Defective Phage Research Articles

A defective phage system reveals bacteriophage T4 replication origins that coincide with recombination hot spots.

Plasmid transduction mediated by bacteriophage T4 has been used to study putative T4 DNA replication origins cloned as inserts in the Escherichia coli plasmid pBR322. Two particular inserts from the T4 genome allow high-frequency plasmid transduction, suggesting that each insert might contain a T4 replication origin. T4 infection of these plasmid-containing cells produces large numbers of defective phage particles that contain long linear concatamers of the plasmid DNA. During a second cycle of infection, these defective phage genomes can be replicated better than normal phage chromosomes present in the same infected cell; consequently, the T4 DNA inserts must be functioning as replication origins. Both of these origins appear to utilize a previously unrecognized mode of T4 replication initiation. Moreover, each origin coincides with a major recombination hot spot in the phage genome, and therefore this mode of replication initiation seems to involve a local stimulation of homologous genetic recombination. From a purely practical standpoint, additional DNA fragments can be cloned in an origin-containing plasmid, allowing isolation of large amounts of any DNA sequence with the glucosylated hydroxymethylcytosine modifications of T4 DNA.

Identification of Genes Involved in the Rhizobium-Legume Symbiosis by Mu-dI (Kan, lac)-Generated Transcription Fusions

The process by which free-living Rhizobium forms nitrogen-fixing nodules on the roots of leguminous plants involves the induction and repression of a number of bacterial genes. To help identify these genes and to determine their mode of regulation, we have transposed the E. coli lac operon, minus its promoter, randomly around the chromosome of R. japonicum, a soybean nodulating bacterium. This was accomplished using the defective phage, mu-dI (Kan, lac) placed onto a self-transmissible, narrow host-range suicide vector, pGS6. Among the mu-dI insertion mutants examined, auxotrophs were detected at frequencies of about 0.3%, and nodulation and nitrogen fixation defective mutants at about 4%. Many of the symbiotically defective mutants were found to have deletions in their indigenous plasmids. Three mutants showed increased β-galactosidase levels when grown in the presence of soybean or kidney bean root-extracts but not in the presence of root-extracts from other legumes or a non-legume, indicating specific plant control of Rhizobium gene expression.

Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy- d- arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from attλ to aroG. A 7.6-kb PstI- HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-lacZ' (α) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of β-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR + background.

DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis.

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.

Loss of plasmids containing cloned inserts coding for novobiocin resistance or novobiocin sensitivity in Haemophilus influenzae

Plasmids pNov1 and pNov1s , coding for resistance and sensitivity to novobiocin, respectively, were readily lost from wild-type Haemophilus influenzae but retained in a strain lacking an inducible defective prophage. The plasmid loss could be partly or wholly eliminated by a low-copy-number mutation in the plasmid or by the presence of certain antibiotic resistance markers in the host chromosome. Release of both phage HP1c1 , measured by plaque assay, and defective phage, measured by electron microscopy, was increased when the plasmids were present. The frequency of recombination between pNov1 and the chromosome, causing the plasmid to be converted to pNov1s , could under some circumstances be decreased from the normal 60 to 70% to below 10% by the presence of a kanamycin resistance marker in the chromosome. This suggested that a gene product coded for by the plasmid, the expression of which was affected by the kanamycin resistance marker, was responsible for the high recombination frequency. Evidence was obtained from in vitro experiments that the gene product was a gyrase.

Mutagenesis of Erwinia carotovora subsp. carotovora with bacteriophage Mu d1 (Apr lac cts62): construction of his-lac gene fusions.

The bacteriophage Mu d1(Apr lac cts62 ) obtained from an Escherichia coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid phage was utilized as a vector for phage mutagenesis in Erwinia carotovora subsp. carotovora. Among ampicillin-resistant transductants. 1.4% were auxotrophs. The synthesis of beta-galactosidase was derepressed upon starvation for histidine in two different his-lac fusion strains.

Bacillus mycoides phages and their satellites

The relationship between large and small particles of phages No. 1M and H17 reproducing simultaneously in one and the same bacterial cell of Bacillus mycoides was studied by the immune electron microscopic technique. The large particles of phages No. 1M and H17 were morphologically identical with phage No. 1 of B. mycoides, whereas only the tails of small particles of phages No. 1M and H17 were morphologically identical with the tail of phage No. 1. Antigens were identified in phages No. 1, No. 1M, and H17 using specific antibodies against phage No. 1, containing only large phage particles, and specific antibodies against phage H17 small heads. It was shown that (i) all structural elements of large particles and tails of small particles of phage No. 1M were antigenically identical with those of phage No. 1; (ii) all structural elements of small and large particles of phage H17, except the inner core of the tail, were antigenically different from phage No. 1; and (iii) the small heads of phages No. 1M and H17 were antigenically identical. Particles of phage No. 1 are morphologically and antigenically identical with the large particles of phage No. 1M and are antigenically different from the large particles of phage H17. Since the tails of small and large particles are antigenically identical in each phage pair (No. 1M and H17), this suggests that in both cases, the genome of a small defective phage codes for the synthesis of head proteins only, whereas its tail is borrowed from the corresponding helper phage. The small phage may therefore be considered as a satellite of the large phage which depends on a helper partner for production of complete particles and whose tail proteins are identical with those of the helper phage.

Mu dX, a derivative of Mu d1 (lac Apr) which makes stable lacZ fusions at high temperature.

We describe defective Mu phage Mu dX (Mu d1 Bx::Tn9 [lac Apr Cmr]) which is useful for insertion mutagenesis and for construction of lac operon fusions in vivo. Mu dX retains the insertion properties of Mu d1 but produces temperature-resistant lysogens and transposes at a reduced frequency. A method is described to convert existing Mu d1 insertions to Mu dX.

Molecular characterization of ilvC specialized transducing phages of Escherichia coli K-12.

A series of lambda defective ilvC specialized transducing phage has been isolated which carry regions of isoleucine and valine structural and regulatory genes derived from the ilv cluster at minute 83 on the linkage map of the chromosome of Escherichia coli K-12. The ilv genes carried by these phages and their order have been determined by transduction of auxotrophs. The ilvC+ lysogen of an ilvC- strain gave rise, after heat induction of the lysogen, to transducing particles which carried the wild-type allele of the cya-marker. Further experiments have shown that the lambda defective ilvC phages were able to cotransduce a rho-15ts mutation as well as a rep-5 mutation. Hence, the order of the clockwise excision of the ilv cluster was found to be ilvC-rho-rep-cya. Enzyme levels in strains carrying the lambda defective ilvC phages indicated the the ilvC gene was not altered by the insertion of lambda into the ilv cluster. The isolation and digestion of lambda defective ilvC DNA by EcoRI and HindIII restriction endonucleases demonstrated that the specialized transducing phages carried part of the genome from the E. coli K-12 chromosome.

Purification and characterization of two phage PBSX-induced lytic enzymes of Bacillus subtilis 168: an N-acetylmuramoyl-L-alanine amidase and an N-acetylmuramidase.

After heat-induction of the defective phage PBSX in a xhi-1479 mutant of Bacillus subtilis 168, the culture lysed rapidly even if the lyt-2 mutation was present (which greatly reduces the amount of the bacterial autolysins). Two lytic enzymes, an N-acetylmuramoyl-L-alanine amidase and an endo-N-acetylmuramidase, were purified from the culture supernatant. The amidase was readily distinguished from the bacterial amidase by its low molecular weight. In addition, it was not inhibited by antibody directed against the bacterial enzyme. These results indicate that PBSX does not rely on the bacterial autolysins to accomplish lysis.

Isolation of an autonomously replicating DNA fragment from the region of defective bacteriophage PBSX of Bacillus subtilis.

We have isolated a 5.4-kilobase fragment of Bacillus subtilis DNA that confers the ability to replicate upon a nonreplicative plasmid. The B. subtilis 168 EcoRI fragment was ligated into the chimeric plasmid pCs540, which contains a chloramphenicol resistance determinant from the Staphylococcus aureus plasmid pC194 and an HpaII fragment from the Escherichia coli plasmid, pSC101. A recE B. subtilis derivative, strain BD224, is capable of maintaining this DNA as an autonomously replicating plasmid. In rec+ recipients, chloramphenicol-resistant transformants do not contain free plasmid. The plasmid is integrated as demonstrated by alterations in the pattern of chromosomal restriction enzyme fragments to which the plasmid hybridizes. The site of plasmid integration was mapped by PBS1-mediated transduction to the metC-PBSX region. A strain was a deletion in the region of defective bacteriophage PBSX differs in the hybridization profile obtained by probing EcoRI digests with this cloned fragment. This same deletion mutant, though proficient in normal recombinational pathways, permits autonomous replication of the plasmid apparently owing to the lack of an homologous chromosomal region with which to recombine. We believe that, like E. coli. B. subtilis contains at least one DNA fragment capable of autonomous replication when liberated from its normally integrated chromosomal site and that this cloned DNA fragment comes from the region of defective bacteriophage PBSX.

Phage P1 mutant with decreased abortive transduction

One amber mutant, named sus50, of phage P1 with decreased abortive transduction was isolated from P1CM cts-1, a thermo-inducible mutant of PlCM, after hydroxylamine mutagenesis. When sus50 phage is grown in Su − host, defective phage particles are produced at a rate 670 times that of infective phage particles and, in addition, many head particles containing sus50 DNA and many phage tails are also produced. In generalized transduction by sus50 mutant phage, the frequency of abortive transduction per infective phage decreased to about one-tenth that of P1CM cts-1 sus + phage, while that of stable transduction appeared to be about 10 times higher than that of P1CM cts-1 sus + phage. Stimulation of stable transduction by uv irradiation was not observed. From revertant analysis of the sus50 mutant, it is suggested that sus50 might be a single mutation, forming defective phage particles as well as decreasing abortive transduction.

Bacteriophage PBSX-induced deletion mutants of Bacillus subtilis 168 constitutive for alkaline phosphatase.

Mutants of Bacillus subtilis with deletions extending from the PBSX prophage, and in some cases removing pro(AB) and metC, have been found to be constitutive for vegetatively synthesized alkaline phosphatase. Such deletions were isolated by selecting for heat-resistant derivatives of a strain carrying a xhi-1479 mutation causing heat-inducibility of the defective phage PBSX. These deletions remove the phoS gene, a regulatory gene for alkaline phosphatase; it is concluded that the phoS gene product exerts negative control on alkaline phosphatase synthesis. Deletion mapping, combined with previously published linkage data, indicates a gene order of PBSX-phoS-pro(AB)-metC.

Gene dosage as a regulatory factor for gene expression. I. In lambda plac5-infected cells.

To study the effect of gene dosage on gene expression, lambda plac5cI857O29P3, a replication defective lambda phage carrying part of the lac operon (containing the lac promotor, operator and z gene) in the b2 region was studied in Escherichia coli strain JC6256 where the lac operon is deleted and at a temperature where the lambda repressor is inactive. In measuring the synthesis of beta-galactosidase, it was possible to separate the effects of the lac promoter from those of the phage promoter. When the synthesis of beta-galactosidase was initiated from the inserted lac promoter in JC6256(lambda +) in the presence of additional cyclic AMP, the rate and level of beta-galactosidase synthesis were directly proportional to the multiplicity of infection (gene dosage). Furthermore, beta-galactosidase synthesis was initiated about 5 min after infection, just as with isopropyl-beta-D-thiogalactoside (IPTG) induction. When the synthesis of beta-galactosidase was initiated from the phage promoter in JC6256 in the absence of additional cyclic AMP, the rate and level of beta-galactosidase synthesis were again linearly proportional to gene dosage. On the other hand, initiation of beta-galactosidase synthesis was delayed until 10 to 20 min after infection. These results suggest that: (i) in the absence of negative controlling factors, the extent of gene expression is proportional to gene dosage; (ii) varying the gene dosage can be used to regulate gene expression.

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction.

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.

An evaluation of the rate constants for the adsorption of the defective phage PBS Z to Bacillus subtilis.

The adsorption of the defective phage PBS Z of Bacillus subtilis has previously been assumed to proceed in two steps, a reversible adsorption of extended phages followed by contraction of the adsorbed particles (Steensma, 1981a). This model, also used for other phages, explained the biphasic character of the adsorption curve, but a discrepancy was found between the calculated and observed concentrations of adsorbed, extended phages. Computer simulations indicated that this might be caused by inhomogeneity of the phage preparations with respect to their adsorption properties and that in that case other models would also fit the experimental data. Discrimination between the models was not possible on the basis of the available information on PBS Z and it was therefore concluded that the values reported previously for the rate constants (Steensma, 1981a) should be used with caution.

Isolation of an autonomously replicating DNA fragment from the region of defective bacteriophage PBSX of Bacillus subtilis.

Isolation of an autonomously replicating DNA fragment from the region of defective bacteriophage PBSX of Bacillus subtilis.

Defective specialized SP beta transducing bacteriophages of Bacillus subtilis that carry the sup-3 or sup-44 gene.

We isolated defective specialized transducing phages of SP beta that carry one of the extracistronic suppressors, sup-3 or sup-44. Lysates containing these phages can be used in a simple spot test to determine whether an auxotrophic mutation can be suppressed. The sup-3 and sup-44 mutations are distinct, in that their suppression patterns differ for the markers hisA1, metC3, and thr-5; and they are not alleles.

In vivo isolation of a pPJ3a-λ cosmid mediated by the Tn2307 transposon

From an E. coli cell harboring plasmid pPJ3b (= pPJ3a::Tn 2301) and infected with phage λ, we have isolated two defective phages having inserted pPJ3a DNA and Tn 2301 in their genomes. One of them has been extensively characterized: it behaves like a cosmid, i.e., upon injection into the cell, its DNA circularizes and replicates as a plasmid (pPJ10); it can be packaged again in λ heads, provided the presence of a phage helper. Furthermore, heteroduplex analysis has shown that in pPJ10, the transposon Tn 2301 is inverted compared to its direction in pPJ3b. We give evidence suggesting that this type of inversion is in part mediated by Tn 2301.