Isolation of a lambda transducing bacteriophage carrying the relA gene of Escherichia coli.

In Escherichia coli the relA and pyrG loci are 99% cotransducible. On the basis of this knowledge, we have isolated lambdacI857S7dpyrG transducing bacteriophages carrying both the pyrG and relA genes. Single lysogens of this bacteriophage show basal levels of ppGpp that are 10-fold higher than normal. Stringent factor is present among the gene products synthesized by lambdadpyrG relA after infection of ultraviolet-killed cells, as analyzed by polyacrylamide gel electrophoresis. The intracellular content of stringent factor, as determined by enzymatic activity, rises 20-fold after induction of a single lysogen of lambdadpyrG relA. As measured by two-dimensional gel electrophoresis, the amount of stringent factor in an exponentially growing strain carrying a pyrG relA plasmid is at least 10-fold greater than in a normal strain. These data constitute strong evidence that stringent factor is the relA gene product.     

Isolation and characterization of lambda specialized transducing bacteriophages carrying Klebsiella pneumoniae nif genes

Seve lambda dnif specialized transducing bacteriophages were isolated from Escherichia coli strains containing plasmids carrying the his-nif region of Klebsiella pneumoniae. These phages collectively carry deoxyribonucleic acid for all of the genes in the nif regulon and adjacent deoxyribonucleic acid of K. pneumoniae. The phages were isolated by using Mu insertions in the nif region to direct the integration of lambda pMu phages in nif via formation of lambda pMu-Mu dilysogens which, upon induction, yielded lambda dnif phages. This procedure should be generally applicable for isolating lambda specialized transducing phages carrying genes from E. coli or other bacteria.     

Isolation and characterization of a lambda transducing bacteriophage carrying the cysE gene of Escherichia coli K-12

A specialized lambda transducing phage carrying the cysE and gpsA genes of E. coli K-12 has been isolated. The transducing phage has been separated from the helper phage on equilibrium gradients and has been shown to be defective. Evidence is presented that the phage kil gene is not expressed.     

Isolation and characterization of specialized transducing lambda phages carrying ribosomal protein genes of Escherichia coli

Summary Insertion in an episome of a kanamycine-resistant element (Tn5) at the polynucleotide phosphorylase gene level, results, after transduction into a wild strain, by the loss of activities specific to polynucleotide phosphorylase. A low phosphorolytic activity is nevertheless detectable in crude extracts, but no longer in extracts slightly purified after heat treatment at 54°C. The part played by other enzymes in these activities is discussed. Bacterial growth is not affected by introduction of the mutation.     

Isolation of specialized transducing bacteriophage lambda carrying genes of the L-arabinose operon of Escherichia coli B/r.

A heat-inducible lysis-defective phage lambda (lambdacI857S7) has been integrated at multiple sites within the L-arabinose region (araCOIBAD) of a strain of Escherichia coli K-12 deleted for the normal lambda attachment site (lambdaattdelta). The lambda phage has become integrated with opposite orientations at two different loci within the aratb gene and with the "normal" orientation (clockwise N-RA-J) at a single site in the araC gene. The burst size, spontaneous-curing frequencies, and number of prophage harbored by each of the ara secondary-site lysogens have been determined. From these secondary-site lysogens it has been possible to generate plaque-forming ara-transducing phage (lambdapara) and defective ara-transducing phage (lambdadara), as well as defective leucine-transducing particles (lambdadleu). The construction and characterization of these lambdaara-transducing phage and their derivatives which carry genetically defined portions of the L-arabinose region are presented.     

Construction and genetic characterization of lambda specialized transducing particles carrying therglB gene ofEscherichia coli K-12

TherglB gene ofEscherichia coli codes for a restriction activity that cleaves the hydroxymethylated DNA of T2 and T4 phages. Earlier mapping data placed the gene at 98.39 min counterclockwise to thehsd operon. Genetic analysis of the in vivo gene fusions with fusion-transducing phages established the location of therglB gene next to thehsdS gene of thehsdRMS cluster. The methodology used in this study could be extended to similar in vivo physical mapping of closely linked genes.     

The isolation and characterization of plaque-forming arabinose transducing bacteriophage lambda.

Non-defective arabinose transducing phage, λpara, were isolated in two steps: first, Escherichia coli strains containing rare insertions of λ DNA into the arabinose C or B genes were selected; and second, these lysogens were induced and transducing phage were selected from the resulting lysates. The approximate location of the bacterial substitution on the phage and the ara gene content of the substitution were determined genetically. The precise location of the substitution was determined by electron microscopy of DNA heteroduplexes.Transducing phage, derived from the strain possessing λ inserted into the araC gene, carried part of the araC gene, the ara regulatory region, and all of the araB gene. Transducing phage, derived from eight independent strains possessing λ inserted in the same orientation and in the same position in the araB gene, carried a portion of the araB gene, the ara regulatory region and all of the araC gene. In these nine cases the ara DNA on the phage was immediately adjacent to the normal phage attachment site, indicating that the transducing phage were formed by the same type of abnormal excision which produces gal or bio transducing λ phage. The relative orientations of ara and phage genes were deduced from the topology of such excisions. One anomalous transducing phage was also characterized.     

A specialized transducing lambda phage carrying the Escherichia coli genes for phenylalanyl-tRNA synthetase.

A lambda phage has been isolated which specifically transduces the Escherichia coli pheS and pheT genes coding for the alpha and beta subunits of the phenylalanyl-tRNA synthetase (PRS). This phage transduces with high frequency (i) several temperature-sensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog. The in vitro PRS activities of such lysogens suggest that the alpha and beta subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes.The transducing lambda phages were also used to infect UV light irradiated cells. The SDS-gel electrophoretic analysis of the proteins synthesized in such cells revealed that the phage codes at least for four different E. coli proteins. Two proteins with molecular weights of 94,000 and 38,000 daltons cross-reacted with an anti PRS serum and were thus identified as the beta and alpha subunits of PRS, respectively. A third protein with a molecular weight of 22,000 daltons is identical with the ribosomal initiation factor IF3 (Springer et al., 1977b). The other protein (Mr 78,000) is still unidentified.     

Isolation and characterization of transducing coliphage fd carrying a kanamycin resistance gene.

The DNA segment (Tn903) with a size of 3100 nucleotide pairs which carries a gene specifying kanamycin resistance derived from a chimeric plasmid pML21 (Hershfield et al., 1976) was transposed to various sites on the filamentous phage fd DNA. Wild type fd can be restored by excision of Tn903 from the resulting hybrid DNA molecule. The fd DNA carrying Tn903 when converted to the mature phage particle, was capable of transducing the kanamycin marker, and its replicative form DNA could be maintained in a bacterial cell like a plasmid.     

Use of lambda pMu bacteriophages to isolate lambda specialized transducing bacteriophages carrying genes for bacterial chemotaxis.

A general method for constructing lambda specialized transducing phages is described. The method, which is potentially applicable to any gene of Escherichia coli, is based on using Mu DNA homology to direct the integration of a lambda pMu phage near the genes whose transduction is desired. With this method we isolated a lambda transducing phage carrying all 10 genes in the che gene cluster (map location, 41.5 to 42.5 min). The products of the cheA and tar genes were identified by using transducing phages with amber mutations in these genes. It was established that tar codes for methyl-accepting chemotaxis protein II (molecular weight, 62,000) and that cheA codes for two polypeptides (molecular weights, 76,000 and 66,000). Possible origins of the two cheA polypeptides are discussed.     

Isolation and characterization of nondefective transducing lambda bacteriophages carrying fla genes of Escherichia coli K-12.

In Escherichia coli K-12, 11 fla genes and a hag gene are located between his and uvrC, making two clusters at map positions 42.5 and 43.0 min. Nondefective transducing lambda phages for these genes were isolated. Low-frequency-transducing donors were constructed starting from lysogens of lambda cI857 in which the prophage is integrated at a secondary attachment site at 44 min on the E. coli map. Two strategies were used to delete the region between the prophage and the fla genes. Deletion mutants of the supD locus between fla and the prophage were isolated by selecting for loss of Su1+, an allele of supD. A strain with a deletion starting within the prophage and ending at a position close to the fla genes was isolated from heat-resistant derivatives of the lysogen. A lysogen of lambda b2 was then constructed in which the prophage had integrated at the site of the defective prophage by means of recombination with residual lambda deoxyribonucleic acid. From low-frequency-transducing lysate of the donor strains thus constructed, either directly or in combination with a procedure that extends the loci transduced, various lambda pfla's were isolated. lambda pflaL1 carries all nine fla genes at 43 min, and lambda pflaH14 carries hag and two fla genes at 42.5 min.     

Isolation and characterization of lambda b221poriCasnA, a plaque-forming specialized transducing phage carrying the origin of replication of the Escherichia coli chromosome

Summary A specialized transducing phage, b221poriCasnA has been isolated carrying oriC the origin of chromosomal replication of Escherichia coli. All phage genes required for lytic growth are retained, thus the phage is capable of lytic growth. The presence of the oriC locus confers upon infecting phage DNA the ability to replicate as a plasmid using only host DNA replication functions. The presence of both oriC and asnA markers has allowed the development of a plaque assay for origin function which can be used to identify mutants at these loci. Comparison of restriction endonuclease cleavage sites present on b221poriCasnA DNA to those on tis parent, b221 rex::Tn10 suggests the steps involved in the formation of the transducing phage.     

Isolation and characterization of a plaque-forming lambda bacteriophage carrying a ColE1 plasmid

A plaque-forming lambdaimm434 bacteriophage carrying the entire genome of colicinogenic factor E1 has been isolated and characterized. This phage, lambdaimm434ColE1, can lysogenize as a stable plasmid within a recombination-deficient Escherichia coli cell that lacks the normal attachment site for lambda phage. Furthermore, it has been found that lambdaimm434ColE1 phage carrying amber mutations in the O and P genes of the lambda genome, i.e., lambdaimm434OamPamColE1, behaves as a plaque-forming phage, and this finding suggests that the ColE1 factor DNA permits replication of the DNA of the plaque-forming phage.     

Genetic and physical studies of lambda transducing bacteriophage carrying the beta subunit gene of the Escherichia coli ribonucleic acid polymerase.

The prophage lambdac1857 was inserted into the bfe gene located near rif (the structural gene for the beta subunit of deoxyribonucleic acid [DNA]-dependent ribonucleic acid polymerase) on the Escherichia coli chromosome. Induced lysates (low-frequency transducing lysates) of such a lysogen contained defective lambda phage particles (lambdadrif+) that can specifically transduce the wild-type rif+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harboring both the wild-type and the mutant rif genes were isolated. Rec+ derivatives of these heterogenotes produce high-frequency transducing lysates that contain lambdadrif+ and normal active phages at a ratio of 1 to 2. The results of marker rescue experiments and of density determination with several transducing phages indicate that most of the late genes are deleted and replaced by a segment of the chromosomal DNA carrying the bfe-rif region. The length of the chromosomal segment seems to vary between approximately 0.5 and 0.6% of the total bacterial DNA among the three independently isolated lambdadrif+ phages. Electron microscopy of heteroduplex DNA consisting of one strand from lambdadrif+-6 and the other from lambdaimm-21 phages directly confirmed that most of the phage DNA of the "left arm" was replaced by the bacterial DNA. The heteroduplex study also demonstrated that the integration of prophage lambda into the bfe region occurred at the normal cross-over point within the phage attachment site.     

Plaque assay for lambda transducing phage carrying the E. coli metB gene

A halo plaque assay has been developed for the detection of nondefective lambda transducing phage carrying functional alleles of the metB gene of Escherichia coli K12. The assay is based upon the production of phage plaques on lawns of metB- bacterial cells which are supplemented with limiting amounts of methionine and upon the subsequent transduction of methionine-starved cells in the lawn surrounding the plaques. The resulting prototrophic transductants give rise to a halo of bacterial growth surrounding the plaque. A precise genotype can be ascribed to the characteristic morphologies of selected haloes. This technique has general application for all biosynthetic markers.     

Specialized transducing bacteriophage lambda carrying the structural gene for a major outer membrane matrix protein of Escherichia coli K-12.

A specialized transducing phage lambda carrying the structural gene for the OmpF protein, an outer membrane matrix protein, was isolated. The phage carries the 20.5--21-min region of the Escherichia coli K-12 chromosome and carries asnS, ompF, and aspC genes.     

Site-specificity of abnormal excision: the mechanism of formation of a specialized transducing bacteriophage lambda plac5.

Molecular mechanism of the specialized transducing bacteriophage lambda plac5 formation has been studied. Phage-bacterial DNA junctions in lambda plac5 DNA are localized and primary structure of regions of the abnormal excisional recombination leading to the phage formation is elucidated; the crossover region proved to be comparable with the central part of attP and attB sites (the core and the adjacent tetranucleotide) in length and degree of homology. Bacterial insert in lambda plac5 DNA is shown to end immediately after Z-Y spacer, the DNA not containing lacY gene segments. The data obtained led to the conclusion of site-specific (homologous) character of abnormal excision upon formation of lambda transducing bacteriophages. Possible mechanisms of the excision are discussed.     

Characterisation of a lambda transducing phage carrying the F conjugation gene traG

Summary A lambda transducing phage carrying the traGSTD genes of the E. coli K12 sex factor F was isolated by an in vivo technique, and characterized in ra complementation tests, by determining its restriction endonuclease fragment sizes, and by measuring heteroduplex molecules. The size and location on the F physical map of the tra transducing segment was thereby determined.Comparison of the proteins synthesized in UV-irradiated cells by this phage and by a derivative carrying the amber traG79 mutation, allowed the traG product to be identified as a protein of molecular weight 100,000. In the same experiments, the sizes of the traT and traD products made by the phage were also measured, being 25,000 and 85,000 daltons respectively.     

Isolation and characterization of mutations in the bacteriophage lambda terminase genes.

The terminase enzyme of bacteriophage lambda is a hetero-oligomeric protein which catalyzes the site-specific endonucleolytic cleavage of lambda DNA and its packaging into phage proheads; it is composed of the products of the lambda Nul and A genes. We have developed a simple method to select mutations in the terminase genes carried on a high-copy-number plasmid, based on the ability of wild-type terminase to kill recA strains of Escherichia coli. Sixty-three different spontaneous mutations and 13 linker insertion mutations were isolated by this method and analyzed. Extracts of cells transformed by mutant plasmids displayed variable degrees of reduction in the activity of one or both terminase subunits as assayed by in vitro lambda DNA packaging. A method of genetically mapping plasmid-borne mutations in the A gene by measuring their ability to rescue various lambda Aam phages showed that the A mutations were fairly evenly distributed across the gene. Mutant A genes were also subcloned into overproducing plasmid constructs, and it was determined that more than half of them directed the synthesis of normal amounts of full-length A protein. Three of the A gene mutants displayed dramatically reduced in vitro packaging activity only when immature (uncut) lambda DNA was used as the substrate; therefore, these mutations may lie in the endonuclease domain of terminase. Interestingly, the putative endonuclease mutations mapped in two distinct locations in the A gene separated by a least 400 bp.