III. BACTERIOPHAGES OR BACTERIAL VIRUSES 1.Introduction and Properties of Phages Viruses Are Obligate Intracellular Parasites

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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. Core tube and sheath • gp 19 is responsible for core tube formation.. • Tail sheath is made up of of gp 18. Collar • Gp 15 forms the neck protein Tail fibers • Each tail fiber has two halves namely A and BC • gp 34 forms A and gp 37 forms BC • gp 38 is involved in union of A and BC • gp 35 and gp 36 are involved in formation of complete tail fibers Tail pins is made up of gp 12 6.Release • The release of progeny takes place by lysis of bacterial cell wall by lysozyme coded by gene ‘t’. It occurs at 22 minutes after phage attachment. • Lysozyme acts on NAM and NAG linkage of cell wall, lead to cell cleavage. • The Lysozyme molecules are brought to the cell surface by gene rII. • Lysis of the bacterial cell releases about 300 bacteriophages.

6 4.LAMBDA PHAGE (λ-Temperate phage) Introduction Normally virulent bacteriophages lyse their host cells during reproductive cycle. Lamda phages carry out lysogenic life cycle. Many DNA phages also establish a different relationship with their host. After adsorption and penetration viral genome does not destroy its host. Instead, viral genome remains with in the host cell and replicates with the bacterial genome. • The phage DNA that is in an integrated form with host chromosome is referred to as prophage. • Lambda phage very effectively uses K12 strain of Escherichia coli Structure of λ phage • It belongs to Sophoviridae family • Head is icosahedral in shape and is about 55nm in diameter • Capsid contains 300 to 600 subunits of capsomers. • Molecular weight is 37, 500 D • Head is joined to tail by head – tail connector • Tail is long, flexible and is about 150x 8nm in size with 25nm long non contractile tail fiber.

λ DNA • DNA is double stranded linear and 17µm in length • Genome has 46, 500 base pairs. • The molecular weight of DNA is 31x106 Daltons. • λ DNA possesses 12 nucleotides as extending units in ether side of the DNA, which are complementary to each other, and it is said to be cohesive ends. Genes of lambda phage and their responsibilities Gene Product WBCDE Synthesis of head proteins F Z U V G H I J Tail proteins K L M aH Attachment site in λ phage Xis Excisionase (exision of λ DNA from host genome) red, gam Site specific recombination int integrase (prophase formation) c I λ repressor cII Activator of C I cIII Protects C I from degradation Cro Inhibition of repressor synthesis (codes for lytic pathway) O, P DNA synthesis N Early control Q Control transcription of phage head and tail genes. S , R Lysis of the host cell. A Maturation (Assembly process).

LIFE CYCLE OF λ PHAGE λ phage is a temperate phage and is capable carrying out both lytic and lysogenic life cycle. 1.attachment and Entry of phage. λ phage attaches to the host cell by tail through maltose receptor. It leads to entry of Lambda DNA in to the host (E. coli K12.

2.Replication First phase in λ DNA replication is circularization of λ DNA via joining of the cohesive ends by host DNA ligase. This process allows supercoiling of DNA. Transcription takes place by making use of host cell RNA polymerase. Transcription is proceeded bi-directionally. The first genes transcribed are N and cIII on the leftward direction and cro and cII on the rightward direction. cII products activates transcription of cI and int gene. Right ward transcription produced O and P Protein, which initiate replication at ori site. λ DNA is replicated by rolling circle mechanism and produced long concatemers that are finally cleaved to give complete genomes.

3. Assembly Virion assembly takes place in the cytoplasm are similar to the same process already described in T4 Phage.

Lysogenic life cycle After attachment, λ DNA is released into the cytoplasm of host cell. Suddenly λ DNA circularized and trascription has commenced, the cII and CIII protein accumulates.The cII protein binds with PRE (RE-Repressor establishment) and stimulates RNA polymerase binding.The cIII protein protects cII from degradation by host enzyme.Lambda repressor cI rapidly synthesized and binds to OR and OL, thus turning off mRNA synthesis, and leads to stoping of cII and CIII protein synthesis. This initiates lysogenic cycle.CII protein stimulates the transcription of int gene and produced integrase enzyme.λ DNA has att site ( attachment) that can base pair with a bacterial attachment site located between the gal operon and biotin operon.

8 Integrase enzyme aid integration and produced a prophage. Now the bacteria perform regular duties without any lysis of host cell but with new character. This is called as lysogenic life cycle. Lambda repressor is coded by cI gene. This repressor protein has 236 aminoacids and folds into a dumb bell shape.

Choice between lytic and lysogenic pathway Cro protein accumulation leads to binding of this protein to OR and OL, turns off the transcription of the Repressor gene. It represses the PRM (RM-Repressor maintanence) function. Lmbda repressor can block cro trancription. There are a race between the production of cro protein and repressor protein. If cro protein binds to OR may block repressor protein synthesis and the lytic cycle started. If the lambda repressor wins the race the circular DNA is inserted in to the host genome and proceeds Lysogenic life cycle.

Outcome of lysogeny – immunity to super infection The prophage integrated along with the bacterial chromosome is carried on for many generations. The presence of prophage confers immunity to the host cell against super infection.

Induction Prophage can be induced to enter lytic cycle under certain conditions. The conversion of lysogenic life cycle to lytic cycle is known as induction. This is achieved by the competiting binding of cro protein to PRE . This process blocks binding of lysogenic protein. There fore, lambda switches to lytic pathway. Since λ repressor can block cro transcription, there is a race between the amount and time of

9 production of λ repressor and cro proteins. At this step, Xis gene codes for excisionase enzyme which removes λ DNA from the host chromosome. λ DNA which is excised then carrier out lytic pathway. Agents responsible for induction are UV, Ritomycin C.

LYSOGENIC CONVERSION Temporate phages may induce phenotypic character of host cell by inserting phage gene into bacterial chromosome. The phenomenon in which prophage can confer new properties on the cells is called lysogenic conversion. This conversion involves alterations in bacterial surface characteristics and pathogenic properties of the host. Examples of lysogenic conversion Synthesis of diphtheria toxin is induced when Corynebacterium diphtheriae is infected with β phage.Clostridium botulinum Synthesis its botulinum toxin because of the availability of new phage gene. Phage infected β-haemolytic Streptococcus produce Erythrogenic toxin . In staphylococcus aureus, lipase activity is lost upon infection with phage L54 a owing to inactivation of the lipase structural gene by insertion of prophage. Pathogenic property of Salmonella is increased when it is infected by an epsilon phage (E15). This phage modify the structure of its outer membrane lipoplysaccharide layer.

5.M13 BACTERIOPHAGE INTRODUCTION M13 phage is a filamentous Coliphage, these bacteriophages are composed of helical capsids and single stranded DNA as a genome. It belongs to the family of Inoviridae. The name M13 derives from city of Munich where it was first isolated by Hotschneider in 1963. M13 designation may also be attributed to Messing who studied the use of M13 as cloning vector. M13 is known as male specific phages as its is capable of infecting only E.coli F+strains . F+ denotes the cell which contains F pilus also known as male cells or donor cells.

M13 phages are known as leaky phages as the phage particles are not released by lysing the host cell after infection and multiplication. Instead, M13 phages are extruded or leaked out through F pilus of the host cell. As a result M13 phages do not form clear plaques on the lawn of host cell (E.coli F+ strain).

Structure of M13 M13 phage is about 900nm long, 9nm in diameter.

10 Capsid Proteins Capsid is made up of one major protein and four minor proteins. The major coat protein is the product of phage gene VIII (g8p). There are 2700 to 3000 copies of protein. The major protein is held together with four minor capsid proteins. Four minor capsid proteins are the product of the genes gIII, gVI, gVII and gIX. Minor capsid proteins are located at the ends of the filamentous M13 particle. gp8 proteins consist of approximately 50 aminoacid residues. It assumes the form of α helix and appears as a shot rod.

Genome M13 has circular single strandeds DNA as a genome. It is 6,407 base pair long. The genome codes for a total of 10 genes and is named using Roman numerals I through X. Genome contains specific restriction sites for Bam H1, Hpa I, NspBII, BanII, AvaI, NaeI, BalI. It also contains unique origin of replication. Functions of various genes are presented in the table.

Gene Protein Function I gp1 II gp2 Nick formation and initiation of rolling circle replication III Gp3 Minor coat protein. Binds to receptor of host cell in F pillus IV Gp4 Forms a channel for movement of M13 phage from the cytoplasm to the exterior. V Gp5 Binding protein (Binds strongly to new (t) strands). VI Gp6 Minor coat protein at the proximal it is associated with PIII VII Gp7 Protein required for assembly VIII Gp8 Major coat protein which binds at the inner surface of plasma membrane IX Gp9 Five copies at end of Bacteriophage and needed for assembly X Gp10 Repressor to control RF formation

Infection cycle of M13 Bacteriophage The filamentous phage infects only F+ cells of Escherichia coli. Attachment and entry • Adsorption of virus requires interaction between pilus and minor coat protein pIII, occurs at one end of the filamentous rod. • As the rod shaped virus penetrated pIII interacts with host cell protein.

11 • This interaction mediates the removal of the major coat protein and allow the entry of viral ssDNA into the cytoplasm.

• Replication of genome There are three stages of replication , which produce (-) strand DNA and (+) strand DNA. -stand serves as a template for (+) strand synthesis. Stage I • Positive ssDNA is converted into DS circular form called Replicative form (RF) with the help of E.coli DNA ligase. • Replicative form (RF) formed during stage I undergoes several steps to synthesize many RF molecules. Stage II Synthesis of RF • RF is initiated when MB gene II protein nicks the outer (+) strand at the origin. • Using E.coli SSB (single strand binding) protein and DNA Pol III, the (+) strand is extended from 3’OH end. • When the new(+) strand reaches the origin, it is cleaved again by geneII protein. • The old (+) strand is freed and its 3’ OH and 5’-P ends are joined by gene II protein. • RF molecule replication is controlled by gene X which acts repressor. Stage III : • The new (+) strand is formed during stage II and this serves as ss molecule. • Gene V protein binds to new (+) strand thereby preventing (-) strand synthesis.

Assembly and Release • Assembly of M13 phage occurs at the inner membrane of the host cell. • The replicated DNA is moved to the cell membrane and g8p forms the capsid. • Minor coat proteins are attacted at the respective ends and the complete M13 phage is extruded out form F pilus. • M13 phages establish permanent infection without lysogeny and produce ~300 particles / infected cell.

12 • Application of M13 Phage • It acts as a cloning vector. • Replicative form (RF) of M13 is the source for the vectors like M13MP1, M13 MP2, M13MP7, M13MP8. • Insertion of Lac Z gene in M13RF leads to M13MP1. • Site specific mutation of M13MP1 derived M13MP2 • Other vectors are constructed from M13MP2 by using restriction enzymes and poly linkers. • Larger volume of DNA segments are packed within M13 phage due to its filamentous nature. • Host cells are not lysed while releasing mature phages because M13 is a leaky phage. • M13 derived vectors also prepared by making use of other plasmids like pUC and are called phagmids eg.pEMBL8.

13 6.MU PHAGE Introduction Mu is a temperate bacteriophage. It is included under the family Myoviridae and the genus Mu like viruses. The term Mu stands for Mutator. Mu phage behaves like a transposable element or transposon. Transposon is a genetic element (ie., DNA) that can move from one location to another in the genome. Tn is also known as mobile genetic element or jumping gene. Mu causes mutations when it transposes. Early studies on Mu were carried out by A.J.Tay in 1960s. Mu DNA is unusual as it is flanked by host DNA at both ends. Mu has the ability to alter host range by altering the orientation of G segment. Mu DNA replicates by transposition .

Structure of Mu phage Mu phage has a complex structure with an icosahedral head 54nm in diameter, a contractile tail 100nm long and 18nm wide, the tail ends in a baseplate with six tail fibers. Three tail pins also available in Mu phage. Morphology is like T4 phage

Mu DNA • Mu DNA is linear ds DNA and is about 37 to 42Kb • Mu DNA is flanked by unequal lengths of host DNA at two ends. Mu Genome: • attL and attR designate left and right attachment sites. • C – Expression of transposase gene and repressor product required for establishment of a lysogen. • A and B – transposition genes • cim – control of immunity which prevents super infection • gam – inhibition of exonuclease digestion of Mu DNA • lys – lysis of the host cell • gin – inversion of G segment

Life cycle of Mu phage • Mu infective cycle may lead to the formation of a lysogen or at rare times may lead to formation of progeny phage particles. • Lysogenic state is maintained via repressor activity of c gene product

14 Mu replication occurs by transposition process. Mechanism of transposition 1. Staggered cuts are made in host DNA by viral enzyme transposase. The site of insertion is not specific. So Mu DNA can be inserted anywhere. 2. Base pairs were thus made separate exposing two single standard ends. 3. Mu ds DNA is attached to the two single stranded (overhangs) ends of the host DNA. 4. The nicks in the ss DNA are ligated and then Mu DNA replicates by semi- conservative method. 5. The replicated Mu DNA then transposes to new sites on the host DNA. Mu phages are capable of infecting E. coli and Serratia marcescens.

Lytic cycle of Mu

Adsorption of Mu The attachment of tail fibers to host LPS receptor triggers tail contraction and it occurs in the presence of Ca 2+ and Mg2+ ions, ATP which pushes the tail core through the cell wall and injects phage DNA in to the cell.

(A) When Mu infects a sensitive host, the linear DNA enters the cell and the Mu DNA (i.e. not including the variable sequences of DNA acquired from the previous host) is inserted into the recipient genome via a non- replicative, "cut and paste" mechanism.

(B) Lysogens of wild-type Mu are quite stable and are not induced by UV or other DNA damaging agents. However, derivatives of Mu with a temperature sensitive repressor -- Mu c(Ts) -- can be induced by shifting the lysogen to 420 C.

(C) When the repressor is inactivated, the A and B proteins are expressed and Mu transposes by a replicative mechanism to 50 - 100 new sites on the chromosome. Meanwhile, late phage gene products are made (including phage heads, tails, lysis proteins, etc). The phage DNA is packaged by a headful mechanism, beginning by cutting the dsDNA in host sequences located about 100 bp from the left end of Mu. The length of Mu DNA is about 37 Kb and about 39 Kb are packaged into each head, so about approximately 2 Kb of host DNA is included on the right end of the packaged DNA. After assembly of the phage, the host is lysed, releasing 50-100 phage particles.

15 Application of Mu 1. Mu variants are created which are formed by deletion of several genes essential for host cell lysis. Such phages can infect suitable host and insert their genome into host DNA but are unable to produce complete phage particles or lyse the host cell. Use: Random mutagenesis of the host genome as a result of Mu infection, thereby allowing the search for unknown genes. 2. Mini Mu variants termed as Mud-lac phages have been constructed in which lac Z gene has been attached to the viral genome. Result: Lac Z gene is activated if the viral genome is inserted near the host promoter which is detected by β-galactosidase activity. Use: Identification of promoters and the genes they regulate under different conditions. 3. Mu phages are also widely employed in gene fusions and inviro cloning. Mud means Mu derivatives.