genes and genomes of mycobacteriophage

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MYCOBACTERIOPHAGES: GENES & GENOMES Varsha 2009BS142M

Bacteriophage:

Bacteriophage Obligate intracellular parasites that multiply inside the bacteria by making use of some or all of the host biosynthetic machinery i.e. viruses that infect bacteria . Apx. 10 31 on earth 1977- ssDNA phage ϕ X174 (5,386bp) ds DNA phage – lambda (48,502bp) 1993- ds DNA tailed mycobacteriophage L5. Seq. genome size - 2.3 to 316 kbp.

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Size distribution of sequenced bacteriophage genomes 30-50 kbp – 50% <10 kbp - 20% 100-200 kbp – 6% Size (kbp)

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Interest began in late 1940s with the isolation of phages that infect M. smegmatis & M. tuberculosis First sequenced Mphage- L5 (1993). 70 sequenced genomes is present in Genbank. 60 genomes are used for comparative analysis. Mycobacteriophage Mycobacterium :- A genus in the family Mycobacteriaceae, G +ve, obligate aerobic, acid fast bacteria occurring as slightly curved or straight rods. Viruses that infect mycobacterial hosts.

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General properties of Mphage Virion morphology Host range & host range determinants Life cycles

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Virion morphology ds DNA- tailed phages Order:- Caudovirales 70 61 9 Family Siphoviridae Myoviridae None is Podoviridae 60 53 7 Siphoviridae – considerable variation Myoviridae – morphologically indistinguishable

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Scott McG - Isometric head (85.9 nm dia.). 50 Siphoviridae - isometric heads (55-60 nm). 3 Siphoviridae - prolate head (Corndog, Che9c & Brujita ). length / width Brujita 2.5 : 1 Corndog 4 : 1 Siphoviridae tail legnth -135-350 nm Cluster A (Solon) -113 nm Cluster H (Predator) – 293 nm Fig :- M.phage Morphotypes Capsid size correlates with genome size .

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Host range & host range determinants Broad host range Narrow host range e.g. D29 e.g. Barnyard Strain discrimination: 33D- b/w BCG strains & M. bovis Why host preferences? Specific cell surface receptors e.g. D4- M.smegmatis – peptidoglycolipid ( mycoside ) D29- M.smegmatis - phospholipid Phlei phage- M.phlei – glycolipid C Particular metabolic requirement after DNA injection Specific phage protection mechanism (immunity & restriction). Cont………

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For M.phage barriers may be absolute or relative. Clustered Regularly Interspaced Short palindromic Repeats. For e.g. BPs & Halo which were isolated on M. smegmatis form plaques on M. tuberculosis at a ⱱ of 10 -5 . CRISPRSs may also play a role in phage resistance. Composed of short direct repeats (21-47 bp) separated by short (32-50 bp) unique spacer sequences. Mycobacterial CRISPRS spacer sequences don’t have similar counterparts in any of the sequenced M.phage genomes, consistent with the idea that phages of these hosts remain unidentified.

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Life cycle Temperate virulent e.g. L5, 12 phages of cluster A e.g. D-29, Approx. one half of the characterized M.phages (36 of 70) have an integration cassette & are candidates for forming lysogens, albeit at relatively low ⱱ.

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Phage genomics Comparative genomic study of 60 cultures have been done (Hatfull et al . ,2010). M. Phage isolated from……………….. Mostly in the vicinity of pittsburg (37/60), remaining 23 from japan, India, and 9 states of U.S. Fig. Plaque formation Agar concentration-0.35% Host- M. smegmatis mc 2 155 Cont………

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Genometrics of 60 sequenced M.phage genomes

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Sequenced M.phage genomes First completely seq. M.phage genome L5. Temperate phage isolated from Japan. Close relative of phage L1, similar restriction pattern but no growth at 45 o C. Both L5 & L1 infect fast growing and slow growing mycobacterial strains although L5 requires high Ca 2+ conc. for infecting slow growing strains.

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Second completely seq . - M.Phage D-29 Isolated from california Considerable ntd seq. similarity to L5 Clear plaque formation. Third completely seq . - TM4 Isolated by induction of a strain of M.avium . Unclear whether the original strain was lysogenic or pseudolysogenic . At ntd seq. level distinct from L5 and D29. Doesn’t encode any integration system or any phage repressor.

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Genomic Diversity High diversity but not uniform e.g. any two particular phages Adjutor and PBI 1 They share extensive ntd seq. similarity over the entire genome lengths with only a few base difference. e.g. Barnyard and Giles Their gene products share only 25% aa identity. To recognize the heterogeneous nature of genome diversity, genomes can be grouped into clusters according to their relationship to each other.

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Genomes Clustering M.Phage genomes grouped into clusters of related genomes. Genomic relationships during clustering is divided into three classes Closely related genomes No relationship Complex relationship Short segments with high degree of similarity Weak relationship in large segments No ntd similarity but same amino acid sequence

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Techniques used for clustering the genomes Dot plot analyses Average nucleotide identities Gene content analyses Pairwise genome analyses

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Dot Plot Analyses Genome show >50% similarity are placed in to same clusters. Out of 60, 55 genomes are divided into 9 clusters. Remaining 5 are not related to each other so placed into different clusters known as Singletons. (TM4, Wildcat, Giles, Omega, Corndog) Based on uniformity between clusters , they are divided into 12 subclusters. Fig: showing dot plot results

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Table: showing the different Clusters, subclusters and singletons . Total no. of groups 21 .

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Average Nucleotide Identity (ANI) Division is based upon the measurement of average nucleotide content in the relative genome. Genomes showing the 60-70% similarity are placed into same cluster. Members of a subcluster may show similarity up to 99.8%. ANI values vary greatly for different clusters and subclusters.

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Table: ANI shared by cluster A members

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Omega shares 6.1 to 8.3 kbp (95%) with Tweety (cluster F ). ANI values between Omega and the Cluster F genomes are fairly high (65.6%-74.3%). Fig:- Dot plot of Omega & Tweety Complexity of Divisions Why Omega is not a Member of Cluster F? Don’t fulfil the criteria of sharing evident sequence similarity spanning 50% of the genomes .

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Gene content analysis A third approach to genome clustering is a gene content analysis based on scoring whether the genomes contain a member of each of the protein Phams. Resulting pattern is in good agreement with the analyses from dot plot and ANI comparisons and supports the overall cluster and subcluster groupings . Fig: Splits Tree representation of mycobacteriophage relationships.

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Pairwise genome analyses It found Correlation between regions of genomes and the gene location. This is especially useful for displaying segments of similarity between more distantly related genomes , as well as revealing departures among more closely related genomes. For example, the subclustering of the Cluster B genomes is clearly illustrated, and the locations of genome differences within each subcluster are delineated.

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Fig : Pairwise alignment of cluster B genome. The stregnth of the relationship is represented by shading according to the colour spectrum with purple being the highest.

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Overall, these comparisons show the closeness of relationships with in Clusters C1, D, E, and G, as well the relatively weaker ones with in Clusters F, H, and I. Disadvantage : presentation is limited to pairwise display comparisons, and thus only a subset of the interesting and complex Relationships can be shown in a single representation. Fig: comparison between Cluster E and Cluster I

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Assembly of ORFs into Phams(Phamily) Phams: group of M.phage genes related to each other as defined by Blast-P and Clustal-W. Two ORFs showing 25% similarity in amino- acid seq. or E-value is of 0.0001 are grouped into same Pham. Size of the pham may vary from 1-454 members. Modular construction of some ORFs lead to their large pham. Average pham size:- no. of mycobacteriophage genomes containing a phamily member. Differences in APS reflects the extent to which these phams provide functions.

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Pham 1406 Largest pham - 454 members 40 subphams Pham 1406-11 (largest subpham- 39 members) Mostly virion, structural proteins, also contain capsid and major tail subunit proteins. Pham 1410 Second largest - 292 members 62 subphams (20 orphams) Pham 1396 Third largest – 269 members 86 subphams Total no. of Phams and subphams -1723 orphams – 773 (45.8% total). Three large Phams………

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Genomic architecture/ Organization M.Phage genome length – 44.4 (Angel) to 164.6 kbp (Myrna). Average length- 73.6 kbp Fig: organization of M.Phage Angel (Cluster G) genome.

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In all Siphoviral morphotypes (53) –synteny among virion structure and assembly genes. Largest gene TMP [tail size. 107 nm (L5) to ~ 300 nm (Predator)]. In Myoviral morphotypes (cluster C) - large capsid and longer genome. Structural genes – not well defined, no Synteny. Large no.of t-RNA genes. tm- RNA encoded by C1 phages. Facts about gene architecture

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Fig: Schematic representation of mycobacteriophage genome architectures .

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Cluster F, G, I – defined ends with short ss DNA extensions and leftmost of structure and assembly genes located close to genome end. In contrast cluster A, E with singletons- Corndog , Giles, Omega, TM4 and Wildcat defined genome ends but additional genes between terminase and the end [4 ( Cjw1 ) to 31 ( corndog )], Lysis genes in cluster A . Normal order of virion structure and assembly genome-Terminase, Portal, Protease, Scaffold, Capsid, presumed Head- Tail joining genes, major tail subunit, TMP, and minor tail proteins but cluster D, F, G,I have largely uninterrupted order.

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Clusters or subclusters - B1, B4, E, F, G, I, Corndog, TM4 -both small and large terminase subunits but A, B2, B3, D, H - no small terminase subunit gene. No Scaffold protein gene in all genomes. Additional genes in structure and assembly gene array-b/w Small and large terminase (E) Immediate following major capsid subunit gene (H ) Large insertions in Singletons Corndog, Omega Lysis genes b/w Terminase gene and left end (A) . Clusters A, E, F, G and singletons Giles, Omega -integration cassettes near centre of genome regardless of genome length differences. In Giles atypical location- left of lysis gene.

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Gene functions and expression Lysis cassette first described for-Ms6. 5 genes(1-5 ORFs) Three implicated in lysis- lysin A, Lysin B, and Holin. All seq. M. phage encode endolysin (lysin A)-unusual and comple x gp of proteins composed of many modules. Modules contain different types of PG hydrolysis motifs. Ms6 lysin B –lipolytic activity. Lysis

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Integration and Prophage maintenance 36 of seq. M. phages (A, E, F, G, I, Omega, and Giles) harbor integration cassettte , composed of - an integrase gene. - attP site -RDF Mostly integrases are of tyrosine recombinase family except subcluster A1 phages, Bxz 2 and Peaches (A2) which encode Serine integrases. In each genome encoding a tyrosine integrase, a putative attP site can be identified owing to a short 25 to 45bp common core region shared between the attP and attB sites in the host chromosome. Frequently, the attB core overlaps a host tRNA gene and this is observed for all characterized M.phages .

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In phages L5,D29,Halo, Ms6,Tweety, and Giles the attB site has been Confirmed experimentally and it has been predicted for Che8, Llij , PMC (which are closely related to Tweety ), Che9d, Omega, Che9c, Cjw1, and 244. Integrase-mediated excisive recombination typically requires an RDF, and the best characterized RDF for the M.phage encoded tyrosine integrases is the 56-residue gp36 Xis of L5. RDF class proteins is highly diverse. In the Cluster G phages, a putative RDF with similarity to other Xis proteins is located near the integrase gene, and a similar situation is observed in singletons Giles and Omega. Che9c (Cluster I) encodes a putative RDF that is related to Giles gp30 but is located over 6 kbp away from the integrase gene.

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Gene Expression and its Regulation Bxb1 gene activity pattern was observed during protein synthesis at the time of lytic growth in M. smegmatis by radiolabled 35 S methionine at various time interval. Fig: Synthesis of Bxb1 proteins after infection of M. smegmatis . Soon after infection, reduction in host gene expression. Gene 62-33.8kDa ( by 15 min.) Gene 45, 47, & 57- 28kDa

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Three types of promoters – A leftwards facing promoter for early lytic gene expression. A leftwards facing promoter for repressor synthesis. Rightwards facing promoters for structure & assembly. Gene location 1. 83-84 intergenic region 2. Upstream 35 bp to promoter 83-84 intergenic region

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Other M.phage gene functions Many genes of M.Phage controlling DNA metabolism have been cloned e.g. thymidylate synthase & ribonucleotide reductase RNR. ( gp 48 & 50 by phage L5). Gene 65 of D29 encodes a structure specific nucleases for forked DNA. Two segments of L5 have been identified, cytotoxic to M.smegmatis . 3 more cytotoxic proteins encoded by genes 77,78,79 interupt cellular processes, still ill-defined. Based on these observations, anti -Staphylococcal drugs have been launched e.g. EUROPIUMCRYPTAT ALLOPHYCOCYANIN So, we can predict a large no. of cytotoxic proteins for tuberculosis bacteria.

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Genome Mosaicism Each genome can be considered as unique combination of individual modules that are exchangeable among the population. Size of modules, rate of exchange and the phage genomes carrying the module vary greatly with phages of different virion morphology, size and host range. This mosaicism is not restricted to phage population but is also prevalent in bacteria acquired by horizontal exchange. This exchange occur can be studied by the sequence of ntd as well as aa . Similar. ntd sequence show recent exchange whereas aa similarity latterly exchange. Example of recent exchange; Cluster B genome

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Fig:Recombination between cluster B M.phage

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Fig: Alignment of PG1, Rosebush, and Qyrzula sequences at the right most recombinant. The box shows a region of interrupted similarity between PG1 and Qyrzula within which recombination could have given rise to the Rosebush recombinant structure

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Single gene Mosaicism Fig: Intercluster transfer of single gene.

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Mechanisms for generating Mosaic Genome Three proposed mechanism s Early model: short conserved boundary sequences serve as targets for genetic exchange. M.phage have not prevalent boundary sequences 2) Illegitimate/ non seq. determined recombination: Recombination events are not targeted and occur randomly. leads to non functional genomic trash and give rise to non viable (exception- appropriate genome size or retaining gene function) progeny. This show multiple low ⱱ events for recombination. Homeologous recombination: mediated by λ red-like recombinase .

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Nearly 2/3 rd of the M.phage genome don’t have genes encoding identifiable recombinase- sufficiently that they are absent or that these activities lie within large no. of genes of unknown function.

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Transposons & other mobile elements Transposons are not present in all phages but not so uncommon. They can contribute to genomic mosaicism. No any transposon / IS elements in sequenced M.phage. MPMEs novel class of M.Phage mobile elements. mainly in cluster F,G & I. atypically small MPME1 – 439 bp MPME2- 440bp

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Another transposon insertion: in Llij (cluster F ) gp 83, related to transposases of IS 200 family, shares 73% aa identity with Norcardia farcinia transposon. No introns but several inteins are reported in variety of genes, five of these are in terminases (encoded by phages- Bethlehem, Cjw1, Kostya, Omega and Pipefish). Also in RDF gene 47-Bxb1 Nucleotidyl transferase gene in Cali, gene3 Gene 51 in Bethlehem

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Summary & Conclusion ☺ M.Phage genomes are genetically highly diverse. ☺ M.Phages can be grouped into clusters according to their sequence relationship. ☺ M.Phage genomes are architecturally mosaic. ☺ Approx. 80% of the M.phage gene phamilies are of unknown function. ☺ M.Phages are sources of genetic novelty including new classes of inteins and mobile elements. ☺ Rich resources for mycobacterial genetics.

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Future issues Newly discovered M.phages isolated on a variety of different mycobacterial strains are needed to fully understand M.phage genetic diversity. The potential for generating new tools for mycobacterial genetics and gaining in sights into mycobacterial physiology is great and many advances await development. Elucidating the function of M.phage genes will provide a fuller understanding of their biology and their evolution.

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☺ Thanx ☺