ISOLATION OF PHAGE P1 REGULATORY MUTANTS.
A repressor protein of bacteriophage P1, encoded by the c1 gene, is responsible for maintaining P1 prophage in the lysogenic state. The product of this gene, c1 repressor protein, binds to specific sites along the P1 genome and allows phage survival by preventing cell lysis. To better understand the genetics of P1, it is important to identify mutations within the c1 gene that prevent this lysogeny maintenance. This study identifies a point mutation at nucleotide #542 yielding a clear-plaque phenotype. Clear-plaque mutants of P1 have previously been identified and reported. However, this point mutation is unique in that it results in an amino acid conversion, within the c1 repressor protein, from asparagine to aspartate.
Key Words: Bacteriophage (phage) P1, c1, lysogen, prophage.
P1 is a temperate bacteriophage (phage). Temperate phage, such as P1, have the ability to exist within the bacterial cell they infect in two different ways. In lysogeny, P1 can exist within a bacterial cell as a prophage in that it exists by replicating with the host chromosome and does not cause cell death. Alternatively, in its lytic phase, P1 can promote cell lysis during growth resulting in host cell death. During lysogeny new phage particles are not produced. In contrast, during lytic growth many new phage particles are assembled and released from the cell. By alternating between these two modes of infection, P1 can survive during extreme nutritional conditions that may be imposed upon the bacterial host in which it exists.
A unique feature of phage P1 is that during lysogeny its genome is not incorporated into the bacterial chromosome as is commonly observed during lysogeny bo other bacteriophage. Instead, P1 exists independently within the bacterial cell, much like a plasmid would. P1 replicates as a 90 kilobase (kb) plasmid in the lysogenic state and is partitioned equally into two new daughter cells during normal cell division (2, 7, 18).
Maintenance of lysogeny requires several factors. Both the c1 and the c4 genes code for repressors of lytic growth. The c1 gene codes for a repressor protein that binds to P1 DNA. By binding to specific regions adjacent to known promotor sites of phage DNA, c1 repressor inhibits lytic growth. The P1c1 repressor is functionally analogous to the c1 repressor of lambda bacteriophage. c1 binds to at least 14 unlinked operator sites which are 17 base pair (bp) sites with the asymmetric consensus sequence 5'ATTGCTCTAATAAATTT3' on the P1 genome (1, 2, 3, 7, 9, 10, 11, 15). These sites can be of two types: monovalent operators with a single 17 bp c1-repressor binding site, and bivalent operators with two overlapping c1-repressor binding sites forming an incomplete palindrome (7, 9, 10, 11, 12).
Expression of the c1 gene, together with the c4 gene, renders the host cell resistant to subsequent super infection by P1 (20). The trans acting c1 repressor binds to newly infecting P1 phage DNA if the c4 gene product prevents the production of ant, the gene product that interferes with c1 repressor function. Another gene, coi, located upstream of the c1 gene, is subject to c1- mediated repression. It is now known that the product of the coi gene binds directly to the c1 repressor, not to DNA, to alter its function and is referred to as a c1 antagonist (1, 10, 11). The relative levels of C1 protein and Coi protein synthesis determine whether P1 will enter into lysogenic or lytic growth.
Another protein, called Lxc (formerly called Bof and located within the ImmT region of P1), enhances binding of C1 repressor to certain operator sites along the DNA and is sometimes referred to as a corepressor. On the other hand, by binding to C1 repressor protein, the gene product Lxc renders C1 repressor less likely to bind to other regions along the DNA (1, 9, 10, 13, 20).
Many prokaryotic systems commonly involve DNA looping (a folding of DNA created by protein-protein interactions) as mechanisms for efficient repression (9). C1 protein regulates its own synthesis (at least partially) by the formation of DNA loops (9). Efficient repression of the c1 gene occurs when the C1 protein, bound to the compressor Lxc, binds to a bivalent operator to stimulate DNA looping (9). In this way, expression of the c1 gene is tightly controlled to meet the needs of the infecting phage.
Different classes of temperate bacteriophage vary in mechanisms for maintenance of lysogeny. For example, the lambda prophage requires the continuous expression of only one gene (c1) and this is enough to prevent entry into the lytic cycle. The c1 gene repressor prevents the synthesis of almost all lambda proteins by binding to two operator sites on the lambda genome and repressing transcription originating from promotors adjacent to these operators.
P1, however, requires the continuous expression of at least two genes, c1 and c4, for maintenance of lysogeny. The c1 gene of P1 codes for a repressor that is functionally analogous to that encoded by the lambda c1 gene. The P1c1 repressor binds to specific phage DNA operator sites adjacent to promotor sites. In addition to the c1 gene, the c4 gene is also needed for maintenance of lysogeny in P1. The c4 gene codes for a repressor that prevents the expression of another gene (ant, for antirepressor) whose product, the ant protein, interferes with c1 repressor function. If P1 is defective in the c4 gene, the antirepressor will be constitutively produced and thus will inactivate the c1 repressor. Inactivated c1 repressor results in loss of regulation of lytic growth.
By growing phage P1 on a lawn of Escherichia coli (E. coli) the two modes of growth can be observed by plaque morphology. A plaque, which is formed by the phage while growing in the host organism, is simply a clear area in the midst of a growing bacterial lawn when bacterial cells have been plated on nutrient agar. Plaques are clear or turbid depending on the mode of growth. If lysis ensues, a clear plaque is formed (clear-plaque phenotype). If both lysis and lysogeny occur, a turbid plaque is formed (turbid-plaque phenotype).
There are two major classes of clear-plaque mutants: virulent (mutants that are able to grow in a lysogen), and non-virulent (mutants that are unable to grow in a lysogen). The inability of a bacteriophage to lyse a bacterial cell that already contains bacteriophage (in a lysogenic growth mode) is a condition referred to as "super infection immunity." Virulent mutants grow in a bacterium already containing a resident prophage. Mutants having alterations within operator sites occasionally exhibit a virulent phenotype because circulating repressors in the lysogen are unable to bind to mutated operator sites. This promotes expression of lytic genes within the superinfecting DNA. Non-virulent mutants generally have alterations in the regulatory genes. For example, P1 mutants that have altered c1 genes are unable to enter into lysogenic growth and exhibit a clear-plaque phenotype. However, these mutants are unable to infect another lysogen since lytic genes are still susceptible to regulation by the resident prophage-encoded repressor.
P1 does not incorporate into the bacterial chromosome during lysogeny. The P1 prophage exists as a plasmid with a copy number of 1-2 copies per host chromosome (14). P1 can therefore be considered from either of two perspectives: as a phage replicating like a plasmid, or as a plasmid with the ability to transfer its DNA through infection. In addition to regulation of lytic growth, there must also be regulation of plasmid replication and segregation.
To study P1c1 DNA, various experiments to isolate and characterize new clear-plaque mutants were performed. A unique clear-plaque mutant is characterized.
MATERIALS AND METHODS
Isolation of clear-plaque mutants
Phage P1 was grown in E. coli mutD. The mutD strain is highly mutagenic causing mismatched base repair. A phage lysate was obtained from phage grown in E. coli mutD. Phage P1 lysate was plated on Shigella dysenteriae type 16 (SH-16), a bacterial strain serving as an indicator for plaque morphology.
The phage lysate ([10.sup.-6] - [10.sup.-7] phage/ml) was plated onto SH-16. One-tenth ml aliquots ([10.sup.-6] and [10.sup.-7] dilution) were plated onto agar plates containing SH-16 and incubated at 42[degrees]C overnight. Plaque-forming units (pfu's) were determined, and the mutation rate was calculated as a ratio of clear plaques to total number of plaques.
Streak purification of clear plaque mutants was performed. Soft agar supplemented with Shigella was poured onto Luria-Bertani (LB) agar plates (16). Clear plaques were picked, diluted, and streaked onto the LB plates over-layed with soft agar and Shigella.
The streak-purification plates were scored for clear and turbid plaques. Four clear-plaque mutants were chosen and high titer phage stocks were prepared. To LB broth, E. coli K175 was added and allowed to grow to log phase. Clear plaques were removed from the streak-purification plates and added to a tube containing 15 ml of log phase E. coli K175 and 0.75 ml of 0.5 M calcium chloride. The culture was incubated in a 37[degrees]C shaker bath 4-5 hrs. Chloroform was added, drop by drop, until it was clear that cell lysis had occurred. The culture was separated from the chloroform by centrifugation and decanting. The culture was serially diluted to [10.sup.-4], [10.sup.-6], and [10.sup.-7] for plating onto LB agar. Plates were incubated at 42[degrees]C overnight. To 10 ml of a log phase E. coli K175/calcium chloride mixture, phage lysate (MOI = 0.1) was added. This culture was again incubated in a 37[degrees]C shaker bath for 4-5 hours and serially diluted. A final phage titer between [10.sup.9] - [10.sup.10] pfu/ml was obtained.
Characterization of clear-plaque mutants
Two methods for characterization of clear-plaque mutants were utilized:
A) Cross-streak test for complementation
LB Plates spread with Shigella were cross-streaked in the following way. Vertical streaks using c1 and c4 control phage stocks were made first. Horizontal streaks, overlapping the vertical streaks, using both mutant strains and control strains, were made. Original and replica plates were incubated overnight at 42[degrees]C. Several possible outcomes are noteworthy and are listed here. Failure of a mutant strain to form lysogens when co-infected with c1 gene would suggest a defective c4 gene. Failure of a mutant strain to complement control strains would indicate a virulent mutant. Mutants complementing all control strains would indicate an altered regulatory gene. The latter would represent a unique class of regulatory mutants not yet identified.
B) Screen for temperature sensitive and/or amber mutants
LB plates were spread with either Shigella or E. coil ([Su.sup.0] -- a nonsupressor strain, [Su.sup.I], [Su.sup.II], and [Su.sup.III] -- three suppressor strains). The four mutant strains tested ([10.sup.9] and [10.sup.8] phage/ml) and three control strains (also [10.sup.9] and [10.sup.8] phage/ ml) were vertically cross streaked onto these plates. One of the control strains, c1.55, was a known amber mutant. All E. coli plates were incubated overnight at 42[degrees]C. Shigella plate were incubated overnight at 32[degrees]C - 34[degrees]C or at 42[degrees]C.
To test for amber mutants, four isogenic strains of E. coli ([Su.sup.0], [Su.sup.I], [Su.sup.II], and [Su.sup.III]) were used as indicator strains. These strains differed only by the presence of an amber suppressor tRNMA gene. [Su.sup.+] strains insert an amino acid in response to an amber docon and may restore activity to any altered regulatory gene resulting from an amber mutation. Phage forming a clear streak on [Su.sup.0] (nonsuppressor strain) but not on [Su.sup.I], [Su.sup.II], or [Su.sup.III] (suppressor strains) are amber mutants.
Cloning c1 mutants
Mutant DNA was amplified by the Polymerase Chain Reaction (PCR). Primers (200ug/ml) were designed flanking the c1 gene and contained a ribosome binding site upstream of the c1 gene start codon (Figure 3). 50 [micro]l of mutant c1 DNA was obtained from PCR. PCR products were separated on a 0.8% agarose gel. A single bright band of 850-900 bp was expected from PCR products. (Figure 1: lanes 1, 1A, 3, and 4).
PCR products were purified using a QiaQuick column (QIAGEN[TM]). A pKK223-3 vector and PCR products were digested with Pst1 and HindIII. The amplified products were ligated into the plasmid pKK223-3 and were subsequently referred to as Psd3cl. These plasmids were transformed into DH5[alpha] competent cells.
A plasmid quick prep and gel analysis were performed on Psd3cl- in order to determine of the plasmid DNA, when digested with the appropriate enzymes (PvuII & Bg/II), produced the expected bands. Two bands of the predicted size (2200 and 3250 bp) were observed (Figure 2). DNA Sequencing
DNA products were sequenced by the Sanger et al method (1977). Four reaction tubes were each prepared with single-stranded DNA template (2.5 [micro]l) for the sequence of interest, radiolabeled primers (1 [micro]l), dideoxynucleotide triphosphates and DNA polymerase (9.5 [micro]l), and four deoxynucleotide triphosphates. The c1 gene was PCR amplified for sequencing using the Perkin-Elmer Model 480. This product was purified on spin columns (Centri-Sep [TM]). DNA samples were sequenced using an ABI model 373 DNA sequencer. Specific primers #303061, #303036, and #301751-T were used (Figure 3). These particular primers were chosen because they were known to be complementary to regions of P1 DNA upstream and downstream from the c1 gene. (Sequences of primers were: #303061:GGTCTAGAGGGATAGGGT, #30306: CTCTTGGACCTTCGTTGT and #30175-T: GGATTGATGTTAACTACCG.)
Results on two mutants are given here. The mutation rates of these clearplaque mutants were calculated to be 4.31 x [10.sup.-2] (a ratio of clear plaques to total plaques). Streak purification of these clear-plaque mutants was carried out three times to ensure that completely isolated clear plaques were obtained. Clear plaques were isolated and used to infect K175 E. coli. These cells were then diluted and plated for calculation of the phage titer. Again, clear plaques from these plated were used to infect K175 E. coli. After dilution and plating, a final phage titer of 2.54 x [10.sup.10] was obtained.
Cross-streaks of all clear-plaque mutants and C1 and c4 (as controls) were performed twice on plates spread with SH-16. Results suggested that two mutants were defective in the c1 gene. All clear plague mutants as well as P1, c1, and c4 (controls) were screened for amber or temperature sensitive mutations. Each mutant and control strain was streaked at two concentrations ([10.sup.8] and [10.sup.9]) onto either SH-16, to determine temperature sensitivity, or E. coli suppressor strains or a nonsupressor strain to detect possible amber mutants. These tests were performed three times and final results indicated that the two mutants were neither temperature sensitive mutants nor amber mutants.
Clear plaque mutants were PCR amplified and checked for the correct size on a 0.8% agarose gel. Results indicated that mutants were between 850-900 base pairs (Figure 1).
The PCR amplification products were digested with PstI and HindIII restriction enzymes and ligated into plasmid pKK223-3 before transformation into DH5[alpha] competent cells. Ampicillin resistant plasmids were isolated and digested with PvuII and Bg/II. A 0.8% agarose gel analysis was performed and bands of 2200 and 3250 base pairs (bps) were expected), indicating plasmid DNA with the P1 DNA insert, but not observed for mutant #1. It was speculated that DNA from mutant #1 failed successfully ligate into plasmid pKK223-3. However, these bands, 2200 and 3250 bps, were observed for mutant #3. Mutant #3 was used for all subsequent experiments.
DNA from mutant #3 was PCR amplified and prepared for sequencing. Three primer sets were used: #303061, #303036, and #301751-T (sequences given earlier). Only primer #303036 gave a sequence that could confidently be compared to the DNA sequence of wt phage. A silent mutation was found at base pair #328 following the start codon) and a point mutation was found at base pair #542 (following the start codon) (Figure 3).
The genome of bacteriophage P1 is extremely complex. P1 has capabilities for both lysogenic and lytic growth. Studies of lytic growth have been hampered because of the instability of the lytic region (termed the L replicon) when cloned into a vector (lambda phage, in this case) (6). Mutations that block lytic growth exist, however they are less well characterized than mutations blocking lysogeny. One hypothesis for this is that P1 may contain more than one lytic replicon and inactivation of all replicons is required to inhibit lytic growth. A second hypothesis is that host functions may complement mutations in P1 regulatory genes. Unless host functions are known, search for mutants defective for lytic growth are hindered (6). Establishment and maintenance of lysogeny has been studied extensively making the latter hypothesis unlikely.
The P1 repressor protein, encoded by the c1 gene, is responsible for maintaining the P1 prophage in the lysogenic state. Binding to specific unlinked regions along its genome, C1 repressor inactivates genes responsible for cell lysis allowing survival of both P1 and its host (1, 2, 3, 9, 10, 11). As a result, P1 can replicate and partition itself to maintain a stable copy number within the host organism. Until recently, partitioning of P1 into daughter cells was thought to be functionally linked to plasmid replication. It is now known that partitioning of P1 can act independently of plasmid replication (19).
Lxc, another protein of phage P1, forms a ternary complex with operator DNA and c1 repressor protein (20). Lxc increases the DNA binding efficiency of the c1 repressor at certain sites, increasing repression of lytic growth while decreasing DNA binding efficiency at other sites (1, 9, 10, 13, 20). Lxc is considered the first known protein with the ability to enhance lytic repression (19). The Lxc protein may also be partially responsible for the super infection immunity of P1 from other bacteriophage (20).
Coi, another protein of P1, binds to c1 repressor preventing c1 repressoroperator interaction (1, 9, 10, 11). It has been suggested that the c1 gene product is inactivated by the coi gene product through a 1:1 protein-protein interaction not mediated by DNA (11).
Experiments with P1 DNA identified a point mutation within the c1 gene of isolated P1 DNA responsible for a clear-plaque phenotype. This mutation at nucleotide #542 resulted in an amino acid conversion from asparagine to aspartate. Previous research has yielded other point mutations responsible for c1 repressor inactivation including a nucleotide change at position #11 resulting in a leucine to proline conversion. Point mutations yielding clear-plaque mutants have also been found 3' to primer #303051 and 3' to primer #301751-T at nucleotide #350, #374, #414, and #428 respectively. These mutations also resulted in amino acid conversions.
Many single nucleotide changes within P1c1 DNA result in the loss of lysogeny. It is therefore important to continue the search for and study of new mutants, as this will eventually lead to an understanding of the genetics of P1. The genes important for establishment and maintenance of lysogeny, and the genes which give rise to host cell death, are common to many bacteriophage. For this reason, a general understanding of one organism could be beneficial in understanding other organisms..
* Research was done in the laboratory of B.R. Baumstark, Department of Biology, Georgia State University, Atlanta, GA 30303
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|Author:||Rivera, Sheila D.|
|Publication:||Georgia Journal of Science|
|Date:||Jun 22, 2001|
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