1. 微生物遺傳與生物技術 (Microbial Genetics and Biotechnology) 金門大學 食品科學系 何國傑 教授

2. Autonomously replicating genetic entities: (2) Bacterial viruses – bacteriophages (or phages for short)

3. Bacteriophages Like all viruses, phages are so small that they can be seen only under the electron microscope, and are essentially genes (on DNA or RNA genome) wrapped in a protein coat (capsid) with or without membrane envelope. There are two types of life cycles: lytic or lysogenic life cycle. Phages are usually detected only by the plaques (the holes) they form on the lawns of susceptible host bacteria. Phages are not alive, they can not multiply outside a bacterial cell. Bacterial colony vs plaque

4. Bacteriophages Icosahedral capsid Filament capsid

5. I. The lytic development cycle of bacteriophage (Fig. 7.2) 1.A phage adsorbs to an actively growing bacterial cell by binding to a specific receptor on the cell surface. 2. The phage injects its entire DNA into the cell, and transcribes RNA usually using host RNA polymerase immediately. Those transcribed soon after infection are called the early genes of the phage and encode mostly enzymes involved in DNA synthesis. The early genes have promoters that mimic those of the host DNA and recognized by host σ factor. 3.Next, mRNAs are transcribed from the rest of the phage genes, the late genes. These genes have promoters that are unlike those of host cell and are not recognized by the host RNA polymerase alone. Most of these genes encode proteins involved in assembly of the head and tail.

6. I. The lytic development cycle of bacteriophage (Fig. 7.2) 4. After phage particle is completed, the DNA is taken up by the head and the tails are attached. 5. Finally, the cells break open, or lyse and new phage are released to infect other sensitive bacteria. 6. Whole process known as lytic cycle, takes less than 1 hour for many phages and hundreds of progeny phage can be produced from a single infecting phage (burst size). 7. Virulent phages - phages that lyse their host during the reproductive cycle

7. I. The lytic development cycle of bacteriophage

8. I. The lytic development cycle of bacteriophage (Fig. 7.2)

9. Transcriptional regulation during development of a typical large DNA phage 1. The purple arrows indicates activation of gene expression 2. The black bars indicates repression of gene expression

10. Phage plaques

11. II. The selected examples The following examples are selected either because they are the basis for cloning technologies or because of the impact they have had on our understanding regulatory mechanisms in general. IIa. Phage T7, a phage-encoded RNA polymerase 1. Phage T7 has a relatively simple program of gene expression after infection, with only two major classes of genes, the early and late genes.

12. II. The selected examples - Phage T7 2.Some genes of about 50 T7 genes are shown in Fig. 7.4 (Genetic map of hage T7). 3.After infection, expression of T7 genes proceeds from left to right, up to and including gene 1.3 are early genes. The genes to the right of 1.3 are transcribed after few minutes’ delay – the late genes. 4. Nonsense and temperature-sensitive mutations were used to identify which of the early-gene products is responsible for turning on the late genes. It turns out the gene 1, which encodes the T7 RNA polymerase.

13. II. The selected examples - Phage T7 5. In fact, transcription of the late genes by the gene 1 product may help pull the DNA into the cell, causing sequential gene expression. 6. The specificity of phage RNA polymerase for their own promoter has been exploited in many applications in molecular genetics: (1) T7 phage based expression vectors, for example pET vectors (for plasmid expression T7): i. Using T7 polymerase and T7 gene10 promoter to express foreign gene in E. coli. Gene10 promoter is a very strong promoter. Hundreds of thousands of copies of T7 head protein must be synthesized in a few minutes after infection. Some pET vectors have strong translation initiation regions and His tag. ii. T7 promoter is made inducible by providing the T7 RNA polymerase only when the foreign gene is to be expression. This is important in cases where the fusing protein is toxic to cell.

14. II. The selected examples - Phage T7 iii. Fig. 7.5 demonstrates the strategy for regulating the expression of genes cloned into a pET vector. (i) To provide a source of inducible T7 RNA polymerase, E. coli strains has been constructed in which phage gene 1 for RNA polymerase is cloned downstream of the inducible lac promoter and integrated into their chromosome. Therefore, it is expressed only if the inducer IPTG is added. This kind of strain often has the DE3 suffix. (ii) The T7 RNA polymerase then transcribes the gene cloned into the pET vector downstream of the T7 late promoter on the cloning vector.

15. Strategy for regulating the expression of genes cloned into a pET vector 1. Gene 1 (for T7 RNA polymerase) is inserted into the E. coli chromosome and transcribed from lac promoter. Gene 1 is expressed only when inducer IPTG is added. 2. The T7 RNA polymerase then transcribes the cloned gene inserted downstream of the T7 late promoter on pET vector. 3. If the protein product of the cloned gene is toxic, the transcription of the cloned gene before induction can be reduced by: (1) The T7 lysozyme encoded by a compatible plasmid, pLysS binds to any residual T7 RNA polymerase in absence of induction and inactivates the RNA polymerase. (2) The presence of lac operators between the T7 late promoter and cloned gene also reduce the transcription of the cloned gene in the absence of IPTG.

16. Strategy for regulating the expression of genes cloned into a pET vector

17. II. The selected examples - Phage T7 [ 6. The specificity of phage RNA polymerase for their own promoter has been exploited in many applications in molecular genetics: (slide 12) (1) T7 phage based expression vectors, for example pET vectors (for plasmid expression T7):] (2) Phage display i. A randomized peptide coding DNA sequence is fused to the T7 head protein-encoded gene (gene 10). Different phages display different versions of the peptide on their surface. ii. The phage that display a version of the peptide which binds to the other protein molecule can then be screened and isolated. iii. The cloned DNA can then sequenced and protein can be determined.

18. II. The selected examples - Phage T4 IIb. Phage T4: Transcription activators, antitermination, a new sigma factor, and replication-coupled transcription 1. Structure of phage T4 (1) Phage T4 is much larger than T7, with over 200 genes. (2) Genes are divided into groups according to the time individual gene expression: immediate-early genes (express immediately after infection), delayed-early and middle genes (express only few minutes after infection), and true-late genes. i. Immediate-early genes and delayed-early genes are transcribed from the same normal σ70 promoter, but delayed-early genes are regulated by an antitermination mechanism (discuss when phage λ is talked).

19. II. The selected examples - Phage T4 Phage T7 and coliphages Todd have short, non-contractle tails without fibers.

20. II. The selected examples - Phage T4 ii. Middle genes are transcribed from their own promoter, which look somehow different fromσ70 promoters in that their - 35 sequence is replaced by a sequence centered at - 30 called Mot box (Fig. 7.9). These promoters required the phage- encoded MotA and AsiA proteins, the products of delay-early genes. AsiA protein binds to region 4 ofσ 70 and inhibits its to the - 35 sequence. AsiA allows MotA to bind to region 4, it can now recognize the - 30 sequence of the middle T4 promoter.

21. II. The selected examples - Phage T4 iii. The products of true-late genes are mostly the head, tail, and tail fiber components of the phage and enzymes and proteins needed to lyse and release the phage. (i) The initiation of transcription of true-early genes is coupled to the replication of DNA. (ii) The promoters of true-early genes have the sequence TATAAATA rather than the - 10 sequence of a typicalσ 70 promoter, and they lack a - 35 sequence. The product of the T4 regulatory gene 55 is an alternate sigma factor that binds to the host RNA polymerase, changing its specificity so that it recognizes only the promoters of true-early genes. This altered sigma seems to lack a region 4 to recognize - 35 sequences. This makes it unable to form open complexes and initiate transcription efficiently.

22. II. The selected examples - Phage T4 (iii) Protein gp33 which binds to β subunit can substitute for the region 4 ofσ 70 to allow open complex formation and the initiation of transcription, only it binds with gp45. (iv) The same strategy use alternate sigma factors to activate the late genes is also employed in bacteria, ex., sporulation of Bacillus subtilis. * Structure of typicalσ70 factor: region 1 – preventing itself from binding to DNA; region 2 – binding to -10 region 3 – involving in both core and DNA binding; region 4 – binding to -35

23. II. The selected examples - Phage T4 (3) Replication-coupled transcription i. In addition to the alternate sigma factor, gp55, and the accessory protein, gp33, the products of other T4 genes are required to turn on the transcription of late genes. Some of these products are also required for replication of phage DNA. ii. Coupling the transcription of the true-late genes to the replication of phage DNA makes sense from a strategic standpoint. Many of the true-late genes encode parts of the phage particle including the head, and the phage heads are not needed until phage DNA is available to be packaged inside them.

24. II. The selected examples - Phage T4 @ Replication-coupled transcription A. 1. The normal situation where the clamp loaders, gp44 and gp62, load the gp45 clamp on the DNA as DNA polymerase (gp43) begins synthesizing an Okazaki fragment at an RNA primer. 2. The gp41 helicase separates the DNA dtrands. 3. After the Okazaki fragment is synthrsized, the gp43 comes of the gp45 clamp, which stays on the DNA and slides to a true- late promoter, contacts gp33 on RNA polymerase (RNA pol) and activates transcription. (gp55, the alternate sigma factor) 4. A new gp45 clamp is loaded onto the DNA as replication continues. B. 1. If infected by a DNA ligase-deficient mutant of T4, nicks persist in the DNA. The gp45 clamp may load at such nicks independent of the other replication proteins and slide on the DNA until it contacts gp33 on RNA pol at a late promoter.

25. II. The selected examples - Phage T4

27. III. Phage DNA replication 1. Phage M13 – a filamentous phage with a circular, single- stranded DNA. M13 is a male-specific phage. They do not lyse the cell when released. (1) While the phage DNA enters the cytoplasm, the major coat protein is stripped off into the host inner membrane. (2) When phage is released, the coat proteins are waiting in the membrane to be assembled.

28. III. Phage DNA replication – M13 (3) The steps in the replication of phage M13 DNA are outlined in Fig. 7.12. i. An RNA primer is used to synthesize the complementary minus strand (in black) to form double-stranded replicative-form (RF) DNA, which is dependent entirely on host functions. Normally the host RNA polymerase recognize only double-stranded DNA, but the single- stranded DNA form a hairpin at the origin of replication. The 5’ exonuclease of DNA polymerase I removes the RNA primer and the nick is sealed by polymerization activity of Pol I and ligase.

29. III. Phage DNA replication – M13 ii. The product of gene II, an endonuclease, nicks the plus strand of the RF and remains attached to the 5’ phosphate at the nick. The host Rep, a helicase, help unwind the DNA at the nick. iii. More + strands are synthesized via rolling-circle replication, and their – strand are synthesized to make more RFs. iv. The gene V product binds to plus strands as they are synthesized, preventing them from being used as template for more RF synthesis and helping package them into phage protein coat. The single-stranded DNA is transferred to the assembling viral particle in the membrane and leaked out of the cell.

30. Phage M13

31. M13 cloning vector (4) M13 cloning vector (Fig. 7.13)