
Full text loading...
Category: Bacterial Pathogenesis; Microbial Genetics and Molecular Biology
Phage Biology, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555816506/9781555813079_Chap02-1.gif /docserver/preview/fulltext/10.1128/9781555816506/9781555813079_Chap02-2.gifAbstract:
This chapter describes the life cycles of selected phages which are the best-studied examples of phages in groups that carry pathogenesis genes. The host DNA-dependent RNA polymerase makes a 20-nucleotide RNA primer at a special sequence. λ phage is carried in Escherichia coli strain K-12 in a latent form called the prophage. In this form, λ DNA is inserted into the continuity of the host chromosome. In the lytic response, E. coli DNA-dependent RNA polymerase starts to transcribe the leftward and rightward promoters, called PL and PR. Theλ R gene product cleaves bonds in the host cell wall, but only after being delivered through the cell membrane to the cell wall by the S gene product. The DNAs of generalized transducing phages have unusual and unexpected properties. The DNAs from P22, P1, and three transducing phages from Listeria have been examined by electron microscopy, and the results are strikingly similar. The pac sequence cannot be too rigidly defined, since this would reduce the possibility for the initiation of DNA packaging on bacterial DNA. The first headful is packaged, giving an overlong molecule with a repetition of the sequence that was packaged first. The second headful begins where the first one left off; thus, the second headful has a different terminal repetition. Many headfuls can be packaged sequentially, giving phage DNA molecules with many different terminal redundancies.
Full text loading...
Electron micrograph of filamentous phage f1 and diagram of the position of each gene in the phage particle. Reprinted from reference 12 with permission of the publisher.
Electron micrograph of filamentous phage f1 and diagram of the position of each gene in the phage particle. Reprinted from reference 12 with permission of the publisher.
Genes and gene products of filamentous phages f1, M13, and fd. pII binds to a sequence (the plusstrand origin) in the intergenic region (IG) of double-stranded DNA (dsDNA) and nicks the plus strand; the original plus strand is displaced by Rep helicase as a new plus strand is elongated from the 3′ end of the nick by host DNA polymerase III, with the minus strand as a template. pX, which is identical to the C-terminal one-third of pII, is required for the accumulation of ssDNA, as is pV. Dimers of pV bind cooperatively to ssDNA, which collapses the circular genome into a flexible rod with the packaging signal exposed at one end of the filament. pVII and pIX are small coat proteins located at the tip of the virus that is the first to emerge from the cell during assembly. pVIII is the major coat protein, several thousand copies of which form the cylinder that encases the ssDNA phage genome. pIII and pVI are located at the end of the virion, where they mediate the termination of assembly and the release of the virion from the cell membrane. pIII is also necessary for phage infectivity. pI may hydrolyze ATP to promote assembly; pXI is identical to the C-terminal one-third of pI; it lacks the cytoplasmic domain and may play a structural role as part of an oligomeric pI/pXI complex. pIV is a multimeric outer membrane channel through which the phage exits the bacterium. Reprinted from reference 37 with permission of the publisher.
Genes and gene products of filamentous phages f1, M13, and fd. pII binds to a sequence (the plusstrand origin) in the intergenic region (IG) of double-stranded DNA (dsDNA) and nicks the plus strand; the original plus strand is displaced by Rep helicase as a new plus strand is elongated from the 3′ end of the nick by host DNA polymerase III, with the minus strand as a template. pX, which is identical to the C-terminal one-third of pII, is required for the accumulation of ssDNA, as is pV. Dimers of pV bind cooperatively to ssDNA, which collapses the circular genome into a flexible rod with the packaging signal exposed at one end of the filament. pVII and pIX are small coat proteins located at the tip of the virus that is the first to emerge from the cell during assembly. pVIII is the major coat protein, several thousand copies of which form the cylinder that encases the ssDNA phage genome. pIII and pVI are located at the end of the virion, where they mediate the termination of assembly and the release of the virion from the cell membrane. pIII is also necessary for phage infectivity. pI may hydrolyze ATP to promote assembly; pXI is identical to the C-terminal one-third of pI; it lacks the cytoplasmic domain and may play a structural role as part of an oligomeric pI/pXI complex. pIV is a multimeric outer membrane channel through which the phage exits the bacterium. Reprinted from reference 37 with permission of the publisher.
Infection cycle of a filamentous phage. Reprinted from reference 43 .
Infection cycle of a filamentous phage. Reprinted from reference 43 .
Replication of filamentous phage DNA. An RNA is synthesized to prime the synthesis of the nonviral (—) strand by DNA polymerase III to form double-stranded replicative-form (RF) DNA. The product of gene II, a site-specific endonuclease, nicks the viral (+) strand of the RF DNA and remains attached to the 5′ phosphate at the nick. More + strands are synthesized by extension of the 3′ hydroxyl terminus of the + DNA strand, and — strands are synthesized with the RNA primer to make more RF DNAs. Late in infection, the gene V product binds to the + strands as they are synthesized, preventing them from being used as templates for more RF synthesis and baring the packaging signal so that they can be packaged and extruded from the cell. Reprinted from reference 43 with permission of the publisher.
Replication of filamentous phage DNA. An RNA is synthesized to prime the synthesis of the nonviral (—) strand by DNA polymerase III to form double-stranded replicative-form (RF) DNA. The product of gene II, a site-specific endonuclease, nicks the viral (+) strand of the RF DNA and remains attached to the 5′ phosphate at the nick. More + strands are synthesized by extension of the 3′ hydroxyl terminus of the + DNA strand, and — strands are synthesized with the RNA primer to make more RF DNAs. Late in infection, the gene V product binds to the + strands as they are synthesized, preventing them from being used as templates for more RF synthesis and baring the packaging signal so that they can be packaged and extruded from the cell. Reprinted from reference 43 with permission of the publisher.
Bacteriophage λ. Bar, 100 nm. Provided by Robert Duda and Roger Hendrix.
Bacteriophage λ. Bar, 100 nm. Provided by Robert Duda and Roger Hendrix.
Genetic map of bacteriophage λ. Clusters of genes encoding products with similar functions and promoters with the transcripts that they direct are indicated at the top. The pR′ transcript continues from right to left because the genome is circular. Reprinted from reference 6 with permission of the publisher.
Genetic map of bacteriophage λ. Clusters of genes encoding products with similar functions and promoters with the transcripts that they direct are indicated at the top. The pR′ transcript continues from right to left because the genome is circular. Reprinted from reference 6 with permission of the publisher.
Antitermination of transcription in phage λ directed by the N gene product. (A) In the absence of the N gene product, transcription at the leftward and rightward promoters terminates at the first leftward and rightward terminators. (B) Synthesis of pN modifies RNA polymerase to read through the first terminators into the leftward recombination genes or into the rightward replication genes and the regulatory gene Q. (C and D) Mechanism of antitermination by pN, showing only rightward transcription. (C) In the absence of pN, transcription initiated at P R terminates at the terminator t R . (D) When pN has been made, it binds with E. coli Nus factors to the nutR site on mRNA and to RNA polymerase, causing RNA polymerase to read through t R 1 . Reprinted from reference 43 .
Antitermination of transcription in phage λ directed by the N gene product. (A) In the absence of the N gene product, transcription at the leftward and rightward promoters terminates at the first leftward and rightward terminators. (B) Synthesis of pN modifies RNA polymerase to read through the first terminators into the leftward recombination genes or into the rightward replication genes and the regulatory gene Q. (C and D) Mechanism of antitermination by pN, showing only rightward transcription. (C) In the absence of pN, transcription initiated at P R terminates at the terminator t R . (D) When pN has been made, it binds with E. coli Nus factors to the nutR site on mRNA and to RNA polymerase, causing RNA polymerase to read through t R 1 . Reprinted from reference 43 .
Replication of λ phage DNA. Reprinted from reference 43 .
Replication of λ phage DNA. Reprinted from reference 43 .
Satellite phage P4 (small head) and temperate coliphage P2 (large head).The electron micrograph was taken by the late Robley C.Williams.
Satellite phage P4 (small head) and temperate coliphage P2 (large head).The electron micrograph was taken by the late Robley C.Williams.
Interactions between satellite phage P4 and its helper phage, P2. P4 infects a P2 lysogenic strain. P4 inhibits the P2 repressor and activates P2 late gene expression. The P4 Sid (size determination) protein directs the production of a smaller head from P2 head proteins. This smaller head packages the shorter P4 DNA rather than P2 DNA. Therefore, mostly P4 phage are released, which the cells then lyse. Reprinted from reference 43 .
Interactions between satellite phage P4 and its helper phage, P2. P4 infects a P2 lysogenic strain. P4 inhibits the P2 repressor and activates P2 late gene expression. The P4 Sid (size determination) protein directs the production of a smaller head from P2 head proteins. This smaller head packages the shorter P4 DNA rather than P2 DNA. Therefore, mostly P4 phage are released, which the cells then lyse. Reprinted from reference 43 .
Integration of X DNA into the chromosome of E. coli. (A) The Int protein promotes recombination between the phage attachment site, attP, in λ DNA and the bacterial attachment site, attB, in the host chromosome. The common core sequence of the two sites is shown in black. (B) Gene order in the prophage. The cos site is where the λ DNA is cut for packaging and recircularization after infection. The locations of the int, xis, A, and J genes in the prophage are shown. This prophage gene order is a circular permutation of the order shown in Fig. 6 . The E. coli gal and bio genes are on either side of the prophage DNA in the chromosome. Reprinted from reference 43 .
Integration of X DNA into the chromosome of E. coli. (A) The Int protein promotes recombination between the phage attachment site, attP, in λ DNA and the bacterial attachment site, attB, in the host chromosome. The common core sequence of the two sites is shown in black. (B) Gene order in the prophage. The cos site is where the λ DNA is cut for packaging and recircularization after infection. The locations of the int, xis, A, and J genes in the prophage are shown. This prophage gene order is a circular permutation of the order shown in Fig. 6 . The E. coli gal and bio genes are on either side of the prophage DNA in the chromosome. Reprinted from reference 43 .
Formation of a λdgal transducing particle. A rare mistake in recombination between a site in the prophage DNA (in this case between A and J) and a bacterial site to the left of the prophage results in the excision of a DNA particle in which some bacterial DNA, including the gal genes, has replaced the phage DNA. Reprinted from reference 43 .
Formation of a λdgal transducing particle. A rare mistake in recombination between a site in the prophage DNA (in this case between A and J) and a bacterial site to the left of the prophage results in the excision of a DNA particle in which some bacterial DNA, including the gal genes, has replaced the phage DNA. Reprinted from reference 43 .
Example of generalized transduction. A generalized transducing phage infects a Trp+ bacterium, and in the course of packaging DNA into heads, the phage mistakenly packages some bacterial DNA containing the wild-type genes tor tryptophan synthesis (trp + ) instead of its own DNA. In the next infection, this transducing phage injects the trp + bacterial DNA instead of phage DNA into the tryptophan-requiring (Trp− ) bacterium. If the incoming DNA recombines with the chromosome, a Trp+ recombinant transductant may arise. Reprinted from reference 43 .
Example of generalized transduction. A generalized transducing phage infects a Trp+ bacterium, and in the course of packaging DNA into heads, the phage mistakenly packages some bacterial DNA containing the wild-type genes tor tryptophan synthesis (trp + ) instead of its own DNA. In the next infection, this transducing phage injects the trp + bacterial DNA instead of phage DNA into the tryptophan-requiring (Trp− ) bacterium. If the incoming DNA recombines with the chromosome, a Trp+ recombinant transductant may arise. Reprinted from reference 43 .
Electron micrograph of L. monocytogenes temperate phage U153 ( 14 ), which is a close relative of phage A118 ( 27 ).
Partial denaturation of DNA molecules from L. monocytogenes generalized transducing phage U153. DNA molecules were heated in alkali ( 18 ) to denature only the most AT-rich regions. The molecules were aligned by their denatured regions to show that their endpoints are not all at the same positions ( 26 ). (A) Histogram showing the positions of AT-rich regions for the individual maps shown in panel B. The vertical scale (0 to 100) shows the percentage of molecules with a denatured area at each of 1,000 positions along the map. (B) Partial denaturation maps of 77 U153 DNA molecules. Each horizontal line represents a molecule, and black rectangles show the measured positions of denatured sites arising from AT-rich regions. The molecules were manipulated (by reversal or by shifting to the left or right) such that the denatured sites could be aligned. They are shown on a scale chosen to encompass the various molecules, with an average size of 42.6 kb.
Partial denaturation of DNA molecules from L. monocytogenes generalized transducing phage U153. DNA molecules were heated in alkali ( 18 ) to denature only the most AT-rich regions. The molecules were aligned by their denatured regions to show that their endpoints are not all at the same positions ( 26 ). (A) Histogram showing the positions of AT-rich regions for the individual maps shown in panel B. The vertical scale (0 to 100) shows the percentage of molecules with a denatured area at each of 1,000 positions along the map. (B) Partial denaturation maps of 77 U153 DNA molecules. Each horizontal line represents a molecule, and black rectangles show the measured positions of denatured sites arising from AT-rich regions. The molecules were manipulated (by reversal or by shifting to the left or right) such that the denatured sites could be aligned. They are shown on a scale chosen to encompass the various molecules, with an average size of 42.6 kb.
Complete denaturation, followed by renaturation, of generalized transducing phage U153 DNA from L. monocytogenes. Adouble-stranded circular molecule with single-stranded ends is produced, as described previously ( 29 ). In the case of U153, the length of the double-stranded circular DNA is 41.1 kb, on average, and the single-stranded tails average 1.5 kb.
Complete denaturation, followed by renaturation, of generalized transducing phage U153 DNA from L. monocytogenes. Adouble-stranded circular molecule with single-stranded ends is produced, as described previously ( 29 ). In the case of U153, the length of the double-stranded circular DNA is 41.1 kb, on average, and the single-stranded tails average 1.5 kb.
Model for headful packaging that generates a circularly permuted and terminally redundant collection of DNA molecules.
Model for headful packaging that generates a circularly permuted and terminally redundant collection of DNA molecules.