Genetics of bacteriophage phi 80--a review

The genetic maps of bacteriophage lambda and lambdoid phage phi 80 are compared. The gene organization of phi 80 is very similar to that of lambda, as shown by isolation and characterization of many am, ts and c (clear) mutants of the phage. In general, the essential genes located in the same position on the genetic map of the phages lambda and phi 80 fulfill the same functions. These include the gene clusters coding for the head and tail proteins, genes for DNA synthesis, and the genes controlling lysogeny and late gene expression. The specific regulatory features of phi 80 in relation to the N function of lambda are discussed, but they require further clarification. The two phages differ in immunity specificity, host range, conversion property and temperature sensitivity.     

DNA sequence analysis. Terminal sequences of bacteriophage phi80.

Sequences of the cohesive ends and the 3'-terminal regions of phi80 DNA have been determined. Sequences of the cohesive ends were obtained through the use of two standard methods. The first method involved the incorporation of all four labeled deoxyribonucleotides into the phi80 cohesive ends using DNA polymerase I. The DNA was then partially digested with micrococcal nuclease or pancreatic DNase. The products were separated by two-dimensional electrophoresis and characterized by composition, 3'-terminal, and nearest neighbor analyses. The second method involved partial incorporation using one, two, or three labeled deoxyribonucleotides followed by similar analyses. Sequences of the double-stranded regions adjacent to the cohesive ends were determined by three new methods. These methods were: (a) the DNA was specifically labeled at the 3' terminus and then partially degraded. Labeled oligonucleotide products were sequenced by their mobilities on various separation systems. (b) The cohesive ends were enlarged by limited degradation with exonuclease III. After this treatment, the DNA was partially repaired with labeled nucleotides, digested, and the products were analyzed. (c) A synthetic ologonucleotide primer was bound to phi80 DNA which had been repaired with DNA polymerase I, and then partially digested with lambda-exonuclease. The primer was extended into the region of interest by partial repair with labeled nucleotides. The extended primer was isolated and analyzed.     

Genes and regulatory sequences of bacteriophage phi X174

Fragments of the DNA of bacteriophage phi X174 were inserted in the plasmids pACYC177 and pBR322, in order to test the in vivo effects of separate phage genes and regulatory sequences. The phi X174 inserts were identified by recombination and complementation with phage mutants, followed by restriction enzyme analysis. The genes B, C, F and G can be maintained stably in the cell even when there is efficient expression of these viral genes. Recombinant plasmids with the complete genes D and E can only be maintained when the expression of these genes is completely blocked. Expression of complete H and J genes could not yet be demonstrated. The intact gene A was apparently lethal for the host cell, as it was never found in the recombinants. The genes F and G are expressed, even when they are not preceded by one of the well characterized viral or plasmid promoter sequences. Screening of the nucleotide sequence of phi X174 gives two promoter-like sequences just in front of the two genes. Viral sequences with replication signals (the phi X174 (+) origin of replication, the initiation site for complementary strand synthesis and the incompatibility sequence) appeared to be functional also when inserted in recombinant plasmids. A plasmid with the phi X (+) origin can be forced to a rolling circle mode of replication. The A protein produced by infecting phages works in trans on the cloned viral origin. The (-) origin can function as initiation signal for complementary strand synthesis during transduction of single-stranded plasmid DNA. The intracellular presence of the incompatibility sequence on a plasmid prevents propagation of infecting phages.     

New genes in the left arm of the bacteriophage phi 80 chromosome.

We describe the isolation and partial genetic characterization of 247 amber (suppressor-sensitive) mutants of temperate bacteriophage phi 80 of Escherichia coli. Of these 247 mutants, the mutations of 201 mapped to the left arm of the phi 809 chromosome and the mutations of 39 mapped to the right arm of the genome. Complementation tests among these and previously described left arm mutants defined five additional genes in the left arm of the chromosome. The positions of these genes are consistent with the hypothesis that four of them represent functions essential for phi 80 tail assembly and one represents a capsid assembly function, probably the major coat protein. The identification of these genes brings the phi 80 genome into closer correspondence with the organization of the phage lambda genome. Two- and three-factor crosses performed between mutants with defects in each of the previously identified genes and mutants with defects in the five new genes allowed us to construct a consistent, roughly additive recombination map of the left arm of the bacteriophage phi 80 genome.     

Cleavage of bacteriophage phi 80 CI repressor by RecA protein

We have purified the CI repressor protein of bacteriophage phi 80. Its N-terminal amino acid sequence and its amino acid composition agree with those predicted from the nucleotide sequence of the cI gene. The phi 80 CI repressor was cleaved at a Cys-Gly bond by the wildtype RecA protein in the presence of single-stranded DNA and ATP or its analogues. This cleavage site is different from other repressors such as LexA, lambda CI and P22 C2, which were cleaved at an Ala-Gly bond. The phi 80 CI repressor was cleaved at the same site by the RecA430 protein, but was not cleaved by the RecA1 protein. This effect of the bacterial recA mutations on cleavage is consistent with the fact that prophage phi 80 in recA430 cells can be induced by irradiation with ultraviolet light, while the prophage in recA1 cells cannot.     

Physical mapping of the immunity region of phage phi 80

Mapping of the sites of cleavage of five restriction endonucleases (BglI, BglII, EcoRV, KpnI and PvuII) in the immunity region of bacteriophage phi 80 DNA is described. The order of the 27 restriction sites was established. This helped to localize the phi 80 cI gene within the 640 bp fragment of the immunity region. Recombinant plasmids carrying phi 80 "kil" function were constructed. The function is suppressed by the cI-coded repressor. The deletion AB43 which is present in the phi 80 vir mutant is located within the phi 80 DNA fragment carrying the cI gene.     

Comparative properties of bacteriophage phi6 and phi6 nucleocapsid.

Nonionic detergent treatments released a nucleocapsid from the enveloped bacteriphage phi6. The nucleocapsid sedimented at nearly the same rate as the whole phage in sucrose density gradients, but the buoyant density in Cs2S04 changed from 1.22 g/cm3 for the whole phage to 1.33 g/cm3 for the nucleocapsid. The detergent completely removed the lipid and 5 of the 10 proteins from the phage. Surface labeling of the phage and nucleocapsid with 125I revealed that protein P3 was on the outer surface of the whole phage and P8 was on the surface of the nucleocapsid. Both the phage and the nucleocapsid were stable between pH 6.0 and 9.5. Low concentrations of EDTA (10-4 M) dissociated the nucleocapsid but had no effect on the whole phage. The nucleocapsid contained all three double-stranded RNA segments, as well as RNA polymerase activity.     

Proteins of bacteriophage phi6.

We investigated the protein composition of the lipid-containing bacteriophage phi 6. We also studied the synthesis of phage-specific proteins in the host bacterium Pseudomonas phaseolicola HB10Y. The virion was found to contain 10 proteins of the following molecular weights: P1, 93,000; P2, 88,000; P3, 84,000; P4, 36,800; P5, 24,000; P6, 21,000; P7, 19,900; P8, 10,500; P9, 8,700; and P10, less than 6,000. Proteins P3, P9, and P10 were completely extracted from the virion with 1% Triton X-100. Protein P6 was partially extracted. Proteins P8 and P9 were purified by column chromatography. The amino acid composition of P9 was determined and was found to lack methionine. Labeling of viral proteins with [35S]methionine in infected cells indicated that proteins P5, P9, P10, and P11 lacked methionine. Treatment of host cells with UV light before infection allowed the synthesis of P1, P2, P4, and P7; however, the extent of viral protein synthesis fell off exponentially with increasing delay time between irradiation and infection. Treatment of host cells with rifampin during infection allowed preferential synthesis of viral proteins, but the extent of synthesis also fell off exponentially with increasing delay time between the addition of rifampin and the addition of radioactive amino acids. All of the virion proteins were seen in gels prepared from rifampin-treated infected cells. In addition, two proteins, P11 and P12, were observed; their molecular weights were 25,200 and 20,100, respectively. Proteins P1, P2, P4, and P7 were synthesized early, whereas the rest began to increase at 45 min post-infection.     

Genetic characterization of a phi80 transducing bacteriophage carrying the histidine operon of Salmonella typhimurium.

A phi 80 transducing phage, phi 80imm lambdadhis, carrying the Salmonella his-gnd region, was characterized by immunity studies, tonB deletion analysis, and marker rescue analysis. Phi 80imm lambdadhis retains the phage immunity region of the phi 80-lambda hybrid phage from which it was derived. Bacterial genes replace most late phage genes. Deletion analysis shows the prophage gene order to be immlambda-his-gnd and indicates the orientation of the his operon to be hisOGDCBHAFIE-gnd. The structure of phi 80imm lambdadhis is remarkably similar to two independently isolated phi 80 phages that carry the his-gnd region of Escherichia coli and that, like phi80imm lambdahis, were derived by directed gene transposition to the tonB locus. A derivative of phi 80imm lambdadhis that is phi 80 immune is also reported.     

Excision and duplication of su3+-transducing fragments carried by bacteriophage phi 80. I. Novel structure of phi 80sus2psu3+ DNA molecule.

DNA molecules of phi 80sus2psu3+ and phi 80dsu3+ isolated by Andoh and Ozeki (1968) were studied by the electron microscope heteroduplex method. The phi 80sus2psu3+ and phi 80dsu3+ DNA lengths were found to be 108.7 and 103.3% of the phi 80 DNA, respectively. The phi 80sus2psu3+/phi 80 heteroduplex shows an insertion loop of 8.7% of the phi 80 DNA which migrates from 7.7 to 9.7%, as measured relative to the left (0%) and right (100%) termini of the mature phi 80 DNA molecule. The region of loop migration occupies the central region of the phi 80 head gene cluster. The presence of su3+-containing Escherichia coli DNA of 6.7% phi 80 unit flanked by two homologous regions of phage DNA of 2.0% of phi 80 unit gives rise to a movable insertion loop. In phi 80dsu3+, from which phi 80sus2psu3+ was derived, 50.5% of the phi 80 DNA at the left arm was replaced by E. coli DNA containing the su3+ gene, equivalent to about 53.8% phi 80 unit in length. The phi 80sus2psu3+/phi 80dsu3+ heteroduplex appears as a double-stranded molecule that bifurcates into two clearly visible single-stranded regions, rejoins, bifurcates, and rejoins again. The middle double-stranded stretches of 6.7% phi 80 unit correspond to the E. coli DNA inserted in phi 80sus2psu3+. Therefore the transducing fragment carried by phi 80sus2psu3+ originates from the inside region of the transducing fragment of defective phage phi 80dsu3+ by at least two illegitimate recombination events.     

Cleavage of single-stranded DNA by the A and A* proteins of bacteriophage phi X174.

The purified A protein and A* protein of bacteriophage phi X174 have been tested for endonuclease activity on single stranded viral phi X174 DNA. The A protein (55.000 daltons) nicks single-stranded DNA in the same way and at the same place as it does superhelical RFI DNA, at the origin of DNA replication. The A* protein (37.000 daltons) can cleave the single-stranded viral DNA at many different sites. It has however a strong preference for the origin of replication. Both proteins generate 3'OH ends and blocked 5' termini at the nick site.     

Genetic map of the Staphylococcal bacteriophage phi11.

Ten sus mutants of the staphylococcal bacteriophage phi 11, each a representative from a different complementation group, have been used in three-factor cross experiments. The results of these crosses indicate a circular genetic map for phi 11. Functional studies of the mutants have been limited to electron microscopic examinations of lysates after prophage induction (or infection). One gene is an early gene, five genes are concerned with tail formation, and three are concerned with head formation. The tenth gene is possibly a head gene. The contribution by phi 11 to the genomic content of the plasmid-phage hybrid phi 11 de has been investigated. Phi 11 de contains most of the late genes and appears to be missing a continuous phi 11 segment that includes the early gene flanked by two late genes.     

Order of assembly of the lower collar and the tail proteins of Bacillus subtilis bacteriophage phi 29.

Extracts obtained after restrictive infection of Bacillus subtilis with mutants in cistron 11 of bacteriophage phi 29 are complemented in vitro by extract donors of the lower collar protein (p11). Purified 11- heads, containing the major capsid protein (p8), the fiber protein (p8.5), the upper collar protein (p10), and the virus DNA, can be also complemented in vitro to produce infective virus. This result suggests that 11- heads are intermediates in phage phi 29 morphogenesis. The order of assembly of the lower collar protein p11 and the tail protein p9 was determined in vitro in two complementation steps. The results obtained indicate that the lower collar protein is assembled before the tail protein.     

Nucleotide sequence analysis of DNA. X. Complete nucleotide sequence of the cohesive ends of bacteriophage phi80 DNA

The base sequence of the cohesive ends of bacteriophage φ80 DNA has been shown to be identical to the base sequence of the cohesive ends of bacteriophage lambda DNA.