III. BACTERIOPHAGES OR BACTERIAL VIRUSES 1.Introduction and properties of phages Viruses are obligate intracellular parasites. The complete virus particle is termed as virion. Phages are bacterial eaters. Bacteriophages are viruses that infect bacteria.. Bacteriophages were discovered independently by Frederick.W. Twort (1915) and Felix d’Herelle (1917). Phages possesses genes coding for a variety of viral proteins but phages use ribosomes, protein-synthesizing factors etc., of the host cell. Properties of phages/ Functions performed by phages for survival 1. Phages have Icosahedral / isometric/ helical capsid. These protect the nucleic acid from environmental chemicals. 2. Phages Deliver their nucleic acid in to the bacterial cell cytoplasm / Periplasmic space. 3. Phages convert an infected bacterium to a phage- producing system this yields a number of progeny phages. 4. Progeny phages were released from an infected bacterium by cell lysis. 5. Some of the phages convert phenotypic character of the bacterium and are called Lysogenic phages. Bacteriophages have the capability to perform two types of life cycle .They are i) Lytic cycle Progeny viruses are released by lysis of host cell. The phage that carries out lytic life cycle is termed as virulent phage. ii)Lysogenic cycle In this cycle, host cell will not produce any progeny viruses. Phage DNA is inserted in to the chromosome of the host, the bacterial cell continues their normal function. Phage, which is capable of lysogenic cycle, is termed as temperate phage. 2.CLASSIFICATION OF BACTERTIOPHAGES Bacteriophages are classified based on their morphology and genome. Maximum number of phages having DNA as a genetic material. There are about thirteen families of viruses infecting bacteria are presented in recent ICTV (International Committee on Taxonamy of Viruses)classification system. Summary of ICTV classification is presented here along with their common morphology. Refer Picture for morphology for viruses. Single stranded DNA Phages Inoviridae Inovirus Enterobacteria phage M13 Plectrovirus Acholeplasma phage MV-L51Mycoplasma Microviridae Microvirus Enterobacteria phage ØX174 Spiromicrovirus Spiroplasma phage 4 1 Bdellomicrovirus Bdellovibrio phage MAC1 Chlamydiamicrovirus Chlamydia phage 1 Double stranded DNA Phages Caudovirales Myoviridae "T4-like viruses"(1) Enterobacteria phage T4 "P1-like viruses" Enterobacteria phage P1 "P2-like viruses" Enterobacteria phage P2 "Mu-like viruses" Enterobacteria phage Mu "SP01-like viruses" Bacillus phage SP01 "H-like viruses" Halobacterium virus H Siphoviridae "-like viruses" Enterobacteria phage "T1-like viruses" Enterobacteria phage T1 "T5-like viruses" Enterobacteria phage T5 "L5-like viruses" Mycobacterium phage L5 "c2-like viruses" Lactococcus phage c2 "M-like viruses" Methanobacterium virus M Podoviridae "T7-like viruses" Enterobacteria phage T7 "P22-like viruses" Enterobacteria phage P22 "29-like viruses" Bacillus phage 29Bacteria Tectiviridae Tectivirus Enterobacteria phage PRD1 Corticoviridae Corticovirus Alteromonas phage PM2 Plasmaviridae Plasmavirus Acholeplasma phage L2 Lipothrixviridae Lipothrixvirus Thermoproteus virus 1 Rudiviridae Lipothrixvirus Sulfolobus virus SIRV1 2 Fuselloviridae Fusellovirus Sulfolobus virus SSV1 "Sulfolobus SNDV-like viruses" Sulfolobus virus SNDV The DSRNA Phages Cystoviridae Cystovirus Pseudomonas phage 6 Negative Stranded SS RNA Viruses Leviviridae Levivirus Enterobacteria phage MS2 Allolevivirus Enterobacteria phage 3.T4 BACTERIOPHAGE Phages that infect coliform group of bacteia are generally called as coliphages. These phages also termed as ‘T’ series phages). There are seven coliphages in the T series (T1 to T7). Of these, T2, T4 and T6 are known as T-even Coliphages. Others phages are termed as odd phages. The best-known member of large virulent bacteriophages is T4. It is included under the family Myoviridae Structure of T4 Phage T 4 phage is a complex virus. It shows binal symmetry. It consist of three parts namely Head, Collar and Tail. Head • Head is called as capsid protein. It is in the form of an Icosahedral shape. The structure is described as an elongated, bipyramidal, hexagonal prism. • It is about 95nm long and 65nm wide. • The head contains DNA, divalent cations (Mg2+, Ca2+), three internal proteins and ATP. • The capsid is made up of 20, 000 capsomers. Neck It is also known as head-tail connector or collar and it has been attached with whiskers. Tail • The tail consists of an outer contractile tail sheath and an inner core or tube. • The tail is about 80nm long and 18nm in diameter. • The tail consists of 24 rings. Each protein ring contains 6 subunits. • It is connected to the collar at upper end and base plate at the lower end. Base plate The base plate is hexagonal and has a tail pin at each corner. Tail fibers • A long thin tail fiber arises from each of the six corners of the base plate. 3 • Each tail fiber is about 130nm long and 2nm in diameter • The tail fibers recognize specific receptor sites on the host cell wall during attachment. • A single tail fiber is composed of two half fibers. • The A half fiber (proximal) is attached to the base plate and BC half fibre (distal) has the attachment to host receptor. DNA Genome of T4 phage is Double stranded DNA and is 500µm long and has a molecular weight of 120x 106 Daltons. Peculiar properties of T4 DNA. Unusual Base • Cytosine of T4 phage DNA undergoes modification to form Hydroxyl Methyl Cytosine (HMC). • Glucosylated and modified form of HMC is referred to as modified base or unusual base of T4 DNA. • This protects the phage DNA from the action of host-encoded restriction endonucleases. Terminal redundancy • T4 DNA is terminally redundant • It is the unique feature of DNA where same base pairs are found at both ends. Circular permutation This is a property in which terminally redundancy bases are present at different locations with in the genome. Responsibilities of the genome. 22 genes of the T4 phage are involved in DNA replication, 34 genes synthesize structural proteins and 19 genes are involved in assembly. T4 Phage Multiplication cycle or life cycle of T4 Phage or Lytic life cycle of phage T4 multiplication cycle can be divided in to two periods. 1. Eclipse period where nucleic acid replication and protein synthesis occurs and 2. Maturation period where morphogenesis (Assembly) of phage 4 particles occurs. T4 phage is also called as virulent phage. It undergoes lytic life cycle when it infecting coliforms (eg. Escherichia coli) 1.Attachment or Adsorption Attachment begins when the tail fibers contact specific receptor sites on Cell wall. LPS, teichoic acid, flagella and pili can serve as receptors for attachment. Pinning Adsorption occurs in two stages, an early reversible stage when the phage is attached to the cell by means of tips of tail fibers. The phage moves on the cell surface, until it is pinned through tail pins. Pinning represents the irreversible stage of adsorption. 2. Entry or Penetration of nucleic acid: The process includes tail contraction, penetration, unplugging and injection of genome. Tail contraction • The tail sheath contracts by using ATPase enzyme activity, becoming shorter and thicker. • The tail sheath is reorganized from 24-ring structure to 12 ring structures. • The hexagonal base plate is converted into a wedge shaped structure. • Now the phage behaves like a microsyringe . Penetration and unplugging • The energy produced during contraction of tail sheath pushed the core tube through the cell wall. • The core tube initially passes only through the cell wall and does not penetrate plasma membrane. • It comes in to contact with a membrane component, phosphatidyl glycerol resulting in unplugging. • Unplugging results in release of phage DNA in to the host cell. Soon after entry of phage genome host cell DNA, RNA and protein synthesis are stopped . 3. Breakdown of bacterial chromosome • The chromosome undergoes unfolding and loses its compactness. This is the result of relaxation of super helical DNA by phage encoded helix- destablishing protein (Phage encoded DNase). • The bacterial DNA is cleaved at its ends by exonuclease and broken down in to fragments by endonuclease. • The free nucleotides remain with in the cytoplasm of the host cell and are used for phage replication. Arresting of host cell development • T4 phage brings about complete arrest of host cell nucleic acid synthesis and protein synthesis. • Thus DNA replication, transcription and translation of the host cell is inhibited. 5 4. Synthesis of protein and replication of phage DNA The phage utilizes host-synthesizing machinery (ribosome, RNA polymerase) to synthesize its proteins. Both early and late proteins are produced. Early genes code for enzymes and late genes code for structural proteins. • T4 DNA replication begins about 6minutes after infection • The rate of DNA synthesis increases and reaches a maximum at 10 to 12 minutes. • Each step of DNA synthesis begins with the synthesis of RNA primer. • T4 DNA polymerase then extends the DNA strand, using the single strand as a primer by addition of nucleotides that are complementary to the parental strand. This process is referred to as polymerization. • This enzyme also contains proofreading activity, which corrects mismatched base pair. • Both strands replicate discontinuously in 51→31 direction and results in the formation of ‘Okazaki fragment’. • These fragments are joined together by T4 DNA ligase and the replication proceeds continuously to form multi-genome length molecules called concatamers. Terminal redundancy is responsible for this process. • Concatemeric DNA is cleaved to form unit length T4 DNA by the enzyme terminase. • At the end of this step about 80 DNA molecules of phage accumulate in the host cell. 5. Assembly or Morphogenesis Head assembly (gp means gene product) gp 22 protein is responsible for core formation . gp 21 is for polyhedral capsid synthesis. Complete icosahedron is formed by using gp 2 and 4 proteins. Finally mature head is formed by filling of prohead with replicated DNA and adding gp 16, gp 17 proteins. Tail assembly • gp 5, gp 6, gp 7 are involved in base plate formation • Maturation of base plate involves gp 48, gp 54.