Bacteriophages Lecture Notes PDF

BACTERIOPHAGES  LEARNING OUTCOMES At the end of this lecture, students will be able to: Explain the structure and function of selected phages, particularly in regard to replication mechanisms. Describe how bacteriophage λ vectors are used to clone DNA. Give examples of vectors...

BACTERIOPHAGES  LEARNING OUTCOMES At the end of this lecture, students will be able to: Explain the structure and function of selected phages, particularly in regard to replication mechanisms. Describe how bacteriophage λ vectors are used to clone DNA. Give examples of vectors used to clone long pieces of DNA, and evaluate the strengths and weaknesses of each type.  BACTERIOPHAGES Are viruses that infect only bacteria (such as T4, lambda, P1, M13 etc.). The nucleic acid inside the coating, called the phage genome (either double- or single-stranded DNA or RNA,) in a bacteriophage. Surrounded by a protective coat (capsid) made up of protein molecules. 2 main types of phage structure head and tail (eg. λ) filamentous (eg. M13)  STRUCTURE OF BACTERIOPHAGES T4 a linear double-stranded DNA genome enclosed in a capsid and attached to a tail. capsid is an elongated icosahedron. very elaborate tail structure including a collar at the base of the head and a rigid tail core surrounded by a contractile sheath.  STRUCTURE OF BACTERIOPHAGES Lambda a linear double-stranded DNA genome of 48.5 kb, a capsid, and a tail. capsid is shaped like an icosahedron tail is a thin flexible tube that ends in a small conical part and a single tail fiber  STRUCTURE OF BACTERIOPHAGES M13 a circular single-stranded DNA genome of 6407 nucleotides surrounded by five phage-encoded proteins chromosome is coated by a single layer of ~2700 subunits of gene VIII encoded protein (gpVIII) giving it a filamentous appearance  LIFE CYCLE The phage must be able to recognize a bacterium in which it can multiply by binding to the bacterial cell surface. The phage must inject its genome and the genome must be protected from the bacterial nucleases in the cytoplasm. The phage genome must be replicated, transcribed, and translated to a large number of genomes, capsid proteins, and tail proteins. Complete phage particles are then assembled and the phage must be released from the bacterium.  LIFE CYCLE Has 2 developmental pathways: Lytic (productive cycle) Lysogenic (non-productive cycle) The process of a phage infecting a bacterium and producing progeny is referred to as a lytic infection. The capable of maintaining their chromosome in a stable, silent state within the bacteria is called lysogeny infection.  LYTIC INFECTION Lytic or virulent phages are phages which can only multiply on bacteria and kill the cell by lysis at the end of the life cycle. Infection is a 3-step process:- - attachment to bacterium and injection of DNA into the cell - the phage DNA is replicated - new phage particles are assembled and released from the bacterium. Lysis of the host and release of ~100 progeny particles/cell The average yield of phages per infected bacterial cell is known as burst size.  ECLIPSE PHASE The interval between the entry of phage nucleic acid into bacterial cells and the release of mature phage from the infected cell. No infectious phage particles can be found either inside or outside the bacterial cell during the eclipse phase. This phase is devoted to the synthesis of phage components and their assembly into mature phage particles.  ASSAY FOR LYTIC PHAGE Plaque assay Lytic phage is classified as a plaque assay. Plaque is a clear area which results from bacteria lyses. Each plaque arises from a single infectious phage. The infectious particle that gives rise to a plaque is called a pfu (plaque forming unit).  LYSOGENIC INFECTION Phage DNA inserted into the host chromosome at a specific site. The integrated form of phage DNA is called prophage (provirus). A host cell containing a prophage is called a lysogenic bacterium / lysogen. Most of the phage functions are switched off. Phage DNA molecules can be retained in the host bacterium for several cell divisions. Entry and exit of the DNA from the chromosome are site-specific genetic recombination events catalysed by the lambda integrase protein. It may be induced to undergo lytic development. VIRULENT VS TEMPERATE PHAGES Virulent phages do not integrate their genetic material into the host cell chromosome and usually kill the host cells (lytic infection) (e.g. T-phages of E.coli). Temperate phages can be integrated into the host DNA, causing LYSOGENY. Phage that is capable of both a lytic and lysogenic pathway is called temperate phage (e.g: P1 & lambda).  SIGNIFICANCE OF BACTERIOPHAGES Transduction is responsible for the transfer of drug resistance, especially in Staphylococci Lysogenic conversion is responsible for the acquisition of new characteristics Random insertion into bacterial chromosome can induce insertional mutation Epidemiological typing of bacteria (phage typing) Lambda phage is a model system to study the latent retroviral latent infection in mammalian cells.  SIGNIFICANCE OF BACTERIOPHAGES Phages are commonly used in genetic engineering where they serve as cloning vectors Genes libraries and monoclonal antibodies are maintained in phages They are responsible for natural removal of bacteria from water bodies  LAMBDA ( λ) BACTERIOPHAGE Its DNA is a double stranded linear molecule of about 49 kb. The ends of the genomic DNA are single-stranded and are cohesive, i.e. they are complementary to one another. The two cohesive ends - known as cos sites - are 12 nucleotides in length. As linear DNA is injected into the host, it circularise via the cohesive termini (cos sites). A temperate phage - Lysogenic or temperate phages are those that can either multiply via the lytic cycle or enter a lysogenic cycle. λ - A TEMPERATE PHAGE  λ BACTERIOPHAGE The cos site is important during λ infection cycle because:- allow linear DNA injected into host to be circularised act as recognition sequence for an endonuclease to produce complete single λ genomes  ROLLING CIRCLE REPLICATION To begin rolling circle replication, a cut is made in a double stranded circle of DNA. It produces a 3'OH and 5' P end. Nucleotides are added to the 3' end to synthesize the DNA, causing the 5' end to pull out of the circle and produce a linear double stranded DNA molecule. When the 3' OH end is extended, the 5' end can be displaced in a manner similar to the strand displacement reaction. Synthesis on this strand is similar to leading strand synthesis as well. The displaced strand can, in turn, serve as an template for replication as long as a suitable primer is available. Synthesis on this strand is similar to lagging strand synthesis. ROLLING CIRCLE REPLICATION  LAMBDA GENOME PACKAGING Genome size 48.5-50 kb Only about 60% of the phage genome is necessary for lytic growth  All the genes coding for the capsid components are grouped together in the left hand third of the molecule. Genes controlling the integration into host genome are clustered in the middle Genes associated with gene regulation and immunity to superinfection (N, cro, cI) followed by DNA synthesis (O, P), late function regulation (Q) and host cell lysis are to the right. Clustering of related genes is very important for controlling the λ genome because it allows genes to be switched on and off as a group.