Isolation and characterization of specialized lambda transducing bacteriophage carrying the metBJF methionine gene cluster.

Secondary attachment site lysogens of Deltaatt(lambda)Deltappc-argECBH strains of Escherichia coli with lambdacI857 integrated into the bfe gene (88 min) were isolated. Of 20 such lysogens examined, 2 produce lysates with transducing phage containing the metBJF gene cluster (87 min). Reintroduction of the ppc-argECBH chromosome segment (which lies between the bfe and met genes) into these strains virtually abolishes the production of met transducing phage. All of the phage examined have lost essential genes from the left arm of the lambda chromosome. Approximately 85% of the phage appear to have the same genetic composition, containing the metBJF gene cluster, but not the closely linked gene cytR, and having lost phage genes G and J. Analytical CsCl density gradient centrifugation of five representatives of this major class of phage shows four of them to have identical densities (lighter than lambda), while the fifth cannot be resolved from lambda. The four apparently identical phage were isolated from three separate lysates, which suggests the existence of preferred sites for illegitimate recombination on the bacterial and phage chromosomes. Three specialized transducing phage that carry cytR in addition to metB, metJ, and metF have also been studied. Each of these viruses has a different amount of phage deoxyribonucleic acid. Two of them have less deoxyribonucleic acid than lambda, whereas the third has about the same amount. The metB, metF, and cytR genes of the transducing phage have been shown to function in vivo. The phage-borne metB and metF genes are subject to metJ-mediated repression.     

Construction of improved bacteriophage phi 105 vectors for cloning by transfection in Bacillus subtilis

A series of improved phage vectors have been constructed, based on Bacillus subtilis bacteriophage phi 105, which can be used to clone genes in B. subtilis by direct transfection of protoplasts. The new vectors, designated phi 105J23, phi 105J24, phi 105J27 and phi 105J28, show frequencies of plaque formation that are equal to those of wild-type phi 105. This represents at least a 10-fold improvement over phi 105J9, the vector used in previous cloning experiments. Two of the new vectors phi 105J27 and phi 105J28 incorporate a mutation, cts-52, that renders the prophage temperature inducible. This has made it possible to devise a rapid small-scale procedure for screening progeny phage for the presence of inserted DNA. The usefulness of the new vectors is illustrated in the accompanying paper by cloning more than 20 B. subtilis sporulation genes.     

Induction and preliminary characterization of a novel halophage SNJ1 from lysogenic Natrinema sp. F5

Halophage SNJ1 was induced with mitomycin C from Natrinema sp. strain F5. The phage produces plaques on Natrinema sp. strain J7 only. The phage has a head of about 67 nm in diameter and a tail of 570 nm in length and belongs morphologically to the family Siphoviridae. The phage is strongly salt dependent; NaCl concentration affects the integrity of SNJ1, phage adsorption, and plaque formation. The optimal NaCl concentration for phage adsorption and plaque formation is 30% and 25%, respectively.     

Characterization of a bacteriophage that carries the genes for production of Shiga-like toxin 1 in Escherichia coli.

The Shiga-like toxin 1-converting bacteriophage H-19B was recently shown to carry the structural genes for the toxin and was shown to have DNA sequence homology with phage lambda. We present evidence that the linear genome of bacteriophage H-19B has cohesive termini which become covalently associated during prophage integration. Integration occurs through a site on a 4-kilobase-pair EcoRI fragment located near the center of the bacteriophage chromosome. The relationship between bacteriophages H-19B and lambda was examined by Southern hybridization. Homologous regions were mapped on the respective chromosomes which corresponded to the regions of the J gene, the int-xis area, and the O and P genes of phage lambda. The H-19B tox genes were mapped to the right of the O and P gene homology, which was far away from the phage attachment site. We concluded that H-19B is a lambdoid bacteriophage. Unlike other toxin-converting bacteriophages, the toxin genes were not located adjacent to the phage attachment site. It appeared that the Shiga-like toxin 1 genes were not picked up by a simple imprecise prophage excision. H-19B could, however, have acquired chromosomally located toxin genes by a series of events involving deletion and duplication followed by aberrant excision.     

Phage Growth and Deoxyribonucleic Acid Synthesis in Escherichia coli Infected by a Thymine-Requiring Bacteriophage.

Mathews, Christopher K. (Yale University, New Haven, Conn.). Phage growth and deoxyribonucleic acid synthesis in Escherichia coli infected by a thymine-requiring bacteriophage. J. Bacteriol. 90:648-652. 1965.-Cultures of Escherichia coli B infected with a mutant strain of phage T4 which cannot induce the formation of thymidylate synthetase produce deoxyribonucleic acid (DNA) at about two-thirds the rate of cultures infected with the parent strain. Under certain conditions the yield of viable phage observed with the mutant is one-third of that brought about by the wild-type strain. Addition of thymine increases both DNA synthesis and phage production in cells infected by the mutant. It is suggested that the ability to induce thymidylate synthetase formation in infected cells confers a selective advantage on the wild-type strain.     

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.     

Chromosomal relocation of prophage-associated bacterial genes

Taylor, M. W. (Stanford University, Stanford, Calif.), and C. Yanofsky. Chromosomal relocation of prophage-associated bacterial genes. J. Bacteriol. 91:1469-1476. 1966.-Two distinguishable colony types, rough-edged and smooth-edged, were observed when tryptophan auxotrophs of Escherichia coli were transformed to tryptophan independence with DNA from the hybrid nondefective transducing phage i(lambda)h(phi80)T(1) (S)tryp A(+)B(+), and with the helper phage lambdai(434). P1kc transduction experiments with cells of the two types of colonies as genetic donors showed that the i(lambda)h(phi80)T(1) (S)tryp A(+)B(+) prophage was located at different regions of the E. coli chromosome. In cells of rough-edged colonies, the prophage was linked to the tryp-cys region, its normal location, whereas in cells of smooth-edged colonies the prophage was associated with the gal region. When transformation experiments were performed with a T(1) (R)tryp(-) deletion mutant as recipient, and phage lambdai(434) as helper, prophage localization was only detected at the gal region. Localization of (lambda)h(phi80)T(1) (S)tryp A(+)B(+) prophage near gal does not appear to be due to the formation of a recombinant phage carrying tryp A(+)B(+), but is due to some type of interaction between the genomes of i(lambda)h(phi80)T(1) (S)tryp A(+)B(+) and the helper phage. When conditions comparable to those used in transformation studies were employed in transduction experiments, including the use of helper phage, two classes of transductants with either cys or gal linkage were also observed. To examine whether the location of the prophage on the E. coli chromosome had any effect on the ability of the prophage-associated tryp A(+) and tryp B(+) genes to function or respond to different repression conditions, specific activities of the A and B subunits of tryptophan synthetase specified by the phage genome were measured. Similar values were obtained regardless of the location of the prophage-associated tryp genes. Furthermore, the prophage-associated tryp genes, free from their normal operator region, permitted enzyme formation which was unaffected by repression or derepression conditions.     

Genetic control of morphogenesis of Pseudomonas aeruginosa transposable phage D3112

The influence of ts mutations in the early and late genes of transposable phage D3112 on phage morphogenesis was studied. The mutations in the early genes A, B and C were shown to suppress morphogenesis of D3112. Six genes (D, E, F, G, H and I), located from 14 to 29 kbp of the phage physical map, control morphogenesis of phage head. Five genes (J, K, L, M and N), clustered in the 29-36 kbp region of the map, control morphogenesis of tail. The similarity of genetic organization of the Escherichia coli transposable phage Mu and the Pseudomonas aeruginosa phage D3112 is discussed.     

Genetic diversity in temperate bacteriophages of Streptococcus pyogenes: identification of a second attachment site for phages carrying the erythrogenic toxin A gene.

Bacteriophage T12, the prototypic bacteriophage of Streptococcus pyogenes carrying the erythrogenic toxin A gene (speA), integrates into the bacterial chromosome at a gene for a serine tRNA (W. M. McShan, Y.-F. Tang, and J. J. Ferretti, Mol. Microbiol. 23:719-728, 1997). This phage is a member of a group of related temperate phages, and we show here that not all speA-carrying phages in this group use the same attachment site for integration into the bacterial chromosome. Additionally, other phages in the group use the same serine tRNA gene attachment site as phage T12 and yet do not carry speA. The evidence suggests that recombination between phage genomes has been an important means of generating diversity and disseminating virulence-associated genes like speA.     

Genetic complementation by cloned bacteriophage T4 late genes.

Bacteriophage T4 containing nonsense mutations in late genes was found to be genetically complemented by four conjugate T4 genes (7, 11, 23, or 24) located on plasmid or phage vectors. Complementation was at a very low level unless the infecting phage carried a denB mutation (which abolishes T4 DNA endonuclease IV activity). In most experiments, the infecting phage also had a denA mutation, which abolishes T4 DNA endonuclease II activity. Mutations in the alc/unf gene (which allow dCMP-containing T4 late genes to be expressed) further increased complementation efficiency. Most of the alc/unf mutant phage strains used for these experiments were constructed to incorporate a gene 56 mutation, which blocks dCTP breakdown and allows replication to generate dCMP-containing T4 DNA. Effects of the alc/unf:56 mutant combination on complementation efficiency varied among the different T4 late genes. Despite regions of homology, ranging from 2 to 14 kilobase pairs, between cloned T4 genes and infecting genomes, the rate of formation of recombinants after T4 den:alc phage infection was generally low (higher for two mutants in gene 23, lower for mutants in gene 7 and 11). More significantly, when gene 23 complementation had to be preceded by recombination, the complementation efficiency was drastically reduced. We conclude that high complementation efficiency of cloned T4 late genes need not depend on prior complete breakage-reunion events which transpose those genes from the resident plasmid to a late promoter on the infecting T4 genome. The presence of the intact gene 23 on plasmids reduced the yield of T4 phage. The magnitude of this negative complementation effect varied in different plasmids; in the extreme case (plasmid pLA3), an almost 10-fold reduction of yield was observed. The cells can thus be said to have been made partly nonpermissive for this lytic virus by incorporating a part of the viral genome.     

Gene responsible for superinfection exclusion of heteroimmune corynebacteriophage.

Wild-type beta and gamma corynebacteriophages are heteroimmune and infect lysogens of each other productively. Unlike their wild-type counterparts, the bin mutants of each phage are excluded in lysogens carrying the heteroimmune phage. The wild-type phages overcome exclusion by means of the bin gene product which appears to act as an antirepressor. When repression is lifted, exclusion of bin mutants is abolished (N. Groman and M. Rabin, J. Virol. 28:28-33, 1978; J. Virol. 36:526-532, 1980). It has not been clear whether the excluding compound is the immune repressor itself or one whose synthesis is positively regulated by repressor. We have isolated beta exclusion mutants (xcl) that as prophage exhibited normal immune repression but no longer excluded gamma-bin mutants. Furthermore, we have shown that an xcl phage with an active immune repressor acted in trans to continue the positive regulation of exclusion by a second xcl+ prophage whose immune repressor was inactivated. From these results it was concluded that there is a gene distinct from the imm gene which is directly or indirectly responsible for exclusion. The xcl gene, mapped in prophage crosses, was located between imm and bin, that is, in the regulatory region of the phage genome. The simplest hypothesis compatible with the established observations is that beta immune repressor regulates the expression of the xcl and bin genes, the former positively and the latter negatively. It is likely that an analogous regulatory model applies to gamma phage since it has already been shown that both beta and gamma have bin alleles.     

Effects of palindromes on in vivo DNA replication and mutagenesis in bacteriophage phi X174 RF DNA.

Bacteriophage phi X174 mutants with insertions of palindromic DNA sequences are rapidly outgrown by competing wild-type phage (Müller & Turnage, J. Mol. Biol. 189: 285). The basis for this defect was investigated and found to be due to an exclusion event early in the infectious cycle, in which phage genomes with palindromic inserts were preferentially excluded by wt phage. In addition, we have obtained further evidence for a palindrome induced genetic instability. Both defects are dependent on palindrome size and sequence, consistent with a model which involves formation of cruciforms, or cruciform-like structures. We propose that formation of unusual DNA secondary structures reduces the effectiveness of replicative form (RF) DNA to interact with limiting replication factors or membrane binding sites, possibly because of interaction with the host recombination system.     

Location of a ColE1 deoxyribonucleic acid region that affects the plaque-forming ability of lambda-ColE1 hybrid bacteriophage.

The plaque-forming ability of a hybrid phage between plasmidColE1 and phage lambda carrying amber mutations in genes O and P was inhibited by the presence of ColE1 in suppressor-deficient Escherichia coli cells. ColE1 deoxyribonucleic acid regions concerned with this inhibition were examined by using various deletion and transposon insertion derivatives of ColE1, and it was found that the presence of the deoxyribonucleic acid region extending between 420 and 613 base pairs upstream from the initiation site of ColE1 deoxyribonucleic acid replication (J. Tomizawa, H. Ohmori, and R. E. Bird, Proc. Natl. Acad. Sci. U. S. A. 74: 1865--1869, 1977) was essential for this function.     

Insertion of the Tn3 transposon into the genome of the single-stranded DNA phage M13.

The transposable genetic element Tn3, which carries an ampicillin (Ap) resistance determinant, has been translocated from a ColE1-Apr plasmid, RSF2124, to the genome of the filamentous single-stranded DNA phage M13. The site orientation of the inserted element has been determined for one such phage, M13::Tn3-15. The insertion is within the intergenic space separating genes 2 and 4 and containing both the viral strand and complementary strand origins. The lengths of both the filamentous phage and the duplex replicative form (RF) DNA are 1.7--1.8 times those of M13 phage and replicative form DNA. Both plaque formation and transduction of sensitive cells to ampicillin resistance by M13::Tn3-15 are sensitive to purified antibodies to the M13 major coat protein.     

The J gene of bacteriophage phi X174: in vitro analysis of J protein function.

The J protein of phi X174 is a small, highly basic protein and is a component of the phage capsid. We have investigated the role of J protein during single-stranded viral DNA synthesis and phage morphogenesis by using an in vitro system composed of purified viral and host components (Aoyama et al., Proc. Natl. Acad. Sci. U.S.A. 80:4195-4199, 1983). The characterization of the products made in the presence and absence of J protein shows that J protein is not required for viral DNA synthesis, but is required for the packaging of DNA into infectious phage. The ability of J protein to bind to double-stranded DNA as well as single-stranded DNA and other interactions with DNA suggest a model in which J protein binds to double-stranded, replicative form DNA and enters the phage prohead by remaining bound to viral DNA as it is encapsidated.     

Genetic comparison of bacteriophage PS17 and Pseudomonas aeruginosa R-type pyocin.

PS17 is a bacteriophage of Pseudomonas aeruginosa that is serologically cross-reactive with phage tail-like bacteriocins called R-type pyocins. In addition to having immunological cross-reactivity, certain genes are functionally complementable between PS17 and R-type pyocins. To compare the genetic structures of PS17 and R-type pyocins, a physical map of PS17 genes was constructed by cloning phage DNA fragments on RSF1010-derived vector plasmids. The head and tail gene clusters were tandemly arrayed and together occupied about half of the 41-kilobase-pair PS17 chromosome. With use of these phage clones, the following results were obtained with respect to the genetic relationship between PS17 and R-type pyocins: (i) serological cross-reaction between PS17 and pyocin occurred for the major sheath protein and two components of the fiber, (ii) a certain pyocin mutation was complemented by cloned phage fragments, and (iii) the phage DNA fragment carrying sheath and core tube genes was shown to hybridize to the DNA fragment carrying the pyocin R2 genes.     

Head morphologies in bacteriophage T4 head and internal protein mutant infections.

Escherichia coli infected with phage T4 mutants defective in synthesis of the three major internal proteins found in the phage head, IPI-, IPII-, IPIII-, or IP degrees (lacking all three) were examined in the electron microscope for head formation. Infection with IPI- or IPII- does not appear to induce increased aberrant head formation, whereas IPII- or IP degrees infections result in production of polyheads and viable phage. Multiple mutants of the early head formation genes 20, 21, 22, 23, 24, 31, 40 and IP degrees were constructed. Combination with IP degrees increases polyhead formation when head formation is not blocked at a more defective stage but results in a qualitative shift to lump formation in association with gene 22 mutants. Thin-sectioning studies show morphologically similar cores in amber 21 and 21am IP degrees tau particles. These morphological observations, genetic evidence for interaction between ts mutants in gene 22 and the IP mutants, and analysis of the protein composition of tau particles further support the idea that p22 and the internal proteins form an unstable assembly core necessary for an early stage of head formation (M. K. Showe and L. W. Black, 1973).     

Cloning and complementation analysis of the Escherichia coli gpr locus, influencing DNA replication of certain lamdoid phages]

Escherichia coli DNA fragments which suppressed gpr27 and gpr2 mutations in the earlier proposed gprA and gprB genes, respectively, were cloned within phage vectors. Mutations gpr2 and gpr27 restrict DNA replication of some lambdoid phages and are located in the region of dnaK, J genes. DNA-DNA hybridization showed that the cloned fragments correspond to the region where gpr mutations were genetically mapped and, in some cases, do not include dnaK, J genes. These results provide the evidence that gprA and gprB genes may be physically separated from dnaK, J genes.