微生物學(xué)英文教學(xué)課件：chapter9 The Viruses Bacteriophages

1、C H A P T E R 9The Viruses:Bacteriophages9.1 Classification of Bacteriophages Although properties such as host range and immunologic relationships are used in classifying phages, the most important are phage morphology and nucleic acid properties. The genetic material may be either DNA or RNA; most

2、known bacteriophages have double-stranded DNA. Most can be placed in one of a few morphological groups: tailless icosahedral phages, viruses with contractile tails, viruses with noncontractile tails, and filamentous phages. There are even a few phages with envelopes. The most complex forms are the p

3、hages with contractile tails, for example, the T-even phages of E. coli.9.2 Reproduction of Double-Stranded DNAPhages: The Lytic CycleFor a virus to multiply, it must invade a host cell and take over the hosts metabolic machinery. A single virus can give rise to several or even thousands of similar

4、viruses in a single host cell. This process drastically changes the host cell and often causes its death.1. MULTIPLICATION OF BACTERIOPHAGES The multiplication cycle of bacteriophages, like that of all viruses, can be divided into several distinct stages-attachmentpenetration biosynthesis of viral c

5、omponents Maturation Release1. Attachment (adsorption) of the virion to a susceptible host cell.2. Penetration (injection) of the virion or its nucleic acid into the cell.3. Early steps in replication during which the host cell biosynthetic machinery is altered as a prelude to virus nucleic acid syn

6、thesis. Virus-specific enzymes are typically made.4. Replication of the virus nucleic acid.5. Synthesis of proteins used as structural subunits of the virus coat.6. Assembly of structural subunits (and membrane components in enveloped viruses) and packaging of nucleic acid into new virus particles.7

7、. Release of mature virions from the cell(1)Attachment of phage to host cell :After a chance collision between phage particles and bacteria, an attachment site on the virus attaches to a complementary receptor site on the bacterial cell. (2)Penetration:During the process of penetration, the bacterio

8、phages tail releases an enzyme, phage lysozyme, which breaks down a portion of the bacterial cell wall. then the bacteriophage injects its DNA (nucleic acid) into the bacterium. (3)Biosynthesis of viral components:Any RNA transcribed in the cell is mRNA transcribed from phage DNA for biosynthesis of

9、 phage enzymes and capsid protein. The host cells ribosomes, enzymes, and amino acids are used for translation Formation of mRNA after infection of cells by viruses of different types. The chemical sense of the mRNA is considered as plus (+). The sense of the various virus nucleic acids are indicate

10、d as + if the same as mRNA, as if opposite, or as +- if double-stranded. Examples are indicated next to the virus nucleic acid. Although examples of viruses containing SS DNA of the - sense are known, none are discussed in the text.(4)Maturation :The phage heads and tails are separately assembled fr

11、om protein subunits, the head is packaged with phage DNA, and the tail is attached (5)Release: Lysozyme, whose code is provided by a phage gene, is synthesized within the cell. This enzyme causes a breakdown of the bacterial cell wall, and the newly produced bactedophages are released from the host

12、cell. The number of newly synthesized phage particles released from a single cell usually ranges from about 50 to 200. 2.one-step growth curve The development of the one-step growth experiment in 1939 by Max Delbrck and Emory Ellis marks the beginning of modern bacteriophage research. In a one-step

13、growth experiment, the reproduction of a large phage population is synchronized so that the molecular events occurring during reproduction can be followed. A culture of susceptible bacteria such as E. coli is mixed with bacteriophage particles, and the phages are allowed a short interval to attach t

14、o their host cells. The culture is then greatly diluted so that any virus particles released upon host cell lysis will not immediately infect new cells. This strategy works because phages lack a means of seeking out host cells and must contact them during random movement through the solution. Thus p

15、hages are less likely to contact host cells in a dilute mixture. The number of infective phage particles released from bacteria is subsequently determined at various intervals by a plaque count . A plot of the bacteriophages released from host cells versus time shows several distinct phases. During

16、the latent period, which immediately follows phage addition, there is no release of virions. This is followed by the rise period or burst, when the host cells rapidly lyse and release infective phages. Finally, a plateau is reached and no more viruses are liberated. The total number of phages releas

17、ed can be used to calculate the burst size, the number of viruses produced per infected cell. The latent period is the shortest time required for virus reproduction and release. During the first part of this phase, host bacteria do not contain any complete, infective virions. This can be shown by ly

18、sing them with chloroform. This initial segment of the latent period is called the eclipse period because the virions were detectable before infection but are now concealed or eclipsed. The number of completed, infective phages within the host increases after the end of the eclipse period, and the h

19、ost cell is prepared for lysis. The one-step growth experiment with E. coli and phage T2 provides a well-studied example of this process. When the experiment is carried out with actively growing cells in rich medium at 37C, the growth curve plateau is reached in approximately 30 minutes. Bacteriopha

20、ge reproduction is an exceptionally rapid process, much faster than animal virus reproduction, which may take hours.There are genetic controls that regulate when different regions of phage DNA are transcribed into mRNA during the multiplication cycle. There are early messages that are translated int

21、o early phage proteins, the enzymes used in the synthesis of phage DNA. There are late messages that are translated into late phage proteins for the synthesis of capsid proteins. This control mechanism is mediated by RNA polymerase.eclipse period Time course of events in phage T4 infectionLatent per

22、iod9.3 Reproduction of Single-StrandedDNA Phages The reproduction of single-stranded DNA phages now will be briefly reviewed. The phage of X174, family Microviridae, is a small ssDNA phage using E. coli as its host. Its DNA base sequence is the same as that of the viral mRNA (except that thymine is

23、substituted for uracil) and is therefore positive; the genome contains overlapping genes. The phage DNA must be converted to a double-stranded form before either replication or transcription can occur. When X174 DNA enters the host, it is immediately copied by the bacterial DNA polymerase to form a

24、double-stranded DNA, the replicative form or RF. The replicative form then directs the synthesis of more RF copies, mRNA, and copies of the DNA genome. The phage is released by host lysis through a different mechanism than used by the T4 phage. The filamentous ssDNA bacteriophages behave quite diffe

25、rently in many respects from X174 and other ssDNA phages. The fd phage, family Inoviridae, is one of the best studied and is shaped like a long fiber about 6 nm in diameter by 900 to 1,900 nm in length. The circular ssDNA lies in the center of the filament and is surrounded by a tube made of a small

26、 coat protein organized in a helical arrangement. The virus infects male E. coli cells by attaching to the tip of the pilus; the DNA enters the host along or possibly through the pilus with the aid of a special adsorption protein. A replicative form is first synthesized and then transcribed. A phage

27、-coded protein then aids in replication of the phage DNA by use of the rolling-circle method. The filamentous fd phages do not kill their host cell but establish a symbiotic relationship in which new virions are continually released by a secretory process. Filamentous phage coat proteins are first i

28、nserted into the membrane. The coat then assembles around the viral DNA as it is secreted through the host plasma membrane. The host bacteria grow and divide at a slightly reduced rate.9.4 Reproduction of RNA Phages Many bacteriophages carry their genetic information as single-stranded RNA that can

29、act as a messenger RNA and direct the synthesis of phage proteins. One of the first enzymes synthesized is a viral RNA replicase, an RNA-dependent RNA polymerase. The replicase then copies the original RNA (a plus strand) to produce a double-stranded intermediate(RNA), which is called the replicativ

30、e form and is analogous to the DNA seen in the reproduction of ssDNA phages. The same replicase next uses this replicative form to synthesize thousands of copies of +RNA. Some of these plus strands are used to make more RNA in order to accelerate + RNA synthesis. Other + RNA acts as mRNA and directs

31、 the synthesis of phage proteins. Finally, + RNA strands are incorporated into maturing virus particles. The genome of these RNA phages serves as both a template for its own replication and an mRNA. MS2 and Q, family Leviviridae, are small, tailless, icosahedral ssRNA phages of E. coli, which have b

32、een intensely studied. They attach to the F-pili of their host and enter by an unknown mechanism. These phages have only three or four genes and are genetically the simplest phages known. In MS2, one protein is involved in phage adsorption to the host cell (and possibly also in virion construction o

33、r maturation). The other three genes code for a coat protein, an RNA replicase, and a protein needed for cell lysis. Only one dsRNA phage has been discovered, the bacteriophage 6 of Pseudomonas phaseolicola. It is also unusual in possessing a membranous envelope. The icosahedral capsid within its en

34、velope contains an RNA polymerase and three dsRNA segments, each of which directs the synthesis of an mRNA. It is not yet known how the dsRNAs are replicated.9.5 Temperate Bacteriophages and Lysogeny Up to this point many of the viruses we have discussed are virulent bacteriophages; these are phages

35、 that lyse their host cells during the reproductive cycle. Many DNA phages also can establish a different relationship with their host. After adsorption and penetration, the viral genome does not take control of its host and destroy it while producing new phages. Instead the viral genome remains wit

36、hin the host cell and replicates with the bacterial genome to generate a clone of infected cells that may grow and divide for long periods while appearing perfectly normal. Each of these infected bacteria can produce phages and lyse under appropriate environmental conditions. This relationship betwe

37、en the phage and its host is called lysogeny. Bacteria having the potential to produce phage particles under some conditions are said to be lysogens or lysogenic, and phages able to enter into this relationship are temperate phages. The latent form of the virus genome that remains within the host bu

38、t does not destroy it is called the prophage. The prophage usually is integrated into the bacterial genome but sometimes exists independently. Induction is the process by which phage reproduction is initiated in a lysogenized culture. It leads to the destruction of infected cells and the release of

39、new phagesthat is, induction of the lytic cycle.Lysogeny Some phages can incorporate their DNA into the host cells DNA., The phage remains latent and does not cause lysis of the host cell. Such a state is called lysogeny.Such phages are called lysogenic phages or temperate phages. The participating

40、bacterial host cells are known as lysogenic cells. Lysogenic cycle of bacteriophage lambda in E.coliThe consequences of infection by a temperate bacteriophage. The alternatives on infection are integration of the virus DNA into the host DNA (lysogenization) or replication and release of mature virus

41、 (lysis). The lysogenic cell can also be induced to produce mature virus and lyse. 噬菌體對(duì)發(fā)酵工業(yè)的影響烈性噬菌體能在短時(shí)間內(nèi)連續(xù)完成繁殖的五個(gè)階段（吸附、侵入、復(fù)制、裝配和釋放） 大腸桿菌T4、T7和X174噬菌體 烈性噬菌體的生活史就是侵染宿主，裂解細(xì)胞后又侵染鄰近宿主細(xì)胞的過程。溫和性噬菌體感染細(xì)胞后，并不馬上引起細(xì)胞裂解，而是以“原噬菌體（Prophage，亦稱前噬菌體）”方式整合在宿主的DNA中，隨寄主繁殖而延續(xù)傳代的噬菌體 不引起同源菌株細(xì)胞裂解，故不易被人察覺。 一旦溶源性細(xì)菌發(fā)生自發(fā)裂解或誘發(fā)裂

42、解，將會(huì)危害發(fā)酵菌株。因此必需采取有效的手段檢測(cè)出溶源性細(xì)菌。敏感的、非溶源性的菌株作為指示菌 待測(cè)菌樣在合適的培養(yǎng)基中培養(yǎng) 對(duì)數(shù)期進(jìn)行紫外線照射，誘導(dǎo)原噬菌體復(fù)制 觀察是否有噬菌斑出現(xiàn)或菌液變清 溶源性細(xì)菌的檢測(cè) 溶源性菌株的命名是在菌株名稱后加括號(hào)，內(nèi)寫攜帶原噬菌體的名稱 E.coli B() 溫和性噬菌體存在狀態(tài) 游離態(tài) ： 游離于細(xì)胞之外，具感染性的完整病毒粒子。整合態(tài) ：溫和性噬菌體都具有雙鏈DNA。 營養(yǎng)態(tài) 原噬菌體自發(fā)或經(jīng)理化因素誘導(dǎo)，脫離細(xì)菌染色體，進(jìn)入營養(yǎng)期。 噬菌體的防治噬菌體的防治 (1)污染現(xiàn)象污染現(xiàn)象發(fā)酵工業(yè)如抗生素、味精、有機(jī)溶劑和釀酒發(fā)酵經(jīng)常會(huì)遭受噬菌體污染 異常

43、現(xiàn)象 碳源和氮源的消耗減慢，發(fā)酵周期延長，pH值異常變化，泡沫驟增，發(fā)酵液色澤和稠度改變，出現(xiàn)異常臭味，菌體裂解和減少，引起光密度降低和產(chǎn)物銳減等。污染嚴(yán)重時(shí)，無法繼續(xù)發(fā)酵，應(yīng)將整罐發(fā)酵液報(bào)廢（即倒罐）。 (2) 污染原因污染原因發(fā)酵菌種本身是溶源性的 好氧發(fā)酵的空氣過濾系統(tǒng)失效或發(fā)酵環(huán)境中存在大量的噬菌體等原因也很容易加劇噬菌體污染。(3) 防治措施防治措施 杜絕噬菌體的各種來源 應(yīng)定期監(jiān)測(cè)發(fā)酵罐、管道及周圍環(huán)境中噬菌體的數(shù)量變化。 控制活菌體的排放 活菌體是噬菌體生長繁殖的首要條件，控制其排放能消除環(huán)境中出現(xiàn)特定的噬菌體。 使用抗噬菌體菌株和定期輪換生產(chǎn)用菌 選育和使用抗噬菌體的生產(chǎn)菌株是

44、一種較經(jīng)濟(jì)有效的手段。定期輪換生產(chǎn)菌種，可以防止某種噬菌體污染擴(kuò)大，并能使生產(chǎn)不會(huì)因噬菌體污染而中斷。噬菌體污染后的補(bǔ)救措施 發(fā)酵早期：升溫殺滅噬菌體，減少培養(yǎng)基中營養(yǎng)成分被破壞，再補(bǔ)充一些促進(jìn)細(xì)胞生長的玉米漿等，重新接入大量種子，就可以繼續(xù)進(jìn)行發(fā)酵。 發(fā)酵中期：噬菌體污染早期大量接入另一菌種的種子液或發(fā)酵液，繼續(xù)進(jìn)行發(fā)酵，以達(dá)到減少損失、避免倒罐的目的。 Summery1. There are four major morphological groups of phages: tailless icosahedral phages, phages with contractile tail

45、s, those with noncontractile tails, and filamentous phages.2. The lytic cycle of virulent bacteriophages is a life cycle that ends with host cell lysis and virion release.3. The phage life cycle can be studied with a one-step growth experiment that is divided into an initial eclipse period within th

46、e latent period, and a rise period or burst .4. The life cycle of the dsDNA T4 phage of E. coli is composed of several phases: Attachment, Penetration, Biosynthesis of viral components, Maturation ,Release.5. Temperate phages, unlike virulent phages, often reproduce in synchrony with the host genome to yield a clone of virus-infected cells. This relationship is lysogeny, and the infected cell is called a lysogen. The latent form of the phage genome w