Phage is a virus that invades bacteria, and it is also the genetic material that endows the biological characteristics of host bacteria.Phage must be parasitized in living bacteria and have strict host specificity, which depends on the molecular structure and complementarity of phage adsorption organ and receptor on the surface of receptor bacteria.Phage is the most common and widely distributed group of viruses.Phages are usually found in places full of bacterial communities, such as soil and animal intestines.
Bacteriophage (phage) is a general term for viruses that infect bacteria, fungi, algae, actinomycetes, spirochetes and other microorganisms. Some of them can cause the lysis of host bacteria, so they are called bacteriophages.At the beginning of the twentieth centurystaphylococcusAnd Shigella[3]。As a kind of virus, bacteriophages have some characteristics of viruses: small in size;Without complete cell structure;It only contains single nucleic acid.It can be regarded as a kind of organism that "eats" bacteria.Phage genomes contain many genes, but all known bacteriophages are bacterial cells that use various factors, amino acids and energy generation systems required for bacterial ribosome and protein synthesis to achieve their own growth and proliferation.Once you leavehost cellPhages can neither grow nor replicate.
Bacteriophage is a kind of virus, which is special in that it uses bacteria as its hostEscherichia coliHostT2 phage 。Like other viruses, bacteriophages are just a mass of genetic material wrapped by protein shells. Most bacteriophages also have "tails" to inject genetic material into the host.Phage is a ubiquitous organism, and it is often accompanied by bacteria.Phages can usually be found in places full of bacterial communities, such as soil and animal viscera.The most abundant bacteriophage in the world is seawater.
Biological characteristics
Announce
edit
The phage is small in size, and its shape includes tadpole shape, microsphere shape and thin rod shape. Tadpole shape is more common.Phage is composed of nucleic acid and protein.Proteins play a role in protecting nucleic acids and determining the shape and surface characteristics of bacteriophages.There is only one type of nucleic acid, namely DNA or RNA, double stranded or single stranded, circular or linear.
type
Announce
edit
protein structure
Icosahedron without tail structure: This bacteriophage is an icosahedron, the outer surface is composed of a regularly arranged protein subunit, the capsid, and the nucleic acid is wrapped inside.
Icosahedron with tail structure: In addition to the head of an icosahedron, this bacteriophage also has a tail composed of a hollow needle structure and an outer sheath, and a base composed of tail filaments and tail needles.
Linear body: This bacteriophage is linear, without obvious head structure, but a coiled structure composed of shell particles.
Most of the known bacteriophages are icosahedron with tail structure, which is because the regular polyhedron is the simplest structure in the polyhedron and is the easiest to build, so the virus likes to use the regular polyhedron structure.However, there are only five kinds of regular polyhedron, namely, regular 4, 6, 8, 12, and 20. Among them, regular 20 is the closest to spherical, that is, in the case of the same volume, it needs less materials and saves more.
Nucleic acid characteristics
Ss RNA: The nucleic acid contained in the bacteriophage is single stranded RNA.
Ds RNA: The nucleic acid contained in the bacteriophage is double stranded RNA.
Ss DNA: The nucleic acid contained in the bacteriophage is single stranded DNA.
Ds DNA: The nucleic acid contained in the bacteriophage is double stranded DNA.
It refers to replication and proliferation in host bacteria, producing many progeny bacteriophages, and ultimately cracking bacteria.Toxic bacteriophageIts proliferation process undergoes three stages: adsorption penetration, biosynthesis and mature release.
The phage nucleic acid entering the bacterial cell first generates early protein through early transcription, and then replicates the descendant nucleic acid, and then carries out late transcription to generate the structural protein of the phage.When the number of progeny bacteriophages reaches a certain level, due to the dissolution of bacteriophage synthetases, bacterial cells suddenly break down, and the released bacteriophages infect other sensitive bacteria again.
2. Mild bacteriophage
The host bacteria did not proliferate after infection.Its genes are integrated intoBacterial chromosomeUp, that isProphageIt replicates with the replication of bacterial chromosomes and distributes to the chromosomes of progeny bacteria with bacterial division.Temperate bacteriophageThere are lysogenic cycles and bacteriolytic cycles, which can occasionally spontaneously or under the influence of some physical, chemical or biological factors, the integrated prophage breaks away from the host bacteria chromosome, enters the bacteriolytic cycle, leading to bacterial lysis, and produces new mature phages.
Discovery History
Announce
edit
Initial stage: 1915-1940
In 1915, Frederick W. Twort became the director of the Brown Institute in London.In his research, Twater tried to find a variant of vaccinia virus for smallpox vaccine, which may replicate in live extracellular media.In an experiment, he inoculated part of smallpox vaccine to a culture plate containing nutrient agar.Although the virus failed to replicate, bacterial contaminants grew rapidly in agar discs.Twot continued his training and noticed that someBacterial colonyIt displays the "look with water" (that is, it becomes more transparent).Such colonies can no longer replicate when further cultured (i.e. bacteria are killed).Twot called this phenomenon transparent transformation.He then proved that infecting a normal bacterial colony with the transparent transformation principle would kill the bacteria.This transparent entity can easily pass through aCeramic filterIt can be diluted one million times, and when it is placed on fresh bacteria, its strength, or titer, will be restored.
Twot published a short article describing this phenomenon, thinking that the explanation for his observation is the existence of a bacterial virus.Due to service inthe First World WarTwot's research was interrupted.After returning to London, he did not continue this research, so he did not make further contributions in this field.
At the same time, Felix d'Herelle, a Canadian medical bacteriologist, was in ParisPasteur InstituteWork.In August 1915, a French cavalry squadron was stationed in Maisons Lafitte on the outskirts of Paris. A serious dysentery caused by Shigella had a devastating impact on the whole army.De Herrell filtered the patient's feces, quickly isolated dysentery bacilli from the filtered emulsion, and cultured them.Bacteria grow continuously and cover the surface of the culture dish.De Herrell accidentally observed a clear dot on which no bacteria grew.He called these things milky plaques, or plaques.De Herrell tracked the whole infection process of a patient to see when the bacteria were most and when the spots appeared.Interestingly, the patient's condition began to improve on the fourth day after infection.
De Herrell called these viruses bacteriophages, and then he invented methods in the field of virology.He diluted the plaque in a limited way to determine the concentration of the virus.His inference is that the appearance of spots indicates that the virus is a particle or a corpuscular.De Herrell also proved that the first step of virus infection is pathogen attachment (adsorption) to host cells.He proved this by mixing the virus with the host cell and coprecipitating it.(He also proved that the virus does not exist in the supernatant) The attachment of a virus only occurs when the bacteria are sensitive to the virus mixed with it, which indicates that the adsorption of a virus on the host cell has a specific range.He also described the release of lysis in clear modern terms.De Herre is one of the founders of modern virology in many aspects.
By 1921, more and more lysogenic bacterial strains had been isolated, and it was impossible to separate the virus from its host in some experiments.This led Jules Bordet of the Pasteur Institute in Brussels to believe that the infectious pathogen described by De Herrell is just a bacterial enzyme that promotes self reproduction.Although this is a wrong conclusion, it is quite close to the idea of prion structure and replication.
In the 1920s and 1930s, De Herrell focused on exploring the application of his research achievements in medicine, but there was no result.The basic research carried out at that time was often influenced by the explanation produced by the strong personality of individual scientists in the field.Obviously, there are many different bacteriophages, some of which are lystic and others are lysogenic, but the relationship between them is still unclear.The important discovery of this period was Max Schlesinger, who proved that the largest diameter of purified bacteriophages (linear dimension) was 0.1 μ m, and the mass was about 4x10 grams. They were composed of protein and DNA, with roughly the same proportion.No one clearly knew how to use this observation in 1936, but it had a significant impact in the following 20 years.
Modern: 1938-1970
Max Delbruck is a physicist trained by Gittingen University.His first job was at the Wilhelm Institute of Chemistry in Berlin, where he actively discussed the relationship between quantum physics and genetics with some researchers.Del Bruck's interest in this field led him to invent a quantum mechanical model of gene.In 1937, he applied for and won a scholarship to study at the California Institute of Technology.One toCalifornia Institute of TechnologyHe began to work with another researcher, Emory Ellis.Ellis was studying a group of bacteriophages - T2, T4, T6 (T-even bacteriophages).Del Bruck soon realized that these viruses were suitable for studying virus replication.These phages are a way to explore how genetic information determines the structure and function of an organism.From the beginning, these viruses were seen as understandingCancer virusAnd even understand how sperm fertilizes eggs and develops into a typical system of new organisms.Eric and Delbruck designed a one-step growth curve experiment.In this experiment, an infected bacterium released a large number of bacteriophages after a half-hour latency period or eclipse period.This test defines the incubation period, when the virus loses its infectivity.This became an experimental example for the bacteriophage research team.
the Second World WarAfter the outbreak, Delbrueck stayed in the United States (at Vanderbilt University) and met the Italian refugee Salvador E. Luria.Luria fled to the United States and took refuge in New York StateColumbia UniversityStudy T1 and T2 bacteriophages.They met at a conference held in Philadelphia on December 28, 1940, and planned the experiment at Columbia University in the following two days.The two scientists will recruit and lead more and more researchers to focus on using bacteria and viruses as a model to understand the process of life.What played a key role in their success was that they were invited toCold Spring Port LaboratoryDo experiments.Such a German physicist and an Italian geneticist had been working together during World War II, traveling around the United States to recruit a new generation of biologists, who were later called bacteriophage research groups.
Shortly thereafter, Tom Anderson, an electron microscientist at RCA Laboratory in Princeton, New Jersey, saw Delbruk.In March 1942, they obtained the first clear picture of bacteriophage.At about the same time, these phage mutants were isolated and identified for the first time.By 1946, Cold Spring Harbor Laboratory had opened the first bacteriophage course. In March 1947, eight people attended the first bacteriophage conference.Molecular biology developed from these slow beginnings.This science focuses on the study of bacterial hosts and their viruses.
The following 25 years (1950-1975) witnessed fruitful virological research with bacteriophages.Hundreds of virologists have published thousands of papers, mainly involving three fields: (a) research on E. coli bacteriolytic infection using T-even phages;(b) UtilizationλLysogenic studies of bacteriophages, and (c) replication and characterization studies of several unique bacteriophages, such asФX174 (single stranded circular DNA), RNA bacteriophage, T7, etc.They are modernMolecular virologyAnd biology.It is impossible to introduce all these scientific literatures one by one in this paper, and only some selective key points can be mentioned.
From 1947 to 1948, biochemical methods were widely used to study the changes in the incubation period of phage infected cells.Seymour Cohen initially studied lipids and nucleic acids with Erwin Chargaff at Columbia University, and then studied tobacco mosaic virus RNA with Wendell Stanley. In 1946, he majored in Delbruck's bacteriophage course at Cold Spring Harbor Laboratory.He used colorimetric analysis to study the influence of DNA and RNA levels in phage infected cells.
These studies show that the synthesis of macromolecules in phage infected cells has undergone dramatic changes: (a) the net accumulation of RNA stops in these cells.[Later, this became the basis for the discovery of multiple RNAs and proved the existence of messenger RNA for the first time.].(b) DNA synthesis stopped for 7 minutes, and then resumed at a rate of 5 to 10 times.(c) At the same time, the research of Monod and Wollman showed that a kind of cellular enzyme after phage infection can induceβ-Galactosidase(galactosidase) was inhibited.These tests divide the incubation period of the virus into two stages: initial stage (before DNA synthesis) and late stage.More importantly, these results suggest that viruses may change the macromolecular synthesis process of infected cells.
By the end of 1952, two experiments had had an important impact on this field.
First, Hershey and Chase used labeled viral proteins (SO) and nucleic acids (PO) to track the attachment of phages to bacteria.They can use a blender to remove the protein coat of the virus and only retain the DNA associated with the infected cells.This enables them to prove that this DNA has all the information needed to regenerate a large number of new viruses.Hershey Chase's experiment and the new DNA structure elaborated by Watson and Crick a year later together constituted the cornerstone of the molecular biology revolution.
The second experiment in virology was conducted by G.R. Wyatt and S.S. Cohen in 1953.They found a new base, namely 5, when studying T-even bacteriophages‘Hydroxymethyl cytosine（hydroxymethylcytosine）。This newly discovered base seems to replace cytosine in bacterial DNA.This led scientists to start studying the synthesis of DNA in bacteria and phage infected cells for up to 10 years.The most critical research shows that viruses introduce genetic information into infected cells.By 1964, Mathews et al. had proved that there was no 5 'hydroxymethyl cytosine in uninfected cells and it must be encoded by viruses.These experiments proposed the early enzymology concept of deoxypyrimidine biosynthesis and DNA replication, and provided clear biochemical evidence that it can encode a new kind of information and express it in infected cells.After detailed genetic analysis of these bacteriophages, the genes encoding these bacteriophage proteins were confirmed, and gene maps were drawn to make the concept more complete.In fact, the genetic analysis of the r Ⅱ and B cistron of T-even bacteriophages has become one of the most fully studied "genetic fine structures".Using phage mutants and extracts to replicate viral DNA in vitro has made an important contribution to our understanding of how DNA replicates itself.Finally, through the detailed genetic analysis of phage assembly, the complementarity of phage mutant assembly in vitro was used to illustrate how organisms use the principle of self-assembly to build complex structures.Genetic and biochemical analysis of bacteriophage lysozyme is helpful to explain the molecular properties of mutation, bacteriophage mutation（Amber mutation）It provides a clear way to study the second site suppressor mutation at the molecular level.The circular arrangement and tail redundancy (causing phage heterozygotes) structure of DNA can explain the circular genetic map of even T phages.
The synthesis of virus and cell protein changed significantly in the cells infected by phage, which was used in early researchSodium lauryl sulfateonepolyacrylamide gel(sodium dodecyl sulfate (SDS) polyacylamide gels), which was discovered dramatically. The results show that the synthesis of viral proteins has a specific sequence, which can be divided into early proteins and late proteins.This transient basic regulation mechanism finally found the sigma factor that regulates RNA polymerase and confers gene specificity.Almost every level of gene regulation (transcription, RNA stability, protein synthesis, protein processing) research is revealed through the original data obtained from phage infectivity research.
Although the research of lysogenic phage has made such remarkable progress, no one can clearly explain lysogenic phage.This situation changed in 1949, when Andre Lwoff of the Pasteur Institute began to study Bacillus megaterium and its lysogenic phages.By using a micromanipulator to divide a single bacterium up to 19 times, no virus was released.When lysogenic bacteria are dissolved externally, no virus is found.But there is often a phenomenon that a bacterium spontaneously dissolves and releases many viruses.It is an important discovery that ultraviolet rays can induce the release of these viruses. This observation can outline a wonderful relationship between a virus and its host.By 1954, Jacob and Wollman of Pasteur Institute had obtained important research results, namely, a lysogenic strain (Hfr,λ）Genetic cross after binding with insoluble receptors leads to the induction of viruses.They call this process zygotic induction.In fact, lysogenic bacteriophage orProphageThe position of (prophase) in the chromosome of its host Escherichia coli can be mapped by standard mating interruption experiment after genetic hybridization.This is a conceptual understandingLysogenic virusThis is one of the most critical experiments for the following reasons: (a) The virus behaves like a bacterial gene on a bacterial chromosome;(b) It is one of the test results that the viral genetic material remains static in the virus due to negative regulation.When chromosomes are transferred from lysogenic donor bacteria to insoluble receptor hosts, the genetic material of the virus is lost;(c) This helps to explain that the induction of enzyme synthesis and phage production, which Jacob and Walman recognized as early as 1954, is the expression of the same phenomenon ".These experiments laid the foundation for the properties of the operon model and coordinated gene regulation.
Although the structure of DNA was described in 1953, and zygote induction was described in 1954, the relationship between bacterial chromosomes and viral chromosomes in lysogenic phenomenon is still called attachment site, which can only be considered from these perspectives at that time.Later, according to the fact that the sequence of bacteriophage markers is different from the replication or growth state in the integration state, Campbell proposed that DNA and bacterial chromosomesλSo far, the real close relationship between virus and its host has been recognized.This causes separationλphageThis is a clear understanding of the immune characteristics of lysozoites and one of the early examples of how genes can be co regulated.yesλThe genetic analysis of phage life cycle isMicrobial GeneticsMajor academic exploration in the field.It deserves detailed research by all molecular virologists and biologists.
such asSalmonella typhimurium(Salmonella typhimurium) P22 is the first example of general transduction, andλPhage is the first example of special transduction.Viruses may carry cellular genes and transfer such genes from one cell to another, which not only provides a method for precise genetic mapping, but also is a new concept in virology.As the genetic factors of bacteria are studied in more detail, it can be clearly seen that research on lysogenic bacteriophages has developed into epime, transposon, retrotransposon, insertion element, retrovirus, hepadnovirus, viroidViroid like (also known as viroid like, which refers to a kind of virus wrapped in plant virus particles), as well as prion research, all of which make the definition and classification of genetic information between viruses and their hosts become blurred.The genetic and biochemical concepts derived from phage research make it possible to further develop virology.The experience and lessons learned from the research of bacteriolytic and lysogenic bacteriophages are often accompanied byAnimal virusThe research has been relearned and revised by people.
Bacterial defense methods
Announce
edit
The main way for bacteria to defend against bacteriophages is to synthesize enzymes that can degrade foreign DNA.These enzymes, known as restriction endonucleases, can cut the viral DNA injected by bacteriophages into bacterial cells.Bacteria also contain another defense system, which uses CRISPR sequences to preserve the genome segments of viruses they have encountered in the past, so that they can block virus replication by RNA interference.This genetic system provides bacteria with a mechanism similar to acquired immunity to fight against viral infection.
application
Announce
edit
As an experimental tool for molecular biology research
Phage is an important material or tool in basic biological research and genetic engineering in genetic regulation, replication, transcription and translation.The transduction function in genetics is to transfer genetic material between two strains of bacteria through bacteriophages.
Used for identification and typing of bacteria
Phage can only infect corresponding bacteria, with high specificity, and can be used for bacterial identification.At the same time, bacteriophages have type specificity, which can be used to type and identify bacteria.Phage can be used toSalmonellaE. coli and typhoid bacteria.
Phage display technology and phage antibody library
Phage display technology Is a powerfulgene expressionScreening technology was first described by American scientist Smith Ml in Science magazine in 1985.The basic principle of phage display technology is to clone the genes of foreign proteins into the genomic DNA of phages, so as to express specific foreign proteins on the surface of phages.
Ellis SEP et al. pointed out that using phage display peptide library can screen and determine the antigen of nematode vaccine, which is a new method for vaccine antigen identification.In recent years, with the gradual increase of diseases caused by epidemic viruses, antiviral peptides are considered to be a very promising method to prevent and treat diseases.Castel G and others pointed out that recombinant peptides specifically expressed by phage display technology can be applied to antiviral research and drug development.
On the basis of phage display technology and PCR cloning technology, British scientists such as Winter took the lead in publishing articles in the journal Naturephage antibody library 。The technology is to recombine the antibody heavy chain and light chain variable region genes with the phage coat protein gene, and combine the antibody fragment Fab or scFv with the phage coat protein tofusion proteinIt is displayed on the surface of phage particles, and then screen and enrich specific antibodies against certain antigens quickly and efficiently, fundamentally changing the traditional process of monoclonal antibody preparation.Krishnaswamy et al. used phage antibody library technology to screen scFv-C1 positive phage antibody against candida albicans HM-1 killing toxin. The specificity of the phage antibody is 60 times higher than that of monoclonal antibody binding antigen.Xing Youshang and others reported the application of phage antibody library technology in biological parasite detection, virus detection, genetically modified product detection, drug residue detection and other fields, pointing out that this technology has natural advantages and bright prospects in the field of inspection and quarantine.Phage antibody library technology will become the main technology of antibody production, and will bring a very broad prospect for human in disease diagnosis, tumor research, autoimmune disease research, gene therapy, disease prevention and pathogenesis.
Used to detect and control pathogenic bacteria
There are many pathogenic bacteria in food and environment. Research shows that phages can detect and control the growth of pathogenic bacteria and putrefactive bacteria in food and environment.Bmvko LYu and others discussed the advantages and disadvantages of bacteriophage detection of pathogenic bacteria, and pointed out that the use of bacteriophage detection of pathogenic bacteria in food safety and processing and manufacturing processes has great application prospects.Jiang Qin and others pointed out that the use of bacteriophages can detect Salmonella in food in real time, quickly and accurately, which is of great significance in public and food hygiene, animal husbandry and veterinary and 121 shore quarantine.Liu Xinyan believes that bacteriophages can not only be used to detect foodborne pathogens, but also be used to kill pathogens in the process of raw material collection, sterilize equipment in the process of production or processing, extend food storage period, and sterilize fresh fruits and vegetables.
The growth and reproduction of bacteriophages in host cells can cause the lysis of pathogenic bacteria, reduce the density of pathogenic bacteria, thus reducing or avoiding the chance of infection or morbidity of pathogenic bacteria, and achieve the purpose of treating and preventing diseases, namely bacteriophage therapy. This therapy has been widely used in veterinary medicine, agriculture, food microbiology and other fields.
(1) Application of bacteriophage therapy in animal husbandry
The domestic breeding industry, especially the chicken industry, is often troubled by the intestinal diarrhea disease of livestock and poultry, which is mainly caused by pathogenic microorganisms such as Escherichia coli and Salmonella.With the emergence of a large number of drug-resistant bacteria, more and more attention has been paid to the treatment of bacterial diseases with related bacteriophages that have strong specificity and are not easy to produce resistance.Smith, Barrow and others can reduce the probability of lambs, piglets and chickens suffering from intestinal diseases caused by Escherichia coli by using bacteriophage therapy.[1]
(2) Application of bacteriophage therapy in aquaculture
The frequent occurrence of explosive diseases has caused huge economic losses to the aquaculture industry,Bacterial diseasePhage therapy has a good application prospect in aquaculture.Park et al. can effectively eliminate pathogenic bacteria by feeding food containing bacteriophages when treating bacterial blood group ascites infection caused by Pseudomonas plecoghssicida and other bacteria.[2]
(3) Application of bacteriophage therapy in the treatment of human diseases
Phage therapy was first applied in the treatment of human diseases.In 1921, Bruynoghe and Maisin took the lead in treating staphylococcalSkin infection。Since then, bacteriophages have been widely used in the treatment of otolaryngology, stomatology, ophthalmology, dermatology, pediatrics and lung diseases.With the emergence of antibiotics, phage therapy has been gradually ignored.Kutter and others reported that bacteriophage therapy has great potential to treat or prevent human diseases, and pointed out that the key to avoid this therapy being ignored is to commercialize bacteriophage therapy through practice and experiment.
With the bacteriaAntibiotic resistancePhage is widely used in many fields to control the growth and expansion of pathogenic bacteria.Phage therapy can avoidIntestinal floraIt is considered as a safe, effective and potential microecological agent that can replace antibiotics to maintain the normal immunity of the body.
