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Size of DNA in phage

Size of DNA in phage



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I have read that DNA(after recombination) is packaged in bacteriophages lambda only if it's between 40000 and 53000 bp long. This constraint can be used to ensure packaging of recombinant DNA.

I don't understand why shorter DNA can not be packaged.

Source : Lehninger Principles of Biochemistry


For lambda:

If the distance between the two cos sites is less than ~37 kb, the resulting phage particle will be unstable. When the DNA is inside the capsid, it exerts pressure on the capsid. Likewise the capsid exerts an inward force on the DNA. If there is not enough DNA inside the capsid, it will implode from the inward force of the capsid. If the distance between the two cos sites is too far (~52 kb), then the capsid will be filled before the second cos is reached. The tail cannot be added because the DNA hanging out of the capsid is in the way and no infectious phage particle is produced.

http://www.microinmuno.qb.fcen.uba.ar/03-Bacteriophage.pdf

In contrast filamentous phage like M13 have no upper size limit, but since they get longer with more DNA they become physically fragile.


Enhancing phage therapy through synthetic biology and genome engineering

Synthetic biology enables efficient phage genome engineering.

Phages with tunable host range and antimicrobial payload delivery enhance efficacy.

Phage design principles may be guided by computational approaches in the future.

The antimicrobial and therapeutic efficacy of bacteriophages is currently limited, mostly due to rapid emergence of phage-resistance and the inability of most phage isolates to bind and infect a broad range of clinical strains. Here, we discuss how phage therapy can be improved through recent advances in genetic engineering. First, we outline how receptor-binding proteins and their relevant structural domains are engineered to redirect phage specificity and to avoid resistance. Next, we summarize how phages are reprogrammed as prokaryotic gene therapy vectors that deliver antimicrobial ‘payload’ proteins, such as sequence-specific nucleases, to target defined cells within complex microbiomes. Finally, we delineate big data- and novel artificial intelligence-driven approaches that may guide the design of improved synthetic phage in the future.


Size of DNA in phage - Biology

Bacteriophages (viruses that infect bacteria) are fascinating organisms that have played and continue to play a key role in bacterial genetics and molecular biology. Phage can confer key phenotypes on their host, for example converting a non-pathogenic strain into a pathogen, and they play a key role in regulating bacterial populations in all sorts of environments. The phage-bacterium relationship varies enormously: from the simple predator-prey model to a complex, almost symbiotic relationship that promotes the survival and evolutionary success of both. While infection of bacteria used in the fermentation industry can be very problematic and result in financial losses, in other senarios phage infection of bacteria can be exploited for industrial and/or medical applications. In fact interest in phage and phage gene products as potential therapeutic agents is increasing rapidly and is likely to have a profound impact on the pharmaceutical industry and biotechnology in general over the coming years. One potential application is the use of phage to combat the growing menace of antibiotic-resistant infections.

Written by eminent international researchers actively involved in the disparate areas of bacteriophage research this book focuses on the current rapid developments in this exciting field. The book opens with an excellent chapter that provides a broad overview of the topics and also highlights the multifaceted nature of bacteriophage research. This is followed by a series of reviews that focus on the current most cutting-edge topics including bioinformatics and genomics, phage in the environment, bacteriophage in medicine, transfer of phage DNA to the host, contribution to host phenotype and much more.

Essential reading for all phage researchers and of interest to molecular biologists and microbiologists working on bacteria in academia, biotechnology and pharmaceutical companies, and in the food and other industries

"This beautifully produced, hardback book is also manageable in size and clearly a significant investment for any forward-thinking university or hospital library. It would be a source of inspiration and scientific excitement for students and researchers in a variety of fields, including medicine and industry." from SGM Microbiology Today (2007)

" . authoritative and useful . " from Doodys (2007)

(EAN: 9781904455141 9781913652333 Subjects: [virology] [bacteriology] [microbiology] [molecular microbiology] [genomics] )


Life Cycle of Mu Phage

Its lifecycle can be summarized in the following steps:

Attachment

Firstly, the tail fibres attach to the receptor site of the host cell surface. By the binding of the tail fibre, there is the conformational change in the base plate of Mu phage. Due to the conformational change in the base plate, the tail’s sheath contracts.

Penetration

By the contraction of the tail’s sheath, the rigid internal material gets into the host cell surface through the cell envelope. The N protein ( non-replicative protein) also gets injected along with the viral genome.

Circularization

The N-protein undergoes circularization once it binds with the viral genome.

Integration

After circularization, early transcription occurs that gives rise to the Repc and Ner repressors and DDE recombinase A (Mu A). These genes help in the integration of the viral genome with the host genome. During this step, the variable ends are cut off from the viral genome.

Early phase

After the non-replicative transposition, the ratio of Repc and Ner repressors decide whether the phage will enter to the lysogenic phage or lytic phage.

  • Repc: It represses the early promoter by establishing latency or lysogeny.
  • Ner: It represses the expression of Repc by promoting the expression of the early genes for the replication of Mu phage.

Middle phase

After the inactivation of Repc, there is an expression of MuA and MuB genes. MuA is the DDE recombinase-A enzyme, and MuB is the target DNA activator B. MuA performs the transposition of viral genome ends and host DNA. The target DNA activator B helps in the replication of viral host DNA, leading to the formation of the two copies. This type of replication is called replicative transposition. This replication can lead to 100 viral genomes after successive rounds.

Late transcription

This phase carries out the expression of the adenine modification enzyme, which makes the viral DNA resistant to the host restriction enzymes by modifying the adenines in the viral DNA.

Biosynthesis and Assembly

The late gene synthesizes the structural genes of Mu phage, which leads to the biosynthesis of virus particles. Then the virus particles like empty capsid, tail fibres etc. get to assemble.

The packaging of virion

Firstly, the bacterial DNA is first cut on the left of the integrated Mu genome for about 50-150bp. Then, a second cut occurs after the filling of phage head. The packaging of viral DNA also occurs on the right side of the Mu genome. Therefore, at different sites of the bacterial genome, the packaging of the Mu genome will occur.

Cell lysis and release of virion

After packaging, the newly synthesized virions release out of the host cell by the help of lye gene that encodes lytic enzymes (responsible for the cell lysis).


Structure of Bacteriophage

The morphology of bacteriophage includes the following components:

  • It is elongated and hexagonal in shape.
  • Head of the bacteriophage possesses a prismoid structure.
  • It is surrounded by an envelope called a capsid.
  • It is produced by the identical protein subunits called capsomeres.
  • It contains around 2000 capsomeres.

Genetic material:

  • It is 50 nm long and can be either DNA or RNA.
  • The structure of genetic material can be linear or circular.
  • It is tightly packed inside the head.
  • It is also called a collar, which connects head and tail.
  • It possesses a circular plate-like structure.
  • It resembles a hollow tube.
  • A tail is surrounded by a protein sheath.
  • It is composed of around 144 protein subunits.
  • The sheath of the bacteriophage is highly contractile.
  • It contains 24 rings.

Tail fibres:

  • These are attached to the base plate.
  • It appears long and thread-like filaments.
  • Tail fibres induce host specificity, or they are host-specific.
  • They are generally found 6 in number.
  • Size: 130x2nm
  • It is also called a tail pin.
  • Spikes recognizes the receptor sites of the host cell.

Life Cycle of Bacteriophage

Lytic and lysogenic cycles are the common phases of the bacteriophage life cycle.


Lytic Cycle

It is also called Virulent or Infectious cycle. It includes the lytic phages. The phages of a lytic cycle can infect or kill the host cell, due to which the cycle is known as a virulent phase. It includes the following steps:

  1. Attachment
  2. Adsorption
  3. Penetration
  4. Replication
  5. Assembly
  6. Cell lysis

Attachment: In this step, a tail pin or spike recognizes the receptor sites on the host bacterial cell and attaches to it. After that, the tail fibres of the bacteriophage attaches to the surface of the bacteria.

Adsorption: In this step, the amino groups of the host cell reacts with the carboxyl group of the bacterial cell and vice versa. After the reaction, the tail fibres secrete a lytic enzyme. This enzyme creates a hole in the host cell through which a viral genome moves into the host cell cytoplasm.

Penetration: This step is also known as “Injection”, in which the phage genetic material penetrates the genetic material of the host.

Replication or biosynthesis: After the penetration, the mRNA moves out of the nucleus to the cytoplasm, and the viral genome degrades the host genetic material. Then mRNA transcribes and translates to form protein capsid, which leads to the biosynthesis of other components.


Assembly: It also refers as “Maturation”. At this stage, all the components of bacteriophage assemble to produce new daughter or progeny virions.

Lysis: At this stage, the virus kills the bacterial cell by cell lysis and releases about 100-200 progeny virions.

Lysogenic Cycle

It is also termed as a Temperate or Non-infectious cycle. The phages that participate in this cycle are known as “Lysogenic phage”. These phages do not kill or infect the bacterial cell, due to which a lysogenic phase is considered as a non-infectious phase. It includes the following steps:

  1. Attachment
  2. Penetration
  3. Incorporation of genetic material
  4. Replication
  5. Cell division
  6. Induction

Attachment: In this step, lysogenic phage first recognizes the receptors site of the host through their spikes. After recognition, tail fibres attach onto the host cell surface.

Penetration: It is also the same as the lytic phase, in which the tail fibres release a lysogenic enzyme to create a pore through which a viral genome goes into the cytoplasm of the host cell.

Incorporation of genetic material: It is also called as “Integration of genetic material”. At this stage, phage DNA incorporates into the genetic material of the host genetic material, which forms a complex (Prophage). Prophage is a temperate phage, which remains non-active, i.e. it is not able to produce new progenies.

This step is simply the recombination of phage and the host’s genetic material, which does not involve the transcription and translation processes.

Replication: During favourable conditions, prophage replicates when the bacterial genome replicates and pass onto the daughter cells.

Cell division: In this stage, a cell divides into two identical daughter cells.

Induction: It has two conditions:

  • The prophage can remain in the dormant state inside the host cell.
  • Secondly, prophage can continue the lytic cycle by the induction of UV-rays or heat treatment.

Significance

  • It helps in the identification, classification and detection of pathogenic bacteria.
  • It acts as a biocontrol agent by killing the bacteria that are involved in soil and water pollution.
  • To study the concept of evolution, it also acts as the modal organisms.
  • Bacteriophages have significant use in genetic engineering.
  • In space microbiology, it is used as a radiation detector.
  • It is significantly used in the treatment of many diseases caused by bacteria, also known as phage therapy.
  • Bacteriophages play a central role to control bacterial plankton growth.
  • In horticulture, these are used in the form of a spray to protect plants and vegetables.
  • Also, acts like biocides such as disinfectant to clean up the environmental surfaces. E.g. In hospitals.

Therefore, a bacteriophage shows a wide range of ecological, molecular and biomedical significance.


II. Polyhedral or cubic phages

-classified into Microviridae, Corticoviridae, Tectiviridae, Leviviridae, and Cystoviridae.

Microviridae phages- icosahedral head, virion size 27 nm, with 12 capsomers, single-stranded DNA (ssDNA)

Example: phage φX174

Corticoviridae phages- no envelope, 63 nm in size, complex capsid, lipids, dsDNA

Example: phage PM2

Tectiviridae phages- no envelope, 60 nm, flexible lipid vesicle, pseudo-tail, dsDNA

Example: phage PRD1

Leviviridae phages- no envelope, 23 nm, poliovirus-like, ssRNA

Example: phage MS2

Cystoviridae phages-with enveloped, icosahedral head, 70-80 nm, lipids, dsRNA

Example: Pseudomonas ɸ6


Acknowledgements

This study was supported by Polish Ministry of Science and Higher Education research grant No. N N401 3550 33 and within the European Union project Operational Programme Innovative Economy 2007–2013 (OP IE) No POIG.01.03.01-02-003/08: “Optimization of the characteristics and production of therapeutic bacteriophages”. These funding bodies did not have any role in the design of the experiments, in the collection, analysis, and interpretation of data in the writing of the manuscript or in the decision to submit the manuscript for publication.


Bacteriophage T4 genome

Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the "cell-puncturing device," combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages-the most abundant and among the most ancient biological entities on Earth.

Figures

Electron micrographs of bacteriophage T4.…

Electron micrographs of bacteriophage T4. The well-recognized T4 morphology was nature's prototype of…

Intrastrand biases (nucleotide skew) in…

Intrastrand biases (nucleotide skew) in the T4 genome. (A) Cumulative values of the…

Functional genome map of bacteriophage…

Functional genome map of bacteriophage T4. The coding capacity of the T4 genome…

Diagram of the relationship between…

Diagram of the relationship between the T4 transcriptional pattern and the different mechanisms…

Logo of T4 promoters. Nearly…

Logo of T4 promoters. Nearly all the sequences in each alignment have promoter…

Logo of T4 RBS. Translation…

Logo of T4 RBS. Translation initiation regions of the annotated T4 GenBank file…

The T4 replisome. A model of a T4 DNA replication fork and the…

Structural components of the T4…

Structural components of the T4 particle. Features of the particle have been resolved…

Three-dimensional image reconstruction of the…

Three-dimensional image reconstruction of the T4 tube-baseplate from cryoelectron microscopy. (A) Stereo image…

Structure of T4 thymidylate synthase.…

Structure of T4 thymidylate synthase. The T4 sequence was aligned with other available…

Phylogenetic tree of thymidylate synthases…

Phylogenetic tree of thymidylate synthases and deoxynucleotide hydroxymethylases. All protein sequences were obtained…


Amplification of DNA by the Polymerase Chain Reaction

Molecular cloning allows individual DNA fragments to be propagated in bacteria and isolated in large amounts. An alternative method to isolating large amounts of a single DNA molecule is the polymerase chain reaction (PCR), which was developed by Kary Mullis in 1988. Provided that some sequence of the DNA molecule is known, PCR can achieve a striking amplification of DNA via reactions carried out entirely in vitro. Essentially, DNA polymerase is used for repeated replication of a defined segment of DNA. The number of DNA molecules increases exponentially, doubling with each round of replication, so a substantial quantity of DNA can be obtained from a small number of initial template copies. For example, a single DNA molecule amplified through 30 cycles of replication would theoretically yield 2 30 (approximately 1 billion) progeny molecules. Single DNA molecules can thus be amplified to yield readily detectable quantities of DNA that can be isolated by molecular cloning or further analyzed directly by restriction endonuclease digestion or nucleotide sequencing.

The general procedure for PCR amplification of DNA is illustrated in Figure 3.27. The starting material can be either a cloned DNA fragment or a mixture of DNA molecules𠅏or example, total DNA from human cells. A specific region of DNA can be amplified from such a mixture, provided that the nucleotide sequence surrounding the region is known so that primers can be designed to initiate DNA synthesis at the desired point. Such primers are usually chemically synthesized oligonucleotides containing 15 to 20 bases of DNA. Two primers are used to initiate DNA synthesis in opposite directions from complementary DNA strands. The reaction is started by heating the template DNA to a high temperature (e.g., 95ଌ) so that the two strands separate. The temperature is then lowered to allow the primers to pair with their complementary sequences on the template strands. DNA polymerase then uses the primers to synthesize a new strand complementary to each template. Thus in one cycle of amplification, two new DNA molecules are synthesized from one template molecule. The process can be repeated multiple times, with a twofold increase in DNA molecules resulting from each round of replication.

Figure 3.27

Amplification of DNA by PCR. The region of DNA to be amplified is flanked by two sequences used to prime DNA synthesis. The starting double-stranded DNA is heated to separate the strands and then cooled to allow primers (usually oligonucleotides of 15 (more. )

The multiple cycles of heating and cooling involved in PCR are performed by programmable heating blocks called thermocyclers. The DNA polymerases used in these reactions are heat-stable enzymes from bacteria such as Thermus aquaticus, which lives in hot springs at temperatures of about 75ଌ. These polymerases are stable even at the high temperatures used to separate the strands of double-stranded DNA, so PCR amplification can be performed rapidly and automatically. RNA sequences can also be amplified by this method if reverse transcriptase is used to synthesize a cDNA copy prior to PCR amplification.

If enough of the sequence of a gene is known that primers can be specified, PCR amplification provides an extremely powerful method of obtaining readily detectable and manipulable amounts of DNA from starting material that may contain only a few molecules of the desired DNA sequence in a complex mixture of other molecules. For example, defined DNA sequences of up to several kilobases can be readily amplified from total genomic DNA, or a single cDNA can be amplified from total cell RNA. These amplified DNA segments can then be further manipulated or analyzed, for example, to detect mutations within a gene of interest. PCR is thus a powerful addition to the repertoire of recombinant DNA techniques. Its power is particularly apparent in applications such as the diagnosis of inherited diseases, studies of gene expression during development, and forensic medicine.


Replication of Bacterial Viruses

DNA Replication of T4-Like Phages

The serologically related E. coli infecting bacteriophages T2, T4, and T6 are commonly called T-even phages. They have a dsDNA genome 170 kbp long whose termini contain repetitions of 3% of the genome. In addition, the genome of T-even phages contains glucosylated hydroxymethylcytosines that protect DNA from endonucleases and confer double-strand stability. The T-even phages encode for their own replication machinery, which makes them good candidates to study the general mechanism of DNA replication.

At early stages after infection, T4 DNA replication starts from only one replication origin. The T4 helicase/primase complex (gp41/gp61), loaded on to DNA by T4 gp59, moves processively in the 5′–3′ direction in lagging strand synthesis at the same time as the primase activity periodically synthesizes the RNA primers to initiate Okazaki fragment synthesis. Leading strand synthesis is initiated by an RNA molecule synthesized by a host RNA polymerase from early or middle promoters. DNA polymerase (gp43) catalyzes DNA synthesis of both strands assisted by gp45, a trimer that acts as a sliding clamp, holding the DNA polymerase tightly to the DNA. gp44/gp62 complex uses the hydrolysis of ATP to drive the binding of gp45 to DNA. Although T4 DNA replication onset depends on the replication origin, most of T4 DNA replication forks are initiated by using intermediates of recombination as DNA primers at random positions throughout the genome. Once the replication fork reaches the 3′-end, the single-stranded portion of the chain that is templating lagging strand synthesis invades an homology region in other DNA molecules because of the terminal redundancy of its ends, accomplishing a recombination-dependent DNA replication pathway called ‘join-copy replication’, that depends on genes expressed from early or middle promoters, and that is initiated from the invading 3′ DNA ends. This promotes the appearance of replicating DNA intermediates containing multiple covalently linked copies of the genome. When an endonuclease cuts at either of the invaded DNA strands, ‘join-cut-copy recombination’ is initiated from the 3′-ends to allow copying of single-stranded segments of an invading DNA. This pathway requires the action of either endo VII or terminase proteins, predominantely synthesized at late infection times, making the join-cut-copy the late pathway for DNA replication, since origin initiation of replication ceases during T4 development ( Figure 1 ).

Figure 1 . Initiation of DNA replication from intermediates of homologous recombination. Figure shows how ssDNA end of parent 2 invades the homologous dsDNA of parent 1. Reproduced from Mosig G, Gewing J, Luder A, Colowick N, and Vo D (2001) Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer. Proceedings of the National Academy of Sciences, USA 98: 8306–8311, copyright (2001) National Academy of Sciences, USA, with permission from National Academy of Sciences.