程中丢失了N-末端的一个片段，缺乏完整的HTH模体，不能结合到宿主菌DNA上，因而不能形成溶原性反应，即Chy1为裂解性噬菌体。GeneMark软件分析，共预测出71个推定基因(GM)，其中有55个基因与glimmer软件分析结果相同，有16个不同的基因，通过比对，发现GM22与GL23， GM08与GL09， GM51与GL55， GM52与GL56具有相同功能，其余基因未能匹配上已知功能的基因。5.2Chy2
⑴Chy2基因组全长为40017bp， G+C=66.73%，基因平均长度为656bp，基因密度为0.951。Chy2有明显的遗传密码子偏嗜性，共发现3个串联重复序列，未发现tRNA。
⑵采用glimmer在线分析软件分析，确定了58个推定基因，对这些基因进行同源检索分析，得到16个已知功能的基因。GL02、GL05、GL06、GL12、GL13、GL15、GL17和GL19为噬菌体的结构基因；GL26、GL27分别编码LysA和LysB，未发现holin基因；GL31为整合酶基因，GL32推定为阻遏蛋白基因，与分枝杆菌噬菌体Halo的gp33基因序列100％相似，Halo是溶原性噬菌体，gp33表达的产物是阻遏蛋白，即Chy2为溶原性噬菌体。GeneMark软件分析，共预测出54个推定基因，其中有44个基因与glimmer软件分析结果相同，有10个不同的基因，通过比对，发现GM15与GL12具有相同功能，其余基因未能匹配上已知功能的基因。
5.3Chy3
⑴Chy3基因组全长为40070bp， G+C=66.86%，基因平均长度为667bp，基因密度为0.97。Chy3有明显的遗传密码子偏嗜性，共发现3个串联重复序列，未发现tRNA。
⑵采用glimmer在线分析软件分析，确定了58个推定基因，对这些基因进行逐个同源检索分析，得到20个已知功能的基因。GL01、GL41、GL43、GL45、GL47、GL48、GL54、GL55和GL58为噬菌体的结构基因，位于整合酶基因(GL29)的两侧；GL33、GL34分别编码LysB和LysA，未发现holin基因和编码阻遏蛋白的基因，即Chy3为裂解性噬菌体。GeneMark软件分析，共预测出56个推定基因，其中有46个基因与glimmer软件分析结果相同，有10个不同的基因，通过比对，发现GM43与GL43具有相同功能，其余基因未能匹配上已知功能的基因。
5.4Chy4
⑴Chy4基因组全长为46639bp， G+C=63.68%，基因平均长度为586bp，基因密度为0.92。Chy4有明显的遗传密码子偏嗜性，未发现串联重复序列，共发现5个tRNA。
⑵采用glimmer在线分析软件分析，确定了73个推定基因，对这些基因进行同源检索分析，得到28个已知功能的基因。GL05、GL10、GL12、GL13、GL20、GL23、GL24和GL25为噬菌体的结构基因；GL06、GL07和GL08分别编码LysA、holin和LysB；GL31为整合酶基因，GL71与编码噬菌体L5阻遏蛋白的gp71相似性为86.3％，L5gp71具有183aa，而Chy4GL71仅含155个aa，是否能编码有活性的阻遏蛋白还未知，即Chy4很可能为溶原性噬菌体。GeneMark软件分析，共预测出70个推定基因，其中有55个基因与glimmer软件分析结果相同，有15个不同的基因，通过比对，发现GM09与GL09，GM23与GL23，GM46与GL47，GM54与GL57具有相同功能，其余基因未能匹配上已知功能的基因。
5.5Chy5
⑴Chy5基因组全长为51214bp，G+C=66.74%，基因平均长度为530bp，基因密度为0.91。Chy5有明显的遗传密码子偏嗜性，未发现串联重复序列，共发现5个tRNA。
⑵采用glimmer在线分析软件分析，确定了88个推定基因，对这些基因进行逐个同源检索分析，得到29个已知功能的基因。GL05、GL10、GL12、GL13、GL20、GL23、GL24和GL25为噬菌体的结构基因；GL06、GL07、GL08分别编码LysA、holin和LysB；GL31为整合酶基因，gene70与分枝杆菌噬菌体Pukovnik编码阻遏蛋白的基因gp72相似性为70.81％，推定为编码阻遏蛋白的基因，是否能编码有活性的阻遏蛋白还未知，即Chy5很可能为溶原性噬菌体。GeneMark软件分析，共预测出86个基因，其中有68个基因与Glimmer3.0软件分析结果相同，有18个不同的基因，通过比对，发现GM09与GL09，GM12与GL12，GM23与GL23，GM55与GL54，GM63与GL62具有相同功能，其余基因未能匹配上已知功能的基因。
6分枝杆菌噬菌体比较基因组学
6.1共线性分析
⑴Chy1同源性最高的两株噬菌体为D29和Che12，可归入ClusterA，同源性基因分布在整个基因组，具有清晰的共线性。Chy1基因组77个基因中有75个基因与D29的基因具有同源性，有60个基因与Che12的基因具有同源性。
⑵Chy2同源性最高的两株噬菌体为BPs和Angel，可归入ClusterG，同源性基因分布在整个基因组，具有清晰的共线性。Chy2基因组58个基因中有57个基因均与这两个噬菌体具有同源性。
⑶Chy3同源性最高的两株噬菌体为BPs和Angel，可归入ClusterG，同源性基因分布在整个基因组，Chy3基因组58个基因组中有56个基因均与这两个噬菌体具有同源性。Chy3基因组前3个基因与BPs和Angel前3个基因匹配，但从第4个基因开始与BPs和Angel的同源基因排列顺序相反。
⑷Chy4同源性最高的两株噬菌体为D29和Che12，可归入ClusterA，同源性基因分布在整个基因组，具有清晰的共线性。Chy4基因组73个基因组中有70个基因与D29具有同源性，有54个基因与Che12具有同源性。
⑸Chy5同源性最高的两株噬菌体为D29和Pukovnik，可归入Cluster A，同源性基因分布在整个基因组，具有清晰的共线性。Chy5基因组88个基因组中有70个基因与D29具有同源性，有57个基因与Pukovnik具有同源性。
6.2进化树分析
对14株分枝杆菌噬菌体基因组进行系统发育分析构建进化树，距离越近，噬菌体间亲缘关系越近。Chy1与D29亲缘关系最近，Chy4与Chy5亲缘关系最近， Chy2与BPs、Angel亲缘关系最近，Chy3与Legendre亲缘关系最近。
结论
5株分枝杆菌噬菌体均属于双链DNA病毒群(群Ⅰ)，尾病毒目，长尾病毒科。Chy1和Chy3为裂解性噬菌体，Chy2为溶原性噬菌体，Chy4和Chy5为可疑溶原性噬菌体，基因组学研究未在噬菌体基因组中发现毒力基因。5株噬菌体均能裂解耐药结核分枝杆菌，Chy3潜伏期短，体外裂菌能力强且能裂解巨噬细胞内结核分枝杆菌，最具有抗结核潜力。本研究初步探明5株新分枝杆菌噬菌体抗结核菌的潜力和遗传背景，为下一步开发利用噬菌体提供理论基础和方法指导。
Objective
Mycobacterium Phage can be used as a tool in the diagnosis, treatmentof tuberculosis and a genetic tool for analysis of mycobacterium genetics.The study of Mycobacterium Phage biological characteristics and genomicshelps us to identify its role in the physiology, genetics, and evolution ofmycobacterium. Here I report the isolation, sequencing and comparativegenomic analysis of five new Mycobacterium Phages for the first time inchina, which are named Chy1, Chy2, Chy3, Chy4and Chy5, respectively.The purpose of this study is to explore the potential of Mycobacterium Phagein TB diagnosis and treatment and identify the genetic background of thefive new Phages in order to provide a theoretical basis for the furtherdevelopment and utilization of them.
Methods
1Mycobacterium Phages were Isolated and Purified from soil.
2Different ways of plaque assay, Phage amplification and harvest werecompared to find the optimized experimental conditions.
3The study of biological characteristics of Mycobacterium Phage
3.1Mycobacterium Phages were purified by PEG8000precipitationand the morphological characteristics were observed by electronmicroscopy.
3.2Mycobacterium Phages were amplified by double-layer agar platemethod in different multiplicity of infection (MOI) to find the optimal MOI.
3.3One step growth experiment was done to find the latent period,burst period and burst size of Mycobacterium Phages.
3.4The host range of Mycobacterium Phages was examined bysingle-spot examination.
3.5The effect of pH values on lysis ability of Mycobacterium Phageswas surveyed.
3.6The lysis ability of Phage cocktail, Chy1, Chy2, Chy3and D29wascompared and the one-way analysis of variance (ANOVA) is used todetermine whether there are any significant differences between the meansof five independent (unrelated) groups.
3.7Murine macroPhages were isolated and cultured, which wereinfected by mycobacterium tuberculosis and divided in to three groups.Group1was added in Chy3, group2was added in D29and group3wasblank control group. Three groups were cultured overnight and the cellswere collected and observed by electron microscopy.
4Mycobacterium Phage genome sequencing
4.1The genome of Phages was extracted and the type of nucleic acidwas identified by restriction enzyme analysis to check the DNA for quantity,quality and a first estimate of the genome size.
4.2DNA was broken up randomly into small segments, which were sequenced using the chain termination method to obtain reads. Multipleoverlapping reads for the target DNA were obtained by performing severalrounds of this fragmentation and sequencing. Computer programs then usedthe overlapping ends of different reads to assemble them into a continuoussequence.
4.3The electron restriction map, which were analyzed by GeneQuestsoftware in DNAStar package, and the actual restriction map were comparedto determine whether the Chy3genome is circular.
5Bioinformatics of Mycobacterium Phage genome
5.1Gene prediction and genome annotation of Mycobacterium Phages
(1) The genome size, base composition, the average gene length and thegene density of five Phages were analyzed using Editseq software inDNAStar package. TRF (Tandem Repeat Finder) software was used tosearch for tandem repeat and tRNAscan software was used to search fortRNA region.
(2) Glimmer and GeneMark were used to predict where theprotein-coding genes of five Mycobacterium Phages are and identified theputative genes.
(3) Sequence similarity analyses were performed with gapped BLASTpalgorithm against the non-redundant database provided by NCBI.
6Comparative genomics of Mycobacterium Phages
6.1A comparative analysis of five Mycobacterium Phages with their two closest relatives respectively was carried out Clustal W and created SVGimages using Perl.
6.2A phylogenetic supertree of14Phage genomes was constructedusing ClustalW and tree view.
Results
1Five new Mycobacterium Phages were successfully isolated andpurified, which were named Chy1, Chy2, Chy3, Chy4and Chy5,respectively. The plaques of five Phages all are transparent, but the plaquesof Chy2, Chy4and Chy5become turbid cultured after48h.
2The optimal method to measure the titer is small plate plaque assay.The best way to amplify Phages abundantly is that Phage and MS in optimalMOI were cultured in double-solid plat and the upper medium was scrapeddown after48h and centrifuged to collect the Phages.
3The biological characteristics of Mycobacterium Phages
3.1Chy1, Chy3, Chy4and Chy5all have an isometric head, and Chy2has a oval head. All the five Phages have long tail, base plate, tail fiber, andall belong to Siphoviridae.
3.2The optimal MOI of Chy1, Chy2, Chy3, Chy4and Chy5are1.4×10~(-6),9.58×10~(-5),1.1×10~(-4),4.5×10~(-5)and1.8×10~(-5), respectively.
3.3The one-step growth cycle of Chy2is270min, in which the latentperiod is210min, the burst period is60min and the burst size is23. Theone-step growth cycle of Chy3is240min, in which the latent period is150 min, the burst period is90min and the burst size is44. The latent period ofChy1, Chy4and Chy5are all more than420min.
3.4The Phages can lyse MS mc~2155, Mycobacterium TuberculosisH37Rv and the majority of clinical drug-resistant strains.
3.5D29has the most efficient sterilization and can kill almost all of MSwithin48hours, the number of MS roses gradually after48h, but still lowerthan the initial bacterial number. The second is the Phage cocktail, which cankill most MS within24hours, and then the number of bacteria graduallyincreased and equal to the number of bacteria with Chy2and Chy3group at96h. Chy3has the most efficient sterilization in the three newly isolatedPhages within48h, but the number of MS in the three groups has nodifference at96h. Chy1has the weakest bactericidal capacity, and can notinhibit bacterial growth within48hours, but the number of MS maintainsaround in109CFU after48h and no longer increases.
3.6Chy1、Chy2and Chy3can lyse Mycobacterium smegmatis mc~2155in solid medium with pH5.0and7.4.
3.7Electron microscopy shows that the number of Mycobacteriumtuberculosis is lower in the Chy3group and D29group, of which thebacterial structure is destroyed more seriously.
4Genome sequencing of Mycobacterium Phages
4.1The genome of five Phages can be digested by restrictionendonuclease EcoRⅠ, Hind Ⅲand BamH I, and the size of the genomes all are between40kb and50kb.
4.2Multiple overlapping reads for the target DNA were obtained andComputer programs then used the overlapping ends of different reads toassemble them into a continuous sequence. Chy1, Chy2, Chy4and Chy5areassembled into linear genome, and Chy3is circular.
5Gene prediction and genome annotation of five MycobacteriumPhages
5.1Chy1
(1) The length of Chy1genome is47198bp, the%GC content is63.68%, the average length of gene is562bp, and the gene density of thegenome is0.92. Chy1has a significant codon usage bias and has five tRNAgenes and no tandem repeats.
(2)77genes (GL) are identified in the Chy1Phage by glimmersoftware, each of which was submitted to BLAST analysis. Of the77genes,29have similarity to proteins with known functions and the rest arehomologous to those with unknown function. GL05, GL10, GL12, GL13,GL20, GL23, GL24and GL25are involved in the virion structure. GL06,GL07, GL08are assigned to be the lyses cassette. GL31is assigned to beintegrase. GL70shared100%homology with repressor gene (gp72) of D29.However, gp72of D29has lost a segment of its N-terminal during evolutionhence lacking HTH motif, which prevent it from binding to DNA, so Chy1islytic Phage.
5.2Chy2
(1) The length of Chy2genome is40017bp, the%GC content is66.73%, the average length of gene is656bp, and the gene density of thegenome is0.95. Chy2has a significant codon usage bias and has none tRNAgenes and three tandem repeats.
(2)58genes are identified in the Chy2Phage, each of which wassubmitted to BLAST analysis. Of the58genes,16have similarity to proteinswith known functions and the rest are homologous to those with unknownfunction. GL02, GL05, GL06, GL12, GL13, GL15, GL17and GL19areinvolved in the virion structure. GL26and GL27are assigned to be the lysescassette. GL31is assigned to be integrase. Gene32shared100%homologywith repressor gene (gp33) of Halo. The product of gp33is an activerepressor protein, so Chy2is lysogenic Phage.
5.3Chy3
(1) The length of Chy3genome is40070bp, the%GC content is66.86%, the average length of gene is667bp, and the gene density of thegenome is0.97. Chy3has a significant codon usage bias and has none tRNAgenes and three tandem repeats. Restriction analysis shows that the genomeof Chy3is digested into two bands by Hind Ⅲ, which is the same to thelinear electron restriction map, but there are two bands in the actualrestriction map digested by EcoRⅠ, and three in the linear electronrestriction map. So Chy3may have a linear genome.
(2)58genes are identified in the Chy3Phage, each of which wassubmitted to BLAST analysis. Of the58genes,20have similarity to proteinswith known functions and the rest are homologous to those with unknownfunction. GL01, GL41, GL43, GL45, GL47, GL48, GL54, GL55andGL58are involved in the virion structure. GL33and GL34are assigned tobe the lyses cassette. GL29is assigned to be integrase, but there is no gene isassigned to be repressor, so Chy3is lysogenic Phage.
5.4Chy4
(1) The length of Chy4genome is46639bp, the%GC content is63.68%, the average length of gene is586bp, and the gene density of thegenome is0.92. Chy4has a significant codon usage bias and has five tRNAgenes and none tandem repeats.
(2)73genes are identified in the Chy4Phage, each of which wassubmitted to BLAST analysis. Of the73genes,28have similarity to proteinswith known functions and the rest are homologous to those with unknownfunction. GL05, GL10, GL12, GL13, GL20, GL23, GL24and GL25areinvolved in the virion structure. GL06, GL07, GL08are assigned to be thelyses cassette. GL31is assigned to be integrase. GL71shared86.3％homology with repressor gene (gp71) of lysogenic Phage L5. However, gp71has188aa, and Chy4gene71has155aa, whether the repressor protein ofChy4has active function is also unknown, so Chy4may be a lysogenicPhage.
5.5Chy5
(1) The length of Chy4genome is51214bp, the%GC content is66.74%, the average length of gene is530bp, and the gene density of thegenome is0.91. Chy5has a significant codon usage bias and has five tRNAgenes and none tandem repeats.
(2)88genes are identified in the Chy5Phage, each of which wassubmitted to BLAST analysis. Of the88genes,29have similarity to proteinswith known functions and the rest are homologous to those with unknownfunction. GL05, GL10, GL12, GL13, GL20, GL23, GL24and GL25areinvolved in the virion structure. GL06, GL07, GL08are assigned to be thelyses cassette. GL31is assigned to be integrase. GL70shared70.81％homology with repressor gene (gp72) of Phage Pukovnik, whether therepressor protein of Chy5has active function is also unknown, so Chy5maybe a lysogenic Phage.
6Comparative genomics of Mycobacterium Phages
6.1Collinearity analysis
(1) The two closest relatives of Chy1are found to be D29and Che12.The homology genes distribute throughout the genome. There is clearsynteny among genes encoding the virion structure and assembly functions.
(2) The two closest relatives of Chy2are found to be BPs and Angel.The homology genes distribute throughout the genome. There is clearsynteny among genes encoding the virion structure and assembly functions.
(3) The two closest relatives of Chy3are found to be BPs and Angel.The homology genes distribute throughout the genome. The genome hasmost genes (56/58) in reverse order, comparing with BPs and Angel.
(4) The two closest relatives of Chy4are found to be D29and Che12.The homology genes distribute throughout the genome. There is clearsynteny among genes encoding the virion structure and assembly functions.
(5) The two closest relatives of Chy5are found to be D29and Pukovnik.The homology genes distribute throughout the genome. There is clearsynteny among genes encoding the virion structure and assembly functions.
6.2The Phage phylogenetic tree is constructed from14completelysequenced Mycobacterium Phage genomes. Chy1and D29have closestrelationship; Chy4and Chy5have closest relationship; Chy2and Chy3havecloser relationship than Chy1, Chy4and Chy5.
Conclusions
Five Mycobacterium Phages all belong to Siphoviridae, containingdouble-stranded DNA. Chy1and Chy3are lytic Phages, Chy2is lysogenicPhage, Chy4and Chy5may be lysogenic Phages. Chy3has a broad hostrange, shorter latent period, the most efficient sterilization in the newlyisolated Phages and can lyse Mycobacterium tuberculosis in macrophages，which makes Chy3have the potential of anti-TB treatment.
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[1] Pedulla ML, Ford ME, Houtz JM, et al. Origins of highly mosaicMycobacteriophage genomes [J]. Cell,2003,113(2):171–82.
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[17] Hendrix RW, Hatfull GF, Smith MC. Bacteriophages with tails: chasing theirorigins and evolution [J]. Res Microbiol,2003,154:253–257.
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[1] Pedulla ML, Ford ME, Houtz JM, et al. Origins of highly mosaicMycobacteriophage genomes [J]. Cell,2003,113(2):171–82.
[2] Hatfull GF, Pedulla ML, Jacobs-Sera D, et al. Exploring theMycobacteriophage metaproteome: Phage genomics as an educationalplatform [J]. PLoS Genet,2006,2(6): e92.
[3] Hatfull GF, Jacobs-Sera D, Lawrence JG, et al. Comparative genomic analysisof60Mycobacteriophage genomes: genome clustering, gene acquisition, andgene size [J]. J Mol Biol,2010,397(1):119–143.
[4] Pope WH, Jacobs-Sera D, Russell DA,et al. Expanding the diversity ofMycobacteriophages: insights into genome architecture and evolution [J].PLoS One,2011,6(1): e16329.
[5] Hatfull GF. Complete genome sequences of138Mycobacteriophages [J]. JVirol,2012,86(4):2382–4.
[6] Bibb LA, Hatfull GF. Integration and excision of the Mycobacteriumtuberculosis prophage-like element, phiRv1[J]. Mol Microbiol,2002,45(6):1515–26.
[7] Laal S, Sharma YD, Prasad HK,et al.Recombinant fusion protein identifiedby lepromatous sera mimics native Mycobacterium leprae in T-cell responsesacross the leprosy spectrum [J]. Proc Natl Acad Sci U S A,1991,88(3):1054–1058.
[8] Oftung F, Mustafa AS, Wiker HG. Extensive sequence homology betweenthe mycobacterium leprae LSR (12kDa) antigen and its Mycobacteriumtuberculosis counterpart [J]. FEMS Immunol Med Microbiol,2000,27(1):87–9.
[9] Harley JB, Scofield RH, Reichlin M. Anti-Ro in Sj gren's syndrome andsystemic lupus erythematosus [J]. Rheum Dis Clin North Am,1992,18(2):337–58.
[10] McCauliffe DP, Lux FA, Lieu TS,et al. Ro/SS-A and the pathogenicsignificance of its antibodies [J]. J Autoimmun,1989,2(4):375–81.
[11] Hatfull GF. Bacteriophage genomics [J]. Curr Opin Microbiol,2008,11(5):447–53.
[12] Serwer P, Hayes SJ, Thomas JA,et al. Propagating the missingBacteriophages: a large Bacteriophage in a new class [J]. Virol J,2007,4:21.
[13] Hendrix, RW. Bacteriophages: evolution of the majority [J]. Theor PopulBiol,2002,61,471–480.
[14] Brussow H, Hendrix RW. Phage genomics: small is beautiful [J]. Cell,2002,108:13–16.
[15] Hendrix RW. Bacteriophage genomics [J]. Curr Opin Microbiol,2003,6:506–511.
[16]. Wilhelm SW, Jeffrey WH, Suttle CA, et al. Estimation of biologicallydamaging UV levels in marine surface waters with DNA and viral dosimeters[J]. Photochem Photobiol,2002,76:268–273.
[17] Hendrix RW, Hatfull GF, Smith MC. Bacteriophages with tails: chasing theirorigins and evolution [J]. Res Microbiol,2003,154:253–257.
[18] Hendrix RW, Lawrence JG, Hatfull GF, et al. The origins and ongoingevolution of viruses [J]. Trends Microbiol,2000,8:504–508.
[19] Hatfull GF, Sarkis GJ. DNA sequence, structure and gene expression ofMycobacteriophage L5: a Phage system for mycobacterial genetics [J]. MolMicrobiol,1993,7:395–405.
[20] Ford ME, Stenstrom C, Hendrix RW, Hatfull GF. Mycobacteriophage TM4:genome structure and gene expression [J]. Tuber Lung Dis,1998,79:63–73.
[21] Morris P, Marinelli LJ, Jacobs-Sera D, et al. Genomic characterization ofMycobacteriophage Giles: evidence for Phage acquisition of host DNA byillegitimate recombination [J]. J. Bacteriol2008;190:2172–2182.
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