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Chapter 7
Bacteriophage
Bacteriophage or phage for short are viruses that infect only bacteria. In contrast to cells that grow from an increase in the number of their components and reproduce by division, viruses are assembled from pre-made components. Viruses are nucleic acid molecules surrounded by a protective coating. They are not capable of generat- ing energy and reproduce inside of cells. The nucleic acid inside the coating, called the phage genome in a bacteriophage, encodes most of the gene products needed for making more phage. The phage genome can be made of either double- or single-stranded DNA or RNA, depending on the bacteriophage in question. The genome can be circular or linear. The protective coating or capsid surrounding the phage genome is composed of phage-encoded proteins. Many important discoveries have been made using phage as model systems. From the discovery that a nonsense codon stopped protein synthesis to the first develop- mental switch to be understood at the molecular level, phage have proven to be very useful. In this chapter, we will look at phage development using T4, l (lambda), P1, and M13 as examples. Each of these phage infect E. coli. We will examine specific dis- coveries using these phage or specific properties of the phage that have made them particularly useful to biologists.
The structure of phage
All phage have a chromosome encased in a capsid that is composed of phage-encoded proteins. For many phage types, the capsid is attached to a tail structure that is also made from phage-encoded proteins. T4 and P1 contain a linear double-stranded DNA genome enclosed in a capsid and attached to a tail (Fig. 7.1a). The T4 genome is 172 kb, while P1 is a smaller phage with a genome of 90 kb. The T4 capsid is an elongated icosahedron. T4 has a very elaborate tail structure including a collar at the base of the head and a rigid tail core surrounded by a contractile sheath. The core and sheath are attached to a hexagonal base plate. Also attached to the tail plate are tail pins and six kinked tail fibers. P1 also has an icosahedral capsid, a tail with a contractile sheath, a base plate, and tail fibers. l contains a linear double-stranded DNA genome of 48.5 kb, a capsid, and a tail (Fig. 7.1b). The finished capsid is again shaped like an icosahedron whereas the tail is a thin flexible tube that ends in a small conical part and a single tail fiber. M13 contains a circular single-stranded DNA genome of 6407 nucleotides sur- rounded by five phage-encoded proteins (Fig. 7.1c). The M13 chromosome is coated
FYI 7.
Discovery of phage
Phage were first described in 1915 by Frederick Twort and 1917 by Felix d’Herelle. Both men discovered phage when the bacteria they were working with lysed. The agent responsible for the lysis was transferable from culture to culture, invisible by light microscopy, and would go through the smallest filter they had. d’Herelle coined the term “bacteriophage”, signifying an entity that eats bacteria.
by a single layer of ~2700 subunits of gene VIII encoded protein (gpVIII) giving it a fil- amentous appearance, the reason M13 is also known as a filamentous phage. At one end of the filament are bound the M13 proteins encoded by genes VII and IX (gpVII and gpIX) and at the other end are bound the M13-encoded gene III and VI pro- teins (gpIII and gpVI).
The lifecycle of a bacteriophage
All phage must carry out a specific set of reactions in order to make more of them- selves. First, the phage must be able to recognize a bacterium that it can multiply in by binding to the bacterial cell surface. Next, the phage must inject its genome and the genome must be protected from the bacterial nucleases in the cytoplasm. The phage genome must be replicated, transcribed, and translated so that a large number of genomes, capsid proteins, and tail proteins, if present, are produced at the same or nearly the same time. Complete phage particles are then assembled and the phage must get back out of the bacterium. Different phage use different strategies to carry out each of these reactions.
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Capsid Capsid
Tail
Conical part
Tail fiber
Collar Core
Sheath
Tail fibers
Tail pins
Tail plate
gpIX
gpIII
gpVI gpVIII
(a) (b)
(c)
Fig. 7.1 The structures of (a) T4, (b) l, and (c) M13.
that a phage head be attached to the phage tail. Next the l DNA is ejected from the phage and taken up by the bacterium. The DNA in the phage head is very tightly packed. If the condensed state of the phage DNA is stabilized, ejection of the DNA does not occur. The addition of small positively charged molecules such as putrescine to the phage counteracts the negatively charged DNA and stabilizes the DNA in the phage head. This implies that the tight packing of the DNA is used to help eject the DNA from the phage particle. When l DNA is put into the capsid, one end known as the left end is inserted first. When the l DNA comes out of the phage head, the right end exits first. Unlike phage T4 (see below), no change in the l tail structure is seen
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5'
Head/tail genes
att
Recombination
Immunity
DNA replication
Lysis
3'
Single-stranded overhang
3'
5'
(a)
(b)
Head genes
Recombination
Immunity DNA replication
Lysis
Tail genes
att
cos
Cut cos site
Cut cos site Fig. 7.2 The structure of the l DNA in the phage capsid (a) and after circularization in the cytoplasm (b). The DNA circularizes via the cos site.
when the DNA is ejected. In addition to LamB, l also uses an inner membrane protein called PstM to gain entry to the cytoplasm. How the l DNA physically traverses the peptidoglycan and periplasm and gets through PtsM is not known.
Protecting the l genome in the bacterial cytoplasm
What protection the phage genome needs in the cytoplasm depends on the physical state of the injected nucleic acid. l contains a linear double-stranded DNA molecule in its capsid. In the bacterial cytoplasm, dsDNA molecules are subject to degradation by exonucleases that need a free end to digest the DNA. The first event that happens to newly injected l DNA is that the DNA circularizes to prevent it from being degraded. l has a specific site on its DNA, termed the cos site, which it uses to circularize the DNA (Fig. 7.2). The cos site is a 22 bp sequence that is cut asymmetrically when the l DNA is packaged (see below). The cut cos site has a 12 bp overhang. There is one cut cos site at the left end of the l genome and another cut cos site at the right end of the l genome (Fig. 7.2a). When the l DNA is injected into the cytoplasm, the cut cos sites at either of the linear l genome anneal (Fig. 7.2b). A host enzyme, DNA ligase, seals the nicks at either end of the cos site generating a covalently closed, circular l genome. The host encoded enzyme, DNA gyrase, supercoils the l molecule.
What happens to the l genome after it is stabilized?
The l genome contains six major promoters known as PL for promoter leftward, P (^) R for promoter rightward, P (^) RE for promoter for repressor establishment, PRM for promoter for repressor maintenance, PI for promoter for integra- tion, and PR¢ for secondary rightward promoter (Fig. 7.3). After the genome is circularized and supercoiled, tran- scription begins from PL and P (^) R. A series of genes known as early genes are transcribed and translated. These gene products are the initial proteins needed for further phage development. E. coli RNA polymerase interacts with PL to give rise to a short mRNA transcript that is translated into the N protein (Fig. 7.4a). E. coli RNA polymerase interacts with PR to give rise to a short mRNA transcript that is translated into the Cro protein (Fig. 7.4a). The N protein is able to extend transcription when RNA polymerase encounters a sequence in the DNA that tells it to stop. For this reason, N is called an anti-termination protein. N allows RNA polymerase to transcribe through the tL and t (^) R1 termination signals resulting in the synthe- sis of longer mRNA transcripts (Fig. 7.4b). The longer transcripts from PR encode the O, P, and CII proteins, and a small amount of another anti-terminator, the Q protein. From P (^) L, CIII, the recombination proteins Gam and Red and a small amount of Xis and Int are made. The N protein anti-terminates by binding to RNA polymerase after a specific base pair sequence, located upstream of the transcriptional termination site, has been
Bacteriophage 109
Fig. 7.3 The location of the six major promoters on the l genome and the direction in which they specify mRNA production.
att
Recombination
int Xis
PI cIII N^ cI (^) cro cII
DNA replication Q
Lysis
cos
Tail genes Head genes
PL (^) P R
PRM PRE
PR'
lysogenic pathway (Fig. 7.5a). For this reason, CI is also known as CI repressor or l repressor. The expression and binding of Cro leads to lytic development. Cro is made from P (^) R and CI is made from either P (^) RE or PRM. Both Cro and CI bind to the same DNA sequences called operators (Fig. 7.5b). l contains two operators that bind Cro and CI. One, called OR, overlaps the PRM and P (^) R promoters. The other, called
Bacteriophage 111
cro
PRM PR
OR3 OR2 OR
N cro
PL
OL
PRM PR
OR
cro
PRM PR
Cro
PRM PR
Cro Cro
Lysogeny
Lytic growth
or
(a)
(b)
(c)
(d)
(e)
N cro
Cro
Lysogeny Lytic growth
Fig. 7.5 CI and Cro are the proteins responsible for the two developmental fates of l. (a) CI leads to lysogeny and Cro leads to lytic growth. (b) Both CI and Cro bind to two operator regions, OR and O (^) L. O (^) R overlaps with both PR and P (^) RM. O (^) L overlaps with PL. (c) O (^) R is required for the switch between developmental pathways. It is composed of three 17 base pair sequences called OR1 , O (^) R2 , and O (^) R3. They are similar in sequence but not identical. (d) CI binds to OR1 first then OR2. It will bind to OR3 but only at very high concentrations. When CI binds to OR, it represses transcription from PR and activates it from P (^) RM. CI binding to OR is actually required for P (^) RM to be activated. CI binding leads to lysogeny. (e) Cro also binds to OR1 , O (^) R2 , and O (^) R3 but in the opposite order from CI. Cro binds to OR3 first then OR2 and at high concentrations OR1. Cro binding to OR3 inhibits PRM and leads to lysogeny.
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Fig. 7.6 The CII protein is the major player in the switch between lytic and lysogenic growth. CII is unstable and rapidly degraded by the host- encoded HflA protease. Inactive CII leads to lytic growth. CII can be protected by the phage- encoded CIII protein. Active CII leads to lysogenic growth.
OL, is behind the PL promoter. O (^) R is a major player in the lytic–lysogenic decision, while OL is not part of the decision. OR is composed of three 17 base pair sequences called OR1 , OR2 , and O (^) R3 (Fig. 7.5c). CI repressor binds to OR1 10 times better than it binds to OR2 or OR3. At increasing con- centrations of CI, it will bind to OR2 and eventually to OR3. When CI is bound to OR, it stimulates the PRM promoter and the production of CI repressor and inhibits the PR promoter and the production of Cro, leading to lysogeny (Fig. 7.5d). Cro also binds to OR1 , O (^) R2 , and O (^) R3 but in the reverse order from CI repressor. Cro binds to OR3 first, then OR2 , and finally at high concentrations to OR1. When Cro is bound to OR, it inhibits the PRM promoter and the production of CI, leading to lytic growth (Fig. 7.5e). This is the basis for either lytic or lysogenic growth. How does the phage switch between these two developmental pathways? The major protein involved in the switch is another phage-encoded protein called CII (pronounced C-two, Fig. 7.6). CII activates the PRE and P (^) I promoters. This leads to the production of repressor and the Integrase protein, which is also needed for lysogeny (Fig. 7.6b). The gene for CII ( cII ) resides just to the right of the cro gene. When l infects a cell, transcription automatically begins from PL and P (^) R using host proteins. Tran- scription from PR leads to production of both the Cro and CII proteins. If CII is active it will lead to production of CI and Integrase and lysogeny. If CII is inactive then Cro will repress PRM , preventing expression of CI and leading to lytic growth. The CII protein is inherently unstable. Several factors influence this feature of the protein. CII is degraded by the bacterial-encoded HflA protease. When cells are actively growing in nutrient-rich conditions, the amount of HflA in the cell is high, leading to degradation of CII and lytic growth. When cell are growing slowly, HflA levels are low, leading to stabilization of CII, production of CI, and lysogeny. In this manner, CII is used to monitor the health of the cell and impact the lytic–lysogenic decision accordingly. It is thought that l wants to produce more phage when cells are healthy, nutrients are plentiful, and the prospect of completing phage development is good. Lysogeny is a better bet when cells are growing poorly. CII is also stabilized by a phage-encoded protein called CIII. CIII is produced from P (^) L by infecting phage.
The l lysogenic pathway
If CII prevails, CI will be produced, initially from the PRE promoter and eventu- ally from the P (^) RM promoter. CI activates PRM ensuring that a continuous supply of CI is made. CI also activates the PI promoter, leading to the production of the Inte- grase protein. The recombi- nation of l DNA into the chromosome occurs at a
CII
Int CI
CII
Lytic growth Lysogeny
Inactive Active
Degradation by HfLA Protection by CIII
int xis cI cro cII
PI PRE
(a)
(b)
DNA replication during the l lytic pathway
After the infecting l DNA has been converted to a double-stranded circular molecule, it replicates from a specific origin using both the phage-encoded O and P proteins and bacterial-encoded proteins. Replication proceeds bidirectionally, much like the E. coli chromosome. This form of replication produces molecules that look like the Greek letter theta and is called theta replication (Fig. 7.9a). Later in lytic development, l switches to a second mode of replication called rolling circle replication. Rolling circle replication of l DNA commences when an endonuclease, en- coded by l exo , cuts one strand of the covalently closed circular double-stranded DNA molecule (Fig. 7.9b). The cut strand is called the plus strand. The 5¢ end of the cut plus strand is peeled away from the intact minus strand. DNA polymerase adds deoxyri- bonucleotides to the free 3¢ OH of the cut plus strand using the intact circular minus strand as the template. This produces new plus strands through a process of continu- ally elongating the original plus strand. The new plus strands are used as a template to synthesize new minus strands. Rolling circle replication produces long DNA mole- cules containing multiple phage genomes called concatamers.
Making l phage
The structure of the fin- ished capsid is determined by the physical characteris- tics of the structural pro- teins that they are made from and the phage and host proteins used for as- sembly. Assembly of the capsids requires at least 10 phage-encoded proteins and two host-encoded pro- teins. The final capsid is made up of eight proteins, E, D, B, W, FII, B*, X1, and X2. Initially, B, C, and Nu (all phage proteins) form a small, ill-defined initiator structure (Fig. 7.10a). This structure is a substrate for the host-encoded GroEL and GroES proteins. GroEL and GroES act on proteins or protein complexes and help remodel them. The major coat protein, E, is added to this structure to form an immature phage head (Fig. 7.10b). The immature phage head is converted to the mature
114 Chapter 7
Fig. 7.9 l has two modes of DNA replication: theta replication (a) and rolling circle replication (b). Theta replication occurs early in infection and rolling circle replication occurs late in infection. Rolling circle replication produces concatamers for packaging into phage heads.
Theta replication
Rolling circle replication
Theta structure
3' 5'
3'
5'
5'
3'
3'
cos 5'
cos
cos
Newly synthesized DNA
ori O (^) P
(a)
(b)
Bacteriophage 115
B C Nu
Convert B B* Fuse E + C Digest to X1, X
GroEL GroES
Terminase
cos cos
D
W + F
Tails
Infective phage
(a) (b)
(c)
(d)
(e)
(f)
E
Nu
Fig. 7.10 The assembly pathway for l. (a) The initiator structure for the head is composed of the B, C, and Nu3 proteins. (b) E, the major head protein, is added to this structure. Nu3 is degraded, B is cleaved to a smaller form (B*), and E and C are fused and cleaved at a new position to form X1 and X2. This forms the immature phage head. (c) The immature phage head is now ready for DNA from a concatamer. The D protein is added to the capsid at this point. (d) Packaging starts at a cos site and proceeds to the next cos site. (e) The DNA is inserted into the capsid and sealed inside by the W and FII proteins. (f) Tails are added to the full capsid to form a phage.
Induction of l by the SOS System
When a l lysogen is treated with ultraviolet light (UV), ~35 minutes later the cells lyse and release phage. What does the UV do to the cell? UV damages the DNA and trig- gers a cellular response called the SOS response to deal with this damage (Fig. 7.11). The RecA protein, which is normally used for homologous recombination, is activat- ed and binds to the LexA protein. The activated RecA complexed to LexA induces LexA to cleave itself. This leads to the activation of a number of genes whose prod- ucts repair the DNA damage in the cell. l has tapped into this system through the CI protein. CI repressor can inter- act with activated RecA, leading to the cleavage of CI. This leads to expression of the phage lytic genes and phage production. The rational for this response is that l does not want to risk staying in a cell that has DNA damage and may not survive.
Superinfection
If a cell is a l lysogen, another l phage that infects is not able to undergo lytic development and produce phage. The incoming phage can inject its DNA, however, the DNA is immediately shut down and no transcription or translation of the l initiates. l lysogens are immune to infec- tion by another l phage particle, which is called superinfection. Superinfection is blocked because the lysogen is continuously producing CI repressor. The lysogen actually produces more repressor than it needs to shut down one phage. This extra repressor binds to the superinfecting phage DNA at OL and O (^) R and prevents transcription from PL and P (^) R.
Restriction and modification of DNA
A simple experiment with l leads to the discovery of how bacteria tell their own DNA from foreign DNA. l is capable of making plaques on two different types of E. coli , E. coli K12 and E. coli C. If l is grown on E. coli K12, it will form plaques on E. coli K12 or E. coli C with equal efficiency. If l is grown on E. coli C, it will form plaques on E. coli C but if it is plated on E. coli K12, only a few phage will form plaques. The efficiency of forming plaques or efficiency of plating (EOP) is decreased by 10,000-fold. This is known as restriction. If the E. coli C grown phage that did plaque on E. coli K12 are replated on E. coli K12, the EOP is 1. This is known as modification. The few phage that survive the replating on E. coli K12 have been modified so that they can effici- ently plate on E. coli K12. While this originated as a curiosity of phage growth, it has proven to be essential for many molecular techniques. Further investigation showed that the protein responsi- ble for restriction, a restriction enzyme or restriction endonuclease , actually recognizes a specific DNA sequence and cleaves the DNA on both strands. The cut or digested DNA is sensitive to nucleases that degrade DNA. The modification part of the system is a protein that specifically modifies the DNA sequence recognized by the re- striction enzyme and prevents the DNA from being digested. E. coli K12 has a restric-
Bacteriophage 117
Fig. 7.11 The SOS response induces l. UV treatment of cells (a) damages the DNA and leaves stretches of single-stranded DNA (b). The single-stranded DNA activates RecA (c). Activated RecA interacts with CI, leading to cleavage of CI and induction of the l lysogen (d). Activated RecA also interacts with LexA and leads to LexA inactivation (e). LexA inactivation leads to expression of a number of genes, including some DNA repair enzymes.
UV
RecA
LexA
Cleaved LexA
Activated RecA
CI
Cleaved CI
Lytic growth Activate DNA repair genes
(a)
(b)
(c)
(d) (e)
tion/modification system and E. coli C does not. This explains the original observa- tion with l growth. If a bacterium carries the restriction enzyme, it must also carry the modification enzyme so that the bacterial chromosome is not digested and degraded. The restriction/modification system allows a bacterium to tell DNA from its own species from foreign DNA. Many different bacteria contain restriction/modification systems that recognize different DNA sequences. The restriction enzymes are purified and used in vitro to cleave DNA at specific DNA sequences, depending on the recog- nition sequence of the enzyme in question. Restriction enzymes are used to cleave and clone DNA fragments as described in Chapter 14.
The lifecycle of M
M13 adsorption and injection
M13 adsorbs to the tip of the F pilus, a hair-like structure on the surface of some bac- teria. It can only infect bacteria that carry an F or F-like conjugative plasmid that en- codes the proteins that make up the F pilus (see Chapter 10). For the filamentous phage, it is known that infection is initiated by the binding of gpIII to the tip of the F pilus. GpIII then interacts with the inner membrane protein TolA. Two additional facts about gpIII suggest a mechanism for phage DNA entry. GpIII contains amino acid sequences that are fusogenic or promote localized fusion of two membranes and gpIII is capable of forming pores in membranes that are large enough for DNA to go through. If each of these properties of gpIII are important for phage entry, then the phage could bind to the F pilus, promote fusion of the membranes, and use gpIII to form holes in the membrane to gain entry into the cytoplasm.
Protection of the M13 genome
The M13 DNA that ends up in the cytoplasm is a circular single-stranded DNA mole- cule. The strand present in phage particle is known as the plus or + strand. After entry into the cytoplasm, the + strand DNA is immediately coated with an E. coli single- stranded DNA binding protein known as SSB. The SSB coating protects the DNA from degradation.
M13 DNA replication
The M13 plus strand is converted to a double-stranded molecule immediately upon entry into E. coli (Fig. 7.12). Synthesis of the complementary strand is carried out en- tirely by E. coli ’s DNA synthesis machinery. The complementary strand is called the minus or — strand. Only the minus strand is used as the template for mRNA synthesis and ultimately it is the template for the translation of the encoded M13 gene prod- ucts. The SSB that coats the plus strand upon entry of the DNA into the E. coli cyto- plasm fails to bind to ~60 nucleotides of the molecule (Fig 7.12c). These nucleotides form a hairpin loop that is protected from nuclease degradation. M13 gpIII from the phage is found associated with the hairpin loop. The hairpin loop is recognized by E. coli RNA polymerase as a DNA replication origin and is used to initiate transcription of a short RNA primer (Fig. 7.12d). The RNA primer is extended by E. coli DNA poly- merase III to create the minus strand (Fig. 7.12e). The RNA primer is eventually re- moved by the exonuclease activities of E. coli DNA polymerase I. The gap is filled in by
118 Chapter 7
FYI 7.
Pathogenicity in Vibrio cholera
Cholera is caused by the bacterium, Vibrio cholera. Many of the genes that make this bacteria pathogenic or disease causing are part of a prophage located in the V. cholera chromosome. This prophage bears striking resemblance to M13 and other filamentous phage. It is possible that the transmission of these pathogenic genes is as simple as the phage moving from one bacterial species to another sensitive bacterial species.
120 Chapter 7
Fig. 7.14 P1 genomes are both circularly permuted and terminally redundant. Terminal redundancy means that the same sequences are present on both ends of one DNA molecule. Circular permutation means that the order of the genes on each DNA molecule is different but every DNA molecule contains the same genes.
approximately 45 minutes after infection, the cells are filled with concatamers of phage DNA, assembled phage heads, and assem- bled phage tails. Now as- sembly of the complete phage must take place. A protein made from the phage genome recognizes a site on the concatamers of phage DNA called the pac site (Fig. 7.15). The protein cuts the DNA, making a double-stranded end. This end is inserted into a phage head. The DNA continues to be pushed inside the head until the head is full, a process termed headfull packaging. Once the first phage head is filled, anoth- er empty phage starts packaging. Experiments indicate that up to five headfulls of DNA can be packaged sequentially from a single pac site at 100% efficiency. An additional five headfulls of DNA can be packaged although the efficiency gradually decreases over these last five headfulls to only about 5%. While each phage head contains the same genes, the gene order changes. This is known as circular permutation of the genome (Fig. 7.14). After the head is full of DNA, a double- stranded cut is made and a tail is attached. This part of phage development is very much an assembly line. P1 is thought to encode an endolysin and holin to use in lysing the cell, similar to those described for l.
The location of the P1 prophage in a lysogen Prophages can be physically located in one of two places in a lysogen. In the case of l, the phage genome is recom- bined into the bacterial chromosome. P1 is maintained in the cytoplasm as a stably inherited extrachromosomal piece of DNA or plasmid (see Chapter 9). P1 contains an origin for DNA replication and once the phage genome is converted to circular, double-stranded DNA, it can be established as a plasmid.
P1 transducing particles
One unusual aspect of P1 development is the formation of transducing particles or phage particles that contain chromosomal DNA instead of phage DNA. The E. coli
Fig. 7.13 M13 is released from the cell without lysing the bacterium. (a) The plus strand, coated with gpV interacts with the membrane through gpVII and gpIX. (b) As the DNA traverses the membrane, the gpV is replaced by gpVIII.
gpVII
gpV
gpIX gpVIII
Membrane
(a)
gpIX gpVII
gpVIII
Membrane
(b) Displaced gpV
Circularly permuted molecules
A B C D E F A B
E F A G C D E F
C D E F A B C D
Terminal redundancy
Bacteriophage 121
chromosome contains many pseudopac sites or sites that can be used to initiate packaging of host chromosomal DNA into maturing phage. These pseudopac sites are used much less frequently than the phage pac sites but they are used. The resulting phage carry random pieces of the chromosome in place of phage genomes. The ability to package any piece of chromosomal DNA instead of phage DNA makes P1 a generalized transducing phage. Transducing particles are used to move pieces of host chromosomal DNA from one strain to another for the purposes described in Chapter 8.
The lifecycle of T
T4 adsorption and injection
For T4, the phage binds to the lipopolysaccharide. The tips of the tail fibers contact the cell first (Fig. 7.16). Once the phage has bound to the cell, the base plate rearranges creating a hole in the base plate. The outer sheath contracts and the internal tube goes through the outer membrane, peptidoglycan, and periplasm and comes close to the cytoplasmic membrane. The DNA is injected and crosses the cytoplasmic membrane in about 30 seconds. Not all phage that have the structure of T4 inject their DNA this way. Some phage such as T7, have tails that cannot contract. The T7 genome is only 40 kb but takes 9 to 12 minutes to cross into the cytoplasm. For T7, a small portion of
pac pac pac
pac pac
Concatomers
(a)
(b)
(c) (d)
Fig. 7.15 P1 packages DNA from a pac site (a) and packages between 7 and 12% more than one P1 genome, until the phage head is full (b and c). Once the phage head is full, a preassembled head is added (d).
Bacteriophage 123
T4 rII mutations and the nature of the genetic code
The study of two genes in T4 has contributed significantly to our understanding of the genetic code and the nature of the gene. In the late 1950s and 1960s the understand- ing of the nature of the gene was in its infancy. The prevailing thought was that the gene was the smallest genetic unit and it was inherited as a unit. The chemical nature of DNA had just been described by Watson and Crick. The relationship between DNA and the gene was not understood. Seymour Benzer used the T4 rII locus and genetic logic to describe several key features of the gene. rII encodes two proteins, A and B. Several thousand point mutations and deletions were isolated in these two genes. These mutations were put to good use. A phage car- rying one mutation and a phage carrying a second mutation were mixed together and
B C D E F A B A B C D E F A B
A B C D E F A B A B C D E F A
C D E F A B A B C D E F A B
A B C D E F A A B C D E F
B B
A B C D E F A B C D E F A
A B C D E F A B C D E F A B
E F A B
A B C D E F A
B C D
A B C A B C
D E F A B D E F A B
3' 3' 5'
5'
3'
5'
5' A B C
C
B B A
A
(a)
(b)
and
DNA replication
Strand invasion
DNA replication
Free ssDNA
Free ssDNA
(c)
(d)
Fig. 7.17 T4 replicates its DNA using both replication and recombination. (a) Linear T4 DNA molecules are injected into the cytoplasm of the host. (b) DNA replication begins at an origin and proceeds bidirectionally to the ends. However, because of DNA polymerase’s requirement for a primer, a piece of the DNA at one end of the molecule cannot be replicated and remains single stranded. (c) This piece of single-stranded DNA can invade duplexed DNA at any place where it has homology, like the initial reaction in recombination. (d) DNA replication of this molecule will lead to concatamers of the phage genome. Depending on where strand invasion takes place, branched molecules can also be formed. T packages its DNA out of the concatamers. The displaced single strands are free to strand invade the concatamer structures.
124 Chapter 7
grown on an appropriate host to determine if any of the offspring had recom- bined back to wild type. Many of these phage crosses were carried out and used to construct a map of where the mutations resided in the rII locus. Several conclusions were drawn from these studies. Deletion mutations were defined as mutations that could not generate wild- type recombinants when crossed with more than one of the other mutations. Deletion mutations were missing a part of the gene. In the thousands of crosses that were carried out, the frequency of obtaining wild-type recombinants was predictable based on the positions of the starting mutations. This led to the conclusion that DNA was a linear molecule across the length of the gene and not a branched molecule. If DNA was branched, then the recombination frequencies should be very different when one mutation resided on one side of a branch and the other mutation resided on the opposite side of the branch. Using the recombination frequencies, it was de- termined that recombination can take place within a gene and not just outside of it as had been thought. This changed the definition of a gene from the unit of heredity that mutated to altered states and recombined with other genes. It is now recognized that the gene is a functional unit that must be intact in the DNA to lead to a specific characteristic or phenotype. Each of these studies shed more light on the behavior of the gene and the nature of mutations. One of the deletions fused the A gene to the B gene such that the A gene was not functional but the B gene was (Fig. 7.18). Some of the point mutations in A could be crossed onto the same phage that carried the deletion. If a point mutation in A did not affect the activity of B in the fused genes then the point mutation was interpreted to be a missense mutation. Missense mutations could cause a change in the genetic code without affecting the production of the protein product. Other point mutations in A did affect the activity of B. These were interpreted to be nonsense mutations or changes that stopped the production of the B protein. These studies led directly to the modern concepts of a gene and how it functions.
Fig. 7.18 The T4 rII locus was used to conduct studies on the nature of the gene. (a) rII is composed of two genes A and B that are normally made into separate proteins. (b) A specific deletion was described from the mapping studies. This deletion (called r1589) fused the A and B genes, leaving A nonfunctional but B functional. (c) Some mutations in A, called missense mutations did not interfere with B function. (d) Other mutations in A did interfere with B function. These mutations were said to be “nonsense” and interfered with the production of B.
FYI 7.
The RNA phage MS
MS2 is a typical phage containing an RNA genome. MS2 binds to the F pilus. It has a genome of 3569 nucleotides and encodes only four proteins: the coat protein, an RNA-directed RNA polymerase, a lysin and an adsorption protein. MS2 has an icosahedral capsid composed mainly of one type of protein with a few molecules of a minor protein in it.
A B
B
X B
A B
X
X
A B
A B
A B
(a)
(b)
(c)
(d)
mRNA
mRNA
mRNA
mRNA
Ribosome
Protein
Protein
Protein
Nonsense mutations
Missense mutations
B still functional
No protein made
rII locus
