Nucleotide sequence of the genome of the bacteriophage alpha 3: interrelationship of the genome structure and the gene products with those of the phages, phi X174, G4 and phi K.

The complete nucleotide sequence of the genome of the circular single-stranded DNA (isometric) phage alpha 3 has been determined and compared with that of the related phages phi X174 and G4. The alpha 3 genome consists of 6087 nucleotides, which is 701 nucleotides longer than the nucleotide sequence of the phi X174 genome and 510 nucleotides more than that of the G4 genome. The results demonstrated that the three phage species have 11 homologous genes (A, A*, B, C, K, D, E, J, F, G and H), the order of which is fundamentally identical, suggesting that they have evolved from a common ancestor. The sequence of some genes and untranslated intergenic regions, however, differs significantly from phage to phage: for example, the degree of amino acid sequence homology of the gene product is averaged at 47.7% between alpha 3 and phi X174 and 46.9% between alpha 3 and G4, and alpha 3 has a remarkable longer intergenic region composed of 758 nucleotides between the genes H and A compared with the counterparts of phi X174 and G4. Meanwhile, in vivo experiments of genetic complementation showed that alpha 3 can use none of the gene products of phi X174 and G4, whereas the related phage phi K can rescue alpha 3 nonsense mutants of the genes B, C, D and J. These sequencing and in vivo rescue results indicated that alpha 3 is closely related to phi K, but distantly remote from phi X174 or G4, and supported an evolutional hypothesis which has been so far proposed that the isometric phages are classified into three main groups: the generic representatives are phi X174, G4 and alpha 3.     

Transfer of genome fragments in double infection of E. coli with T5 and phi X174 bacteriophages.

Double infection of Escherichia coli by two DNA phages (phi X174 and T5) resulted in encapsidation into T5 particles of T5 DNA containing linked fragments of phi X174 DNA. The phi X474 sequences in T5 "hybrid" DNA were detected by RNA-DNA hybridization.     

Nucleotide sequences at the phi X gene A protein cleavage site in replicative form I DNAs of bacteriophages U3, G14, and alpha 3

Gene A protein, a bacteriophage phi X174-encoded endonuclease involved in phi X replicative form (RF) DNA replication, nicks not only phi X RFI DNA but also RFI DNAs of several other spherical single-stranded DNA bacteriophages. The position of the phi X gene A protein nick and the nucleotide sequence surrounding this site in RF DNAs of the bacteriophages U3, G14, and alpha 3 were determined. Comparison of the nucleotide sequences which surround the nick site of the gene A protein in RF DNAs of phi X174, G4, St-1, U3, G14, and alpha 3 revealed that a strongly conserved 30-nucleotide stretch occurred in RF DNAs of all six phages. However, perfect DNA sequence homology around this site was only 10 nucleotides, the decamer sequence CAACTTGATA. The present results support the hypothesis that, for nicking of double-stranded supercoiled DNA by the phi X gene A protein, the presence of the recognition sequence CAACTTGATA and a specific gene A protein binding sequence upstream from the recognition sequence are required. The sequence data obtained so far from phages U3, G14, St-1, and alpha 3 have been compared with the nucleotide sequences and amino acid sequences of both phi X and G4. According to this comparison, the evolutionary relationship between phages G4, U3, and G14 is very close, which also holds for phages alpha 3 and St-1. However, the two groups are only distantly related, both to each other and to phi X.     

Mutagenesis of phi X174 am3 cs70 incorporated into the genome of mouse L-cells

The objective of our work with phi X174 has been to develop a shuttle vector that can be used comparatively in bacterial cells, different types of mammalian cells, and possibly in the various tissues of transgenic mice, with a constant mechanism for detection and analysis of mutations independent of any host-cell type. Toward that end, we have efficiently rescued phi X174 am3 cs70 that is host-silent and stably integrated into the genome of mouse L-cells. The particular mouse L-cell line contains tandem arrays, single copies, and fragments of phi X that, upon restriction enzyme excision, can result in 5 potentially active copies per diploid genome. The excised phi X DNA is recovered by column chromatography, ligated, and transfected into highly competent spheroplasts. The Rescue Efficiency, defined as the number of viable phages produced out of the total number of potentially recoverable copies, is approx. 10(-3). The Recovery Ratio, defined as the Rescue Efficiency for chromosomally-integrated phage DNA divided by the Rescue Efficiency for phi X am3 cs70, is close to one. Mouse L-cells containing the integrated phi X174 am3 cs70 were treated with 20 mM ethyl methanesulfonate. The reversion frequency of am3 among progeny phages rescued from treated cells was 1.4 X 10(-5) (193 revertants in 1.4 X 10(7) phages). This is significantly higher than the 5.8 X 10(-7) reversion frequency of am3 (7 revertants in 1.2 X 10(7) phages) among progeny phages rescued from untreated cells.     

Differential survival and chloramphenicol-insensitive error-prone repair of hydroxylamine-inactivated phi X174 bacteriophage mutants

Features of inactivation, repair and concomitant mutagenesis of hydroxylamine-treated phi X174 bacteriophages are reported here. (1) For reasons unknown, the nonsense phage mutants tested here were far more sensitive to hydroxylamine than the wild-type phage. In contrast, the sensitivities of these same phi X174 mutants to UV-irradiation are indistinguishable. (2) Hydroxylamine-treated amber phages mutated to ochre but not to wild-type particles, i.e., G leads to A transition events were recovered. (3) The repair of phi X174 phages from hydroxylamine-induced damage was error-prone, but unlike UV damage, did not require protein synthesis de novo. Possible mechanisms of these novel features are discussed.     

Intracellular development of Escherichia coli bacteriophages after culturing the infected bacteria under conditions of anabiosis and hypothermia

The intracellular growth of bacteriophages T3, T4 and phi X174 was studied in Escherichia coli cells frozen to -196 degrees C and cooled to 0 degree C at various intervals from the instant of phage infection. The processes of biosynthesis were delayed and the latent period was longer in the growth of cells frozen to -196 degrees C. The levels of RNA and protein biosynthesis as well as the yield of phages decreased when cells were frozen at a later stage of the phage growth. No changes were found in the intracellular growth processes of the phages during the subsequent cultivation of the bacterium when it was infected and then cooled to 0 degree C.     

Regions of incompatibility in single-stranded DNA bacteriophages phi X174 and G4.

The intracellular presence of a recombinant plasmid containing the intercistronic region between the genes H and A of bacteriophage phi X174 strongly inhibits the conversion of infecting single-stranded phi X DNA to parental replicative-form DNA. Also, transfection with single-stranded or double-stranded phi X174 DNA of spheroplasts from a strain containing such a "reduction" plasmid shows a strong decrease in phage yield. This phenomenon, the phi X reduction effect, was studied in more detail by using the phi X174 packaging system, by which plasmid DNA strands that contain the phi X(+) origin of replication were packaged as single-stranded DNA into phi X phage coats. These "plasmid particles" can transduce phi X-sensitive host cells to the antibiotic resistance coded for by the vector part of the plasmid. The phi X reduction sequence in the resident plasmid strongly affected the efficiency of the transduction process, but only when the transducing plasmid depended on primosome-mediated initiation of DNA synthesis for its conversion to double-stranded DNA. The combination of these results led to a model for the reduction effect in which the phi X reduction sequence interacted with an intracellular component that was present in limiting amounts and that specified the site at which phi X174 replicative-form DNA replication takes place. The phi X reduction sequence functioned as a viral incompatibility element in a way similar to the membrane attachment site model for plasmid incompatibility. In the DNA of bacteriophage G4, a sequence with a similar biological effect on infecting phages was identified. This reduction sequence not only inhibited phage G4 propagation, but also phi X174 infection.     

Possible finger structure in gene A protein of Microviridae

Microvirid phages alpha 3 and phi K encode for A protein which functions in initiation of the viral DNA synthesis. By nucleotide sequencing analysis, we have found that each gene A protein has 'finger motif structure' which conserves two cysteine and histidine residues similar to the consensus sequence deduced from more than thirty finger motifs reported from many eukaryotic regulatory proteins. In closely related phages, phi X174 and G4, we have detected the same structures in their gene A proteins also.     

Cloned bacteriophage phi X174 DNA sequence interferes with synthesis of the complementary strand of infecting bacteriophage phi X174.

The insertion of a particular phi X DNA sequence in the plasmid pACYC177 strongly decreased the capacity of Escherichia coli cells containing such a plasmid to propagate bacteriophage phi X174. The smallest DNA sequence tested that showed the effect was the HindII fragment R4. This fragment does not code for a complete protein. It contains the sequence specifying the C-terminal part of the gene H protein and the N-terminal part of the gene A protein, as well as the noncoding region between these genes. Analysis of cells that contain plasmids with the "reduction sequence" showed that (i) the adsorption of the phages to the host cells is normal, (ii) in a single infection cycle much less phage is formed, (iii) only 10% of the infecting viral single-stranded DNA is converted to double-stranded replicative-form DNA, and (iv) less progeny replicative form DNA is synthesized. The reduction process is phi X174 specific, since the growth of the related G4 and St-1 phages was not affected in these cells. The effect of the recombinant plasmids on infecting phage DNA shows similarity to the process of superinfection exclusion.     

Phi X174 E complements lambda S and R dysfunction for host cell lysis.

Hybrid lambda phages which have the E lysis gene of the bacteriophage phi X174 in cis to defective nonsense and deletion alleles of the normal lambda lysis genes S and R have been constructed and shown to be fully competent for plaque-forming ability, which demonstrates that the single-gene, lysozyme-independent lysis system of phi X174 and related phages can serve the lytic function for large complex phages. These hybrid phages are unable to form plaques on a slyD host. Moreover, plaque morphology indicates that in E-mediated lysis the soluble lambda R endolysin can participate in lysis, indicating that the protein E-mediated lesions are not completely sealed off from the periplasm.     

Growth and DNA synthesis of bacteriophage phi x174 in a dnaP mutant of Escherichia coli.

phi X174am3trD, a temperature-resistant mutant of bacteriophage phi X174am3, exhibited a reduced ability to grow in a dnaP mutant, Escherichia coli KM107, at the restrictive temperature (43 degrees C). Under conditions at which the dnaP gene function was inactivated, the amount and the rate of phi X174am3trD DNA synthesis were reduced. The efficiency of phage attachment to E. coli KM107 at 43 degrees C was the same as to the parental strain, E. coli KD4301, but phage eclipse and phage DNA penetration were inhibited in E. coli KM107 at 43 degrees C. It is suggested that the dnaP gene product, which is necessary for the initiation of host DNA replication, participates in the conversion of attached phages to eclipsed particles and in phage DNA penetration in vivo in normal infection.     

Evaluation of an Escherichia coli host strain for enumeration of F male-specific bacteriophages

A method was developed for the selective enumeration of F male-specific bacteriophages in samples of environmental waters. The host strain for the phages, Escherichia coli HS(pFamp)R, has three antibiotic resistance markers, ampicillin on the Famp plasmid, which codes for pilus production, and streptomycin and nalidixic acid on the chromosome. The strain is resistant to coliphages T2 to T7 and phi X174. More than 95% of the phages from environmental samples which plaqued on the host strain were F male specific. The host bacterium had a higher plaquing efficiency than E. coli K-12 Hfr for F-specific phages in stock suspensions and sewage effluents. The F male-specific phage levels in prechlorinated, secondary-treated sewage effluents generally were about 10(3) to 10(4) PFU/100 ml. The levels in the influents to the sewage treatment plants and in septic tank contents were about 10(5) PFU/100 ml. RNA-containing phages composed about 90% of the total F-specific phage population in sewage effluents.     

Sewage coliphages studied by electron microscopy.

Sewage was enriched with 35 Escherichia coli strains, and sediments of enrichment cultures were studied in the electron microscope. They contained up to 10 varieties of morphologically different particles. T-even-type phages predominated in 14 samples. Thirteen phages were enriched, representing the families Myoviridae (seven), Styloviridae (two), Podoviridae (three), and Microviridae (one). Twelve of these corresponded to known enterobacterial phage species, namely, 121, K19, FC3-9, O1, 9266, T2, 16-19, kappa, beta 4, N4, T7, and phi X174. Cubic RNA phages and filamentous phages were not detected. Types 121 and 9266 have previously been observed only in Romania and South Africa. Identification by morphology is usually simple. Our investigative technique is qualitative and will not detect all phages present. Most enrichment strains are polyvalent, and electron microscopy is always required for phage identification. In a general way, electron microscopy seems to be the method of choice for investigation of phage geography and ecology.     

Evaluation of an Escherichia coli host strain for enumeration of F male-specific bacteriophages.

A method was developed for the selective enumeration of F male-specific bacteriophages in samples of environmental waters. The host strain for the phages, Escherichia coli HS(pFamp)R, has three antibiotic resistance markers, ampicillin on the Famp plasmid, which codes for pilus production, and streptomycin and nalidixic acid on the chromosome. The strain is resistant to coliphages T2 to T7 and phi X174. More than 95% of the phages from environmental samples which plaqued on the host strain were F male specific. The host bacterium had a higher plaquing efficiency than E. coli K-12 Hfr for F-specific phages in stock suspensions and sewage effluents. The F male-specific phage levels in prechlorinated, secondary-treated sewage effluents generally were about 10(3) to 10(4) PFU/100 ml. The levels in the influents to the sewage treatment plants and in septic tank contents were about 10(5) PFU/100 ml. RNA-containing phages composed about 90% of the total F-specific phage population in sewage effluents.     

DNA sequences which support activities of the bacteriophage phi X174 gene A protein

The DNA sequence of 30 nucleotides which surrounds the origin of viral strand DNA replication is highly conserved amongst the icosahedral single-stranded DNA bacteriophages. The A gene of these phages encodes a protein which is required for initiation and termination of viral strand DNA synthesis and acts as a nicking-closing activity specifically within this 30-nucleotide sequence. A system of purified Escherichia coli host proteins and phi X174 gene A protein has been developed which specifically replicates in vitro the viral strand of phi X174 from RF (replicative form) I template DNA and yields single-stranded circular DNA products (RF leads to SS(c) DNA replication system). Recombinant plasmids carrying inserts derived from phage phi X174 or G4 DNA which range in length from 49 to 1175 base pairs and contain the 30-nucleotide conserved sequence have been shown to support phi X A protein-dependent DNA synthesis in vitro in this replication system. We report here that insertion of the 30-nucleotide sequence alone into pBR322 allows the resulting recombinant plasmids to support phi X A protein-dependent in vitro DNA synthesis as efficiently as phi X174 template DNA in the RF leads to SS(c) replication system. The 30-nucleotide sequence functions as a fully wild type DNA replication origin as determined by the rate of DNA synthesis and the structure of resulting DNA products. Furthermore, the DNA sequence requirements for nicking of RF I DNA by the phi X A protein and for supporting replication origin function have been partially separated. Homology to positions 1, 29, and 30 of the 30-nucleotide conserved sequence are not required for cleavage of RF I DNA by the A protein; homology to position 1 but not 29 or 30 is required for efficient DNA replication.     

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.     

Alteration of the ATG start codon of the A protein of bacteriophage phi X174 into an ATT codon yields a viable phage indicating that A protein is not essential for phi X174 reproduction

Bacteriophage phi X174 gene A encodes two proteins: the gene A protein and the smaller A protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling circle DNA replication. The role of the A protein in the life cycle of phi X174, however, is unknown. Using oligonucleotide-directed mutagenesis a viable phi X174 mutant was constructed in which the ATG start codon of the A protein was changed into an ATT codon. This mutant, phi X-4499T, does not synthesize A protein. The burst size of phi X-4499T amounted to 50% of that of wild type phi X174. This indicates that A protein, although advantageous for phage reproduction, is not essential during the life cycle of bacteriophage phi X174.     

RNA priming of DNA replication by bacteriophage T4 proteins

Bacteriophage T4 DNA replication proteins have been shown previously to require ribonucleoside triphosphates to initiator new DNA chains on unprimed single-stranded DNA templates in vitro. This DNA synthesis requires a protein controlled by T4 gene 61, as well as the T4 gene 41, 43 (DNA polymerase), 44, 45, and 62 proteins, and is stimulated by the gene 32 (helix-destabilizing) protein. In this paper, the nature of the RNA primers involved in DNA synthesis by the T4 proteins has been determined, using phi X174 and f1 DNA as model templates. The T4 41 and "61" proteins synthesize pentanucleotides with the sequence pppA-C(N)3 where N in positions 3 and 4 can be G, U, C, or A. The same group of sequences is found in the RNA at the 5' terminus of the phi X174 DNA product made by the seven T4 proteins. The DNA product chains begin at multiple discrete positions on the phi X174 DNA template. The characteristics of the T4 41 and "61" protein priming reaction are thus appropriate for a reaction required to initiate the synthesis of discontinuous "Okazaki" pieces on the lagging strand during the replication of duplex DNA.     

The enzymatic replication of DNA.

Enzymatic mechanisms of DNA replication have been investigated using small bacteriophages as probes to illuminate the cellular systems upon which they must rely during infection. Conversion of the circular, single-stranded DNAs of phages M13, G4, and phi X174 to their duplex forms has revealed the participation of diverse ways to start a new chain and a complex DNA polymerase III holoenzyme upon which all these systems depend for chain elongation. The phi X174 system, which is the most exacting and revealing of the host chromosomal replication pattern, includes at least twenty polypeptides for making the viral DNA into a duplex and multiplying the duplex. Resolution and purification of these numerous proteins is in train and their reconstitution into a "replisome"-like structure is envisioned.