T Even T Odd Bacteriophage Fahareen-Binta-Mosharraf MNS

Bacteriophages T4 This is well studied from the phage era. Its structure includes head,collar, and jointed tail fibers. T4 is among the largest phages; it is approximately 200 nm long and 80-100 nm wide. All phages contain a head structure, which can vary in size and shape. Some are icosahedral (20 sides) others are filamentous

Some phages have tails attached to the phage head Some phages have tails attached to the phage head. The tail is a hollow tube. T4 tail is surrounded by a contractile sheath At the end of the tail, phages like T4 have a base plate and one or more tail fibers attached to it

T4 genome T4 has a double-stranded DNA genome that is circularly permuted and terminally redundant. genome can be divided into three parts, encoding early proteins, middle proteins, and late proteins T4 does not have its own RNA polymerase: thus on entry, it produces proteins that modify the specificity of the host RNA polymerase so that it recognizes the phage promoters. Then an antisigma factor, that interferes with recognition of the host promoters.

the DNA of T4 phages is linear both in the virus and within the host cell, the genetic map is circular the ends of each T4 DNA molecule are terminally redundant—that is, a duplication of genetic information occurs at both ends of a single molecule. This phenomenon is the result of the headful mechanism utilized for the packaging of T4 DNA into the T4 capsid. After DNA replication, replica molecules can recombine with each other at their terminal redundancies, creating very long molecules (concatemers) many genomes in length

One headful, however, is 105% of a single genome One headful, however, is 105% of a single genome. Therefore, the packaging process will generate a population of phages carrying all possible circular permutations of the T4 genome

Bacteriophages T7 Linear double-stranded DNA with 39,936 base pairs 92% DNA codes for protein Its genome is smaller than T even phages One of the coded proteins inhibits the host restriction system, it is synthesized before the entire T7 genome enters the cells. T7 have its own RNA polymerase: on entry, it is produced by using the host RNA polymerase as immediate early protein

T7 structure DNA bacteriophages consist of shell of protein subunits arranged to form a polyhedron that encloses the DNA genome, a tubular tail assembly, and tail fibers for attachment to host bacteria. The length of the DNA is 300-500 times the diameter of the head

T7genome T7has a double-stranded DNA genome that is circularly permuted and terminally redundant. the DNA is linear both in the virus and within the host cell, the genetic map is circular the ends of each T7 DNA molecule are terminally redundant—that is, a duplication of genetic information occurs at both ends of a single molecule. This phenomenon is the result of the headful mechanism utilized for the packaging of T7 DNA into the T7 capsid. After DNA replication, replica molecules can recombine with each other at their terminal redundancies, creating very long molecules (concatemers) many genomes in length

T7 genome

Circular Permutation Concept  If the genetic information is represented by ABCDEFGH then a circular permutation would generate molecules ABCDEFGH BCDEFGHA CDEFGHAB  DEFGHABC EFGHABCD and so on.

Circular permutation

Concatemer formation and packaging of headful genome

Terminally redundant concept A linear DNA molecule with the same sequence (genetic information) at each end. If genetic information is represented by ABCDEFGH then a terminally redundant sequence can be for instance ABCDEFGHAB.

General Pattern of T4 Gene Expression It is a general strategy of T4 viruses to defer some gene expression to later stages of infection and progeny virus maturation. The immediate–early genes are transcribed at 5 minutes postinfection. However, at 5 minutes postinfection, DNA replication has also begin delayed–early or middle genes are transcribed at 10 minutes postinfection Expression of the early and middle genes ceases about 12 minutes postinfection. and transcription of the late genes begins

T4 gene expression

Gene Products Early proteins Middle proteins Late proteins Nuclase (breaks down host DNA) Proteins inhibiting host restriction system RNA dependent RNA polymerase (+ss RNA virus) Proteins stopping functions of host RNA polymerase DNA polymerase Helicase Primase Endonuclease DNA ligase Capsid protein Envelope protein Head, tail ,collar, base plate, tail fiber proteins Lysozyme Proteins necessary for self assembly Enzymes involved in maturation Endolysin for host cell lysis

General Pattern of T7Gene Expression Several genes of left end transcribed first by cell RNA polymerase to form immediate early proteins. The functions of IE proteins involve: Inhibiting host restriction system Formation of T7 RNA polymerase Proteins halting action of host RNA polymerase The phage RNA polymerase involved in major transcription process of phages life cycle.

T7 gene expression Genome Gene functions Class I Class II Class III T7 RNAP expression, host interference host DNA digestion, T7 DNA replication T7 particle formation, DNA maturation and host lysis

Gene Products Class Expression time Significance I 4 to 8 minutes after infection Made by using cell RNA polymerase to form immediate early proteins II 6 to 15 minutes after infection Made by using T7 RNA polymerase for phage DNA replication III 15 minutes to lysis Made by using T7 RNA polymerase to form late proteins

Tphage Lifecycle Three steps: Collision between virion and host cells Establishment of specific association of virion with specific receptors on host cell surface After a virion of Tphage attaches to a host cell and the DNA penetrates the cytoplasm, the expression of viral genes is regulated to redirect the host synthetic machinery to thereproduction of viral nucleic acid and protein. Lysis then assembles and releases new virions from the cell

Adsorption: The first step in the infection process is the adsorption of the phage to the bacterial cell. This step is mediated by the tail fibers or by some analogous structure on those phages that lack tail fibers. Phages attach to specific receptors on the bacterial cell such as proteins on the outer surface of the bacterium, LPS, pili, and lipoprotein. This process is reversible. One or more of the components of the base plate mediates irreversible binding of phage to a bacterium

INJECTION SEQUENCE Stages I II III Events Long tail fiber adsorption Tail pin adsorption Tail sheath contraction and tail tube injection Host factor required OmpC, LPS LPS Peptidoglycan layer

LPS Peptidoglycan CM

Penetration: The irreversible binding of the phage to the bacterium through which phage DNA passes through host cell wall into cytoplasm Penetration takes place in two ways phages which have a sheath :the contraction of the sheath and the hollow tail fiber is pushed through the bacterial envelope. phages which have no sheath:Nucleic acid from the head passes through the hollow tail and enters the bacterial cell Some phages have enzymes that digest various components of the bacterial envelope.

Synthesis of Viral component Replicate own DNA Transcribe its own DNA Replicate and recombine its own DNA to form a concatemaric structure Headful packaging

Tphage Genome replication and recombination

DNA Replication of T7 an RNA primer is involved. Rightward: RNA primer is synthesized by T RNA polymerase Leftward: RNA primer is synthesized by Tprimase Both primers are elongated by T DNA polymerase.

DNA Replication of Tphages

Assembly of phage particles The Assembly of Phage Particles A complex self-assembly process. Late mRNA ( produced after DNA replication) directs the synthesis of three kinds of proteins: (1) phage structural proteins, (2) proteins that help with phage assembly and (3) proteins involved in cell lysis and phage release.

T phage release Many phages lyse their host cells at the end of the intracellular phase. The lysis of E. coli by T takes place (after ~ 22 minutes for T even and 30 minutes for T odd) at 37°C, releasing ~ 300 T phage particles per infected bacterial cell. Several T proteins are involved in this process: endolysin that attacks the cell wall peptidoglycan. holin produces a plasma membrane lesion that stops respiration and allows the endolysin to attack the peptidoglycan ,forms holes in the membrane and lysis.

Escaping Host system by T4 The DNA of most T4-related phages contains hydroxymethylcytosine (HMC) instead of cytosine. In most members of the family, the HMC residues are further glycosylated to different extents. These modifications together allow escape from host and phage restriction enzymes and are important for the developmental strategy of these phages The host DNA and host mRNA, present at the time of infection, are rapidly degraded, and the breakdown products are efficiently reused to synthesize phage DNA and RNA