structure of t4 bacteriophage

includes structure and assembly of t4 bacteriophage and brief history about bacteriophage

1. Presented by G.Vijayalakshmi 20PY10 I MSC., Microbiology Department of microbiology Ayya nadar janaki ammal college Sivakasi , Tamilnadu -India

2. Presented to DR.S.Sivasankara Narayani Assistant Professor Department of microbiology Ayya nadar janaki ammal college Sivakasi , Tamilnadu -India

3. Bacteriophage (ubiquitous) HISTORY 1896 Ernest Hanburg Hankin –antibacterial action against cholera and it could pass a very fine porcelain filter. 1898, Gamalega –observed similar phenomenon in bacillus subtilis 1984-samsygina and boni –reported such phenomeon . 1915- ferederick william Twort -presence of a virus D’Herelle (1917) coined the term bacteriophage meaning” bacteria eater” (observed that filtrate from feces culture from dysentry ) The greek word backerion means “bacteria” and phagein means “to eat” He suggested that lytic agent was a virus and named bacteriophage ( twort -d herelle phenomenon) Jules bordet and andre grata –rediscovered it Salvador e.luria –get clear image of a bacteriophage using electron microscope

4. history 1940-delbruck - worked out the lytic mechanism Luria , delbruck and hershey -studied genetic changes occur when viruses infect bacteria 1952-proved DNA That transmits the genetic information Shared their nobel prize for physiology in 1969 1952-norton zinder and joshua loderberg -phage can incorporate its genes into the bacterial chromosome 1980-frederick sanger –awarded nobel prize for his discovery of determing the nucleotide sequence in dna using bacteriophages .

5. BACTERIOPHAGES Introduction A virus that infects Replicates within a bacterium Derived from”bacteria ” and phagein to “devour” Single stranded or double stranded Surrounded by protein capsid (subunits capsomere ) Genomes may encode as few as four genes and as many hundreds of genes.

6. Bacteriophage Unenveloped ,complex symnetry Up to 70% of marine bacteria may be infected by phages 19 families are currently recognized by the ICTV that infect bacteria and archea . 9families infect bacteria only 9 infect archea only, and one ( tectiviridae ) infects both bacteria and archea . Two life cycles , lytic (virulent)or lysogenic (temperate)

7. classification The ICTV classifies the bacteriophage under an order Caudovirales according to morphology and nucleic acid. On the basis of presence of single or double strands of genetic material the bacteriophage are the following types. SS DNA bacteriophage (fd,fi,m13) Ds DNA bacteriophaget2,t4,t6,t1,t3,t5,t7etc) Ss RNA bacteriophage (m12,ms2,r17) Ds RNA bacteriophage

8. Bacterial viruses may grouped into 6 morphological types TYPE A: this most complex type has a hexagonal head,a rigid tail with a contractile sheath and tail fibres . TYPE B: similar to A, this type has a hexagonal head,a rigid tail with a contractile sheath its tail is flexible,and it may or may not have tail fibres . TYPE C: this type is characterised by a hexagonal head and a tail shorter than the head .the tail has no contractile sheath and may or may not have tail fibres TYPE D: this type has a head madeup of large capsomeres,but has no tail TYPE E: this type has a head madeup of small capsomeres but has no tail TYPE F: this type is filamentous

9. Families of bacteriophages

10. MORPHOLOGY Tadpole shaped,with hexagonal head and cylindrical tail. Contains 5 important substructures ( head,head-tailconnector,tail base plateand fibres ) Heads consists of a tightly packed core of ds DNA surrounded by a protein coat or capsid .( elongated,bi -pyramidal) Size 95*65 contains 2000 identical protein subunits( capsomeres )

11. MORPHOLOGY The tail composed of a contractile sheath surrounding the hollow core and contains 24 annular rings (each containing 6 subunits)constitute the sheath It connected to the head with a connector having a collar with attached whiskers Size of the tail is 80*18nm Terminal base plate having prongs or tail fibres attached The base plate -6 spikes as its six corners Former- helpd in recognition of specific receptor sites dsDNA -50 (mu m) DNA-1000 longer than phage Circular and terminally redundant T-even phages -5- hydroxymethylcytosine instead of cytosine Synthesis of phage occur easily.

12. Diseases caused by bacteriophages Bacteriophages only infect bacteria,they do not cause disease in humans. Alter the genome on non- virulent bacterial strains; thus producing more virulent strains examples are: CHOLERA Most strains of cholera are harmless. Responsible for producing harmful cholera strains.

13. Bacteriophages SCARLET FEVER Commonly affects children Signs and symptoms include sore throat, fever and a characteristic red rash. Usually spread by inhalation There is no vaccine of this disease. Most of the clinical features are caused by erythrogenic toxin, a substance produced by the bacterium streptococcus when it is infected by bacteriophage T12

14. Higher classification: Tequatrovirus Scientific name : Escherichiavirus t4 Realm : Duplodnaviria Family : Mycoviridae Order : Caudovirales

15. T4 bacteriophage Ubiquitously distributed in nature Ranging from mammalian gut to soil ,sewage and oceans 130 similar morphological features as phaget4 has been described App 1400 major capsid protein sequence have been correlated with its 3D structure Large enlongated head,contractile tail and a complex baseplate with 6LTF It serves as an excellent model for large icosahedral viruses Herpes virus It was first observed by negative stain electron microscopy

16. Head structure(head proteins) 194 MDa mature head encapsidates Empty capsid - ATP-dependent packaging machine. Prolate head- Procapsid,capsid,polyhead Hoc(highly antigenic outer capsid protein) and soc (small outer capsid protein) Soc ( Mr =10,000) present in the Capsid equivalent to the number of p23 molecules Hoc( mr = 40,000)is lower than that of p23 molecules These proteins are distributed over the surface of the heads of the antibody bound capsid These proteins basically not changed the cryo -electron microscopic structure determination of isometric capsids Dimension of the phage altered the cryo electron microscopy Prolate icosahedron are Tend=13 laevo and tmid =20(86nm wide and 120nm long) Capsid is composed of 930 post translationally modified monomer

17. Cont…… Or 155 hexamers of the major protein gene product 23(gp23*) Gp24* forms pentamers at 11 of the 12 pentameric vertices and is hologus to gp23* with 21% residues 12 th vertex –special vertex (portal protein gp20) Portal protein- initiates head assembly,genome packing ,genome gatekeeper) Rod shaped soc binds between 2 gp23* hexamer Soc maintains the stability of the head Hoc is an elongated molecule protruding from center of gp23* hexamers

18. ` the major Capsid protein gp23* and the vertex protein gp24* similar in mw and sequence Hk97-like capsid protein fold 17% of the structurally aligned residues between t4gp24 and hk97 were identical Structyrally aligned residues (axial and peripheral domain) A domain forms central part of hexameric and pentameric capsomerrs P domain located in capsomeres periphery Several stages of head prohead formation,prohead proteolysis,dna pacaking,expansion of the porlated head and binding of spc and hoc ;

19. Initiation complex –portal protein gp20 mediated by chaperone protein gp40 Prohead core proteins gp21,gp22,gp67,gp68 Initiation proteins IPI,IPII,IPIIIV and gpalt Gp23 and gp24 start to form a shell around the core protein To assemble prohead Gp23 requires 2 chaperone protein –GroEL,gp31 Assembly of prohead -930 copies of gp23 and 55 copies of gp24 Volume of prohead is app 15-20% smaller than mature head Gp21-protease (t4PPase) in prohead Cleaved head is released from membrane into cytosoland gp23*and gp24* leads to larfe conformational expansion of the prohead .

20. gp17 and gp16 complex cleaves the dna and associates the cleaved end Terminase –DNA complex created Dodecameric portal protein gp20 at the vertex of the prohead Gp16 initiates the dna translocation by ATPase activity of gp17 Capsid expands during the DNA pacaking Expansion causes rotation of the gp23*subunits 50%increase of the capsid volume and stabilises capsid and creates binding sites for soc and hoc

21. Cont.. Motor generates forces app 60pn and trabslocation rates up to app 2000bp/s 172 kb t4 genome pacakged in app 5 min in concentric layers’head is filled by DNA , a signal Internal pressure produced by the pacakaged DNA is sensed by portal protein and transmitted to gp17 It then cleaves DNA and pentameric gp17 motor is released from capsid Mature head is completed by attachment of neck proteins gp13,14,2 and gp4 to portal vertex Head is ready to be joined to tail LTFs produce a mature phage

22. TAIL STRUCTURE Tail consists of tail tube surrounded by helical sheath And attachedd to dome shaped baseplate at end from the head 6STFs folded beneath the baseplate Assemblies of tail begins with assemblies of 6 wedge and central hub Form the baseplate and nucleats tail tube around which the sheath assembles Sucrose density graddient centrifugation assay

23. Wedge protein gp11,gp10,gp7,gp8,gp6,gp53,gp25 are assembled to form a wegde 6 wegdes associated around the central hub form a hexagonal baseplate Is completed by attachment of STF sand gp9 at its periphery Gp48 and gp54 to top of the hub Creating platform for polymerization of tail tube (gp19) And tail sheath (gp18) Gp29 controls the length of the tail. Gp3 binds to last row of gp19 thus stabilizing the polymerized tail tube Tail sheath polymerises around the tail tube in parallel with the formation of the tail tube Is completed by the binding of tail terminator gp15 togp3 and the last row of gp18.

24. Structure and basseplate Baseplate undergoes a dramatic conformational change Relaxes high energy dome structure to low energy star shaped structure Analyzing the structure of the component proteins Crystal structure of nine baseplate proteins(gp11,gp10,gp8,gp6,gp25,gp9,gp12,gp5,gp27) Structure fitted into 3D cryoEMreconstructed baseplate for both dome-shaped and star shaped conformations Conformation of the baseplate is changing from the dome-shaped to star shaped during infection process

25. Proteins at the periphery of baseplate (gp11,gp10,gp9)have 3fold symmetry Connect the LTFs to baseplate and transmit signal to baseplate on recognize a host cell surface lipopolysaccharide molecule Attachment of LTFs is gp9 which ia an elongated trimer has 3 domains N- terminal domain forms alpha helical ,triple coiled coil Middle domain consists of 7 stranded beta sandwich C terminal domain -8 stranded antiparallel beta barrel structure It assocciate with a trimeric domain associates with gp7 in the baseplate Hinge between n-terminal and the middle domain provides flexibility to LTFs Change in gp9 initiates the baseplate conformational change result in transfer of t4 genome into the host Gp10 is the largest protein component

26. Tail tube and sheath terminators Polymerized tail tube and sheath capped by terminator protein gp3 and gp15 Prevent depolymerization before the tail attaches to the head Form hexameric rings interact with last row of gp19 and gp18 molecules Gp3 has not determined Gp15 has 4 stranded antiparallel beta sheet and alpha helix facing inner side of the ring Central pore and side surface of gp15 are negatively charged Top and bottom surfaces are positively charged and it interacts with gp14 and gp3 Interaction between gp15 and gp18 different in extended and contracted conformations

27. In contacted tail ,- vely charged side surface of the gp15 hexamer interacts with + vely charged surfaces of c-terminal domain of gp18 molecules Interactions help to maintain the integrity of the tail in its contracted form Gp15 hexamer undergo conformational change during infection Propagated through gp14 and gp13 to portal assembly to allow the release of the genomic DNA

28. LTF COLLAR AND WHISKERS 1450 A long LTFs consist 4 proteins Gp34,gp35,gp36 and gp37 Chaperon protein gp57A is required for trimerization of gp34 ,gp37 Chaperon protein gp38 required proper folding of gp37 Gp34 formed by proximal half of the fibre and interact with adaptor protein gp9 on baseplate Monomeric gp35 forms the hinge Proximal and distal half fibres assemble independently C-terminal part of gp36 binds to n-terminal region of gp37 Distal part of the fibre divided into 11 domains (D1-D11)

29. Cont… Domain 1 and domain2 are a part of gp36 D3-D11 are part of gp37 D10 and D11 of gp37 (was in terms of collar ,needle and head domain) Head domain sits at tip of the distal end and recognizes the receptor binding site on the host cell Gp37 known to bind to lipopolysacharide and as protein saccharide interactions Involve aromatic side chains – Trp and Tyr residues at the tip of gp37 important for host recognition

30. Filled head is attached to the tail via the neck proteins gp13 and gp14 Six 500 A – long trimeric ‘whiskers’ fibres ( gpWac ) are atached to the neck Whiskers have a trimeric coiled coil structure known as ‘ fibritin ’ 50 amino acid residues at the n-terminus of gpWac bind to neck region 30 amino acid residues 0f the c-terminus are a chaperon determines the proper trimerization of the fibritin 6 other fibres also constructed of fibritin form a collar around the neck Fibritin and gp63 (RNA ligase ) promote attachment of the LTFs to the phage It is also important in sensing the optimum environment for phage infection

31. Complementation assays,cross linking analyses,x -ray crystallography and cryoEM,providing a structural model of phage t4