Study On Biology And Genomics Of Mycobacterium Phages

ObjectiveMycobacterium 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.Methods1Mycobacterium 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 Phage3.1Mycobacterium 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 sequencing4.1The 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 genome5.1Gene 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 Phages6.1A 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.Results1Five 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 Phages3.1Chy1, 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^-5and1.8×10^-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 mc²155, 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 mc²155in 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 Phages4.1The genome of five Phages can be digested by restrictionendonuclease EcoRâ… , Hind â…¢and 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 MycobacteriumPhages5.1Chy1(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 â…¢, which is the same to thelinear electron restriction map, but there are two bands in the actualrestriction map digested by EcoRâ… , 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. GL71shared86.3ï¼…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. GL70shared70.81ï¼…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 Phages6.1Collinearity 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.ConclusionsFive 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.