Bacteriophages Help (page 3)
Introduction to Bacteriophages
Viruses that infect bacteria are called bacteriophages or simply phages. The plural form "phages" is used when referring to different species (e.g., lambda and T4 are both phages). When referring to one or more virions of the same species, the word phage is used; thus, a bacterial cell may be infected by one or more lambda phage. The most commonly studied phages have a roughly spherical icosahedral capsid to which a tail is attached. The tail may be long or short, contractile or noncontractile. Other kinds of phage have tailless heads or filamentous structures. The genetic material of most phages is double-stranded DNA (dsDNA), although some single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA) phages are known. Enveloped forms are rare. Other characteristics that are useful for classification include molecular weight, genomic base composition (G þ C content), antigenic specificities of the capsid, and species or strain of susceptible host cells (host range). Host restriction is the ability of a bacteriophage to replicate in only certain strains of bacteria.
EXAMPLE 11.1 Several bacterial species synthesize a site-specific endonuclease enzyme that can digest any foreign DNA containing the specific nucleotide sequence that constitutes the recognition site of the enzyme. According to the restriction and modification model proposed by W. Arber, such a bacterium would also contain a methylase enzyme to modify (by methylation) these same sequences in its own DNA, and thus protect it from digestion by endogenous endonuclease. Foreign DNA from a different source, such as an infecting phage particle, would not have these recognition sites methylated and hence would be destroyed (and thus restricted from surviving in that strain) by the host's endonuclease.
The nucleic acid from a single phage particle typically infects a bacterial cell, replicates itself many times, produces viral proteins to make numerous viruses, and ruptures (lyses) the cell to release several hundred progeny phage. Repetitions of this reproductive process can cause a turbid bacterial culture to rapidly become clear owing to lysis of the host cells. If a dilute solution of phage is plated on a confluent growth of bacterial cells ("lawn") on nutrient agar in a Petri dish, a cleared area, or "hole," will develop around each position where a phage particle was deposited. These holes, called plaques, contain millions of progeny phage that have been released from lysed cells. By counting the number of plaques on a plate, and knowing the amount and dilution of the phage suspension added to the plate, one can estimate the total number of phage particles or the phage titer in the original phage solution.
Bacteriophage Life Cycles
Most phages (such as phage T4 that infects E. coli) have only a lytic cycle in which they kill the host cell in the production of progeny phage. Such phages are said to be virulent. A few phages (such as phage lambda that also infects E. coli) have a lysogenic cycle in which they may either act as a temperate phage (nonvirulent) or enter a lytic cycle.
The first step in the life cycle of phage T4 (Fig. 11.1) involves the adsorption of a virion to a specific receptor site on the surface of the host cell. Any cell lacking this receptor would be immune to infection by T4. Following adsorption, T4 injects its DNA through its tail into the host cell. The empty phage capsid remains outside the bacterium as a ghost (so named because of the empty appearance of the head in electron micrographs). The filamentous phage, M13, is able to penetrate the cell wall and then has its nucleic acid released by host-cell enzymes that digest the coat proteins.
Once the naked phage DNA is inside the cell, different phages may use different strategies to produce progeny particles. Generally, however, the phage DNA initially is transcribed by the host's RNA polymerase into "early mRNAs." Later mRNAs may be synthesized by a phage RNA polymerase that was made from an early mRNA; or perhaps the bacterial RNA polymerase becomes modified to transcribe phage genes preferentially or exclusively. These mRNAs become translated into enzymatic, regulatory, and structural proteins. The regulatory proteins of the phage control the timing at which various phage genes become active. The structural proteins form heads, tails, and other protein parts of the complete phage particle as needed. The phage enzymes mediate replication of many copies of the phage genome, further transcription, and sometimes even the destruction of the host's DNA.
EXAMPLE 11.2 Phage T4 specifies the enzyme hydroxymethylase that modifies the cytosine bases in its own DNA to 5-hydroxymethylcytosine. Such modified bases are resistant to degradation by host-cell nucleases, making the phage more successful during infection.
Several different mechanisms are known for packaging phage DNAs into their protein coats. In E. coli phage T4, rolling-circle replication of its double-stranded DNA produces long, tandemly linked series (concatemers) of phage genomes. It is thought that the end of the concatemer enters the head, followed by enough DNA to fill the head. The concatemer is then cleaved at a nonspecific site. Since the DNA capacity of the head is greater than the length of one phage genome (monomer), the gene order will be different in each linear fragment cut from the concatemer. Terminal regions will be present twice within each monomer (terminally redundant). Since each phage monomer cut from a concatemer begins at a different gene sequence, they collectively form a cyclically permuted set (Fig. 11.2).
In phage lambda (λ), the circular genome is replicated early in the lytic cycle to increase the number of templates for transcription and further replication. Later in the cycle, rolling-circle replication provides the genomes for packaging into the heads of progeny phage. Lambda genomes are also cut from a concatemer, but unlike phage T4, the cuts are made at base-specific sequences known as cos sites (for cohesive site). Linear phage genomes always end with single-stranded termini because they are cut from the concatemer at the cos site by a sequence-specific terminase or Ter system. Ter-cutting requires that two cos sites or one cos site and a free cohesive end (1/2 cos) be present on a single concatemeric DNA molecule. A modified lambda genome that is 79–106% the length of a normal λ phage genome will still be cut by the Ter system and become packaged into phage heads. This is an important property of λ that makes it useful as a vector for genetic cloning.
After assembly of the phage capsids is completed, the lytic protein lysozyme ruptures the cell and releases the progeny phage in a typical burst size of 50–300 infective particles per cell. Most virulent phages follow the general lytic cycle outlined above.
EXAMPLE 11.3 E. coli phage M13 is filamentous and contains a circular, single-stranded DNA molecule. Among all known phages reproducing vegetatively, M13 is the only one that neither kills nor lyses its host cell. Infective progeny phage leave the cell by budding from its surface without causing cell damage. Upon infection, the entire phage particle penetrates the cell wall by being absorbed at the end of a sex (F) pilus. The entry of coat proteins into the cell is another feature unique to this phage. One genetically engineered strain of M13 (M13mp7) contains the promoter (lacP), operator (lacO), and β-galactosidase gene (lacZ) of the E. coli lactose operon. Insertion of a foreign DNA segment into lacZ inactivates the gene and no enzyme is produced. Lac– bacterial cells infected with wild-type M13mp7 would be able to ferment lactose. On EMB agar, lactose-fermenting colonies would appear dark purple. Cells exposed to a M13mp7 phage carrying a foreign DNA insert in lacZ would be unable to ferment lactose; therefore, they grow into colorless colonies. Like lambda, phage M13 has been widely used as a cloning vehicle in genetic engineering.
EXAMPLE 11.4 The temperate phage Mu inserts its DNA obligatorily into its E. coli host chromosome during its lytic cycle. These insertions are at random, and they often inactivate host genes or regulatory sequences. Insertion always results in duplication of a terminal target sequence. Thus, Mu is a giant transposon (page 314) that has acquired phage functions enabling it to be packaged into phage coats and to escape its host by lysis. Transposition is obligatory during Mu DNA replication. Insertion of progeny Mu DNA occurs at various sites throughout the lytic cycle. Various host DNA sequences are always found at the termini of Mu DNA. However, only Mu DNA inserts; the duplicated terminal bacterial sequences are not inserted.
There are two types of lysogenic cycles. In the most common type, typified by E. coli phage lambda (λ), the phage DNA becomes integrated into the host chromosome. In the other type, represented by E. coli phage P1, the phage DNA does not integrate into the host chromosome, but somehow replicates in synchrony with it as a plasmid. Both the integrated and plasmid forms of phage DNA are called prophage.
The establishment of an integrated lambda prophage occurs in four major steps:
- Linear phage DNA is injected into the host bacterial cell; the phage DNA is circularized by base pairing of its terminally redundant tails.
- Some early phage genes are transcribed to produce a few molecules of a repressor protein and an integrase enzyme. The repressor then turns off transcription of phage genes.
- The phage DNA is usually integrated or inserted at a specific site into the host chromosome as a prophage with the aid of integrase.
- The bacterium survives and multiplies; the prophage is replicated along with the host chromosome.
Two conditions favor the establishment of the lysogenic cycle of a temperate phage: (1) depletion of nutrients in the growth medium and (2) high multiplicity of infection (MOI)—i.e., many adsorbed phages per bacterium. Phage can carry out the lytic cycle only in cells that are actively metabolizing. When nutrients are depleted, bacteria degrade their own mRNAs and proteins before they become dormant. When nutrients become available to an uninfected dormant bacterium, it can again resume growth. A phage-infected cell that becomes dormant interrupts the lytic cycle, and usually loses the ability to produce phage. The cell dies. On the other hand, if the cell can become lysogenized (containing a prophage), both the phage and the bacteriumcan survive a dormant period, and the potential for production of phage by induction persists (Fig. 11.3).
If a lysogenic bacterium sustains damage to its DNA, it would be advantageous for the prophage to deintegrate from the bacterial chromosome, enter the lytic cycle, produce progeny phage, and leave that cell. When bacterial DNA is damaged, a protease (RecA protein) of the SOS repair mechanism is activated. This protease cleaves the lambda repressor that has kept the prophage in its inactive state. The prophage DNA becomes derepressed, an excisionase enzyme is synthesized, and the prophage deintegrates from the host chromosome to enter the lytic cycle. This is the process known as prophage induction. If ultraviolet radiation has damaged the host DNA, the ensuing prophage induction is termed UV induction. When a nonlysogenic F–bacterial cell receives prophage from a lysogenic Hfr donor, the recipient cell dies by induction of the lytic phage cycle. This form of prophage induction is termed zygotic induction.
In the lysogenic cycle of phage P1, the prophage is not integrated into the bacterial chromosome. Upon entry into the cell, P1 DNA circularizes and is repressed. It remains as a free, supercoiled, plasmid-like molecule, and replicates once with each cell division so that each daughter cell receives one copy of the prophage.
Transduction is the virus-mediated transfer of DNA from a donor bacterial cell to a recipient cell. There are two types of transduction: specialized and generalized. In both cases, bacterial DNA is incorporated into the genome of a mature virus that then goes on to infect a different bacterial host. Generally, specialized or generalized tranducing phage are defective due to the integration of additional DNA into their genomes. However, they must be normal enough to infect a new cell.
- Specialized Transduction. Specialized transduction occurs when a specific region of the bacterial chromosome becomes integrated into a mature viral particle. There are four distinguishing characteristics of specialized transduction: (1) the only bacterial genes that can be transduced are those very near the site at which the prophage is integrated, (2) only λ-type prophage are involved, (3) it results from defective excision of the prophage from the host chromosome, and (4) recombinant progeny bacteria may be partial diploids. The only site at which lambda phage integrates into the host chromosome (Fig. 11.4) is between the genes for galactose fermentation (gal) and biotin synthesis (bio). The head of the phage can only contain a limited amount of DNA, so if the prophage deintegrates abnormally from the host chromosome (taking some bacterial DNA in place of its own DNA), only the gal or bio genes could be transduced. Thus, all transducing lambda phages are defective in part of their own genome and cannot replicate on their own. A lambda phage that transduces the galactose genes is therefore called λgal or λdg (d = defective; g = galactose). If a gal– cell is infected by λdg (bearing the gene gal+), integration of the defective prophage into the host chromosome produces (for the gal locus) a partial diploid recombinant chromosome. Aberrant excision of the prophage is usually a rare event, so restricted transduction is an event of low frequency in nature. However, high-frequency transduction can be attained under laboratory conditions. If a bacterial cell is doubly infected with a wild-type lambda phage and a λdg phage, the wild-type phage can supply the functions missing in the defective phage, and the progeny will contain about equal numbers of both types. When the lysate is used for transduction, the process is referred to as high-frequency transduction. In many cases, because of its defective genome, λdg fails to be integrated into the host chromosome and therefore is not replicated. At each division, only one of the two progeny cells contains the defective phage genome; this process is called abortive transduction.
- Generalized Transduction. This type of transduction occurs when any region of bacterial DNA is incorporated into the genome of a mature virus particle. The hallmarks of this type of transduction have been extensively studied in the P phages (P1 of E. coli and P22 of Salmonella typhimurium) and are as follows: (1) any bacterial gene can be transduced, (2) the transduction results from a packaging error during phage maturation, and (3) haploid recombinants are produced. Since there is no homology between DNA sequences of the phage and the sequences of their host, there is no preferential site at which the prophage integrates. Any gene can be transduced because the head of the phage can package an entire headful of bacterial DNA. Cotransduction is the process of transducing two or more genes via the same defective phage. Reciprocal crossing over is required to integrate the transduced genes, so recombinant bacteria tend to be haploid rather than diploid. The endogenote segment replaced by the exogenote fails to replicate for lack of an ori (origin of replication) site and becomes lost in the culture through dilution or digestion.
Fine-Structure Mapping of Phage Genes
- Complementation Mapping. Because so little of the phage or bacterial genome consists of nonfunctional sequences, virtually all crossing over occurs within, rather than between, genes. Before the discovery that DNA is genetic material, the gene was thought to be the smallest genetic unit by three criteria: mutation, recombination, and function. In 1954, Semour Benzer set out to determine the limits of these operational units by performing the most definitive fine-structure mapping ever performed on a phage gene. He chose to investigate the rII region of phage T4.When wild-type (r+) T4 infects its host E. coli, it produces relatively small plaques. Many mutations result in a shorter life cycle than the wild type, thus yielding larger plaques. These "rapid lysing" mutants may be classified into three phenotypic groups, depending on their ability to lyse three strains of E. coli (Table 11.1). The sites of rI, rII, and rIII map at noncontiguous locations in the T4 genome. The rII region is about 8 recombination units long, representing approximately 1 % of the phage DNA. Complementation tests were used to determine that the rII region consists of two genes.
- Deletion Mapping. Benzer also found that about 10% of his more than 2000 rII mutants did not backmutate to wild type because they were deletions of various lengths. By infecting cells with two different deletions, wild-type recombinants could be produced if the deletions did not overlap. Wild-type recombinants cannot be produced if the two deletions overlap to any extent. Thus, by a series of crosses Benzer was able to draw a topological map in which deletions were shown to overlap or not overlap. The lengths of the deletions or the degree of overlap or non-overlap is arbitrary at this point, although they can be determined by crosses with point mutations. Having obtained a topological map, it was then possible to assign one or more point mutations to a relatively small segment of a gene by crossing them with deletion mutants because a point mutation cannot recombine with a deletion at the same site. This principle allowed Benzer to group mutants within relatively small regions of each gene. This was accomplished by doubly infecting strain B with a pair of rII point mutants (e.g., rIIa and rIIb) in broth, and then allowing them to lyse the culture. The total number of progeny phage can be estimated by plating dilutions of this lysate on E. coli B and counting the resulting plaques. Wild-type recombinants are scored by plating the lysate on strain K. For every wild-type (r+IIa; r+ IIb) plaque counted on strain K, we assume that an undetected double mutant (rIIa, rIIb) reciprocal recombinant was formed.
EXAMPLE 11.5 If two rII mutants are added to strain K12 in sufficient numbers to ensure that each cell is infected with at least one of each mutant, one of two results is observed. If all the E. coli K12 cells lyse after one normal propagation cycle (20–30 min), we may infer that the mutants were in different functional units (genes). Each mutant was making a different polypeptide chain, and the two chains ''cooperated'' to allow a normal-sized burst to occur. On the other hand, if the two rII phages contain a mutation in the same gene, progeny phage can only be produced by genetic recombination with a frequency dependent upon how closely the two point mutations are linked. In any event, only a few of the cells would be expected to lyse by this mechanism in the same period of time. Thus, the results of complementation are easily distinguished from those of recombination. In 1954 Benzer found that all of the point mutations in the rII region mapped into two genes (A and B).
The smallest reproducible recombination frequency that Benzer observed between two sites in the rII region was about 0.02%, corresponding to approximately 1/400 of a genetic region whose total length is only 8 recombination units. Thus, it was concluded that the genes of the rII region each contained hundreds of possible mutation sites, and that recombination could occur between the closest of these mutant sites. Benzer reasoned that the smallest distance within which recombination occurs might be as small as adjacent nucleotide pairs. The smallest segment of DNA that mutated could cause a phenotypic effect was found to be as small as five nucleotides or smaller. We now know that a mutation in a single nucleotide base pair is sufficient to cause a mutant phenotype.
A surprising finding of this work was that the point mutants were not randomly located in the rII region; a few locations (called hot spots) in both genes had many more mutations than elsewhere (over a hundred in a couple of positions vs. about 1 to 10 elsewhere).
Practice problems for these concepts can be found at:
- Kindergarten Sight Words List
- First Grade Sight Words List
- 10 Fun Activities for Children with Autism
- Signs Your Child Might Have Asperger's Syndrome
- Theories of Learning
- A Teacher's Guide to Differentiating Instruction
- Child Development Theories
- Social Cognitive Theory
- Curriculum Definition
- Why is Play Important? Social and Emotional Development, Physical Development, Creative Development