DNA cleavage of lambda and phi 80 transducing phages carrying the argA, argECBH, argF and argI operons of Escherichia coli K-12 with the restriction endonucleases EcoRI, SmaI and HindIII.

DNA isolated from each of the seven arginine transducing phages lambdaargA2cI857susS7, phi80ppc argECBH, phi80argF, phi80argF ilambdacI857, lambdaargF2, lambdaargF23 and lambdaargI valScI857susS7 has been specifically cleaved by the restriction endonucleases EcoRI, SmaI and HindIII. The DNA fragments resulting from single, and in some cases, double endonuclease digests were separated by electrophoresis in agarose and also in polyacrylamide gel. The electrophoretic patterns thus obtained were compared with those produced by digestion of DNA isolated from the corresponding lambda and phi80 parental phages. The majority of cleavage sites produced by the action of these restriction enzymes on arginine transducing DNA have been physically mapped.     

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.     

New transducing phage with RNA polymerase beta- and beta'-subunit genes derived from a hybrid phage lambda att80: isolation, genetic analysis and physical mapping]

A hybrid lambda att 80 phage with the genetic structure lambda (A-J) phi 80 (att-int-xis) imm lambda..cI857s7 is shown to be a convenient vector for creating transducing phages. On the one hand, the restriction analysis indicates that it has 3 restriction sites for EcoRI in comparison with 5 and 9 sites for parental phages lambda and phi 80 respectively. On the other hand, its buoyant density is less than that of phage lambda and under centrifugation it is easier separated from the phage transducing particles. When lambda att 80 prophage was excluded from the bfe locus of Escherichia coli, transducing phages with genes of two RNA polymerase beta-subunits (rpoB and rpoC) were isolated. To identify the latter, a convenient genetic test was worked out. A physical map of lambda att 80 drifd 35 transducing phage, carrying rpoB and rpoC genes has been constructed using endonucleases EcoRI and HindIII. A comparison of this map and the corresponding maps of transducing phages lambda drifd 18 and lambda drifd 47, studied earlier, led to the discovery of two integration sites of phage lambda within the locus bfe spaced apart by about 1800 nucleotide pairs. At all the sites both phages (lambda and lambda att 80) have integrated in the locus bfe in the counter clockwise order.     

Isolation of specialized lambda transducing bacteriophages for flagellar genes (fla) of Escherichia coli K-12.

Specialized transducing lambda phages carrying the region III flagellar genes (fla) of Escherichia coli K-12 were isolated by a new method. A strain carrying both a cryptic lambda prophage near the his genes and a deletion of the attlambda gene was used as a starting strain. The lysogen of lambdacI857pga18-bio69 was isolated in which the prophage was integrated within the lambda cryptic genes by means of recombination with the residual lambda DNA. The strains with deletions starting within the prophage and ending in these fla genes were selected from among the heat-resistant survivors of the lysogen. They were then infected with heat-inducible and lysis-defective lambda phages and, thus, specialized transducing phage lines for hag and fla were obtained. High-frequency transfer lines of rare phages carrying the fla genes were isolated by inducing a strain carrying a heat-inducible lambda prophage near the his genes and selecting by transduction of a fla deletion strain. Preliminary characterization of these transducing phages is also reported.     

Isolation of Specialized Transducing Bacteriophages for Gluconate 6-Phosphate Dehydrogenase (gnd) of Escherichia coli

Specialized transducing phages for gluconate 6-phosphate dehydrogenase (gnd), a constitutive enzyme in Escherichia coli, have been isolated using a method previously described for other genes. The gnd-his region, carried on an F' episome, was first transposed to tonB. Rare phages carrying gnd were selected, by transduction, from phi80 lysogens of these strains; one phage also carried his (phi80gndhis). From the transductants, high-frequency transducing lysates were obtained; low multiplicity of infection then yielded defective lysogens. tonB deletion analysis of the phi80dgndhis lysogen shows the order of genes in the prophage to be imm80...hisOGD...gnd; according to a marker rescue experiment most phage late genes have been replaced by bacterial deoxyribonucleic acid. A heat-inducible, lysis-defective lambda-phi80 hybrid derivative of phi80dgndhis has been prepared.     

Insertion of bacteriophage SP beta into the citF gene of Bacillus subtilis and specialized transduction of the ilvBC-leu genes.

We isolated a strain of Bacillus subtilis in which the SP beta c2 prophage is inserted into the citF (succinate dehydrogenase) gene. Defective specialized transducing particles for the ilvBC-leu genes were isolated from phage-induced lysates of this lysogen. We isolated a group of phages that differ in the amount of genetic material they carry from this region. Also, we incorporated mutant ilv and leu alleles into the genomes of several transducing phages. Our phage collection enables us to identify the cistron of new ilv and leu mutations by complementation analysis. In this process we discovered a fourth leu cistron, leuD. Characterization of the phages confirmed the published gene order: ilvB-ilvC-leuA-leuC-leuB; leuD lies to the right of leuB.     

Specialized Transducing Phages Derived from Phage P22 That Carry the proAB Region of the Host, SALMONELLA TYPHIMURIUM: Genetic Evidence for Their Structure and Mode of Transduction

Two independently isolated specialized transducing phages, P22 pro-1 and P22pro-3, have been studied. Lysates of P22pro-1 contain a majority of transducing phages which can go through the lytic cycle only in mixed infection; these defective phages transduce by lysogenization in mixed infection and by substitution in single infection. A few of the transducing phages in P22pro-1 lysates appear to be non-defective, being able to form plaques and to transduce by lysogenization in single infection. Transduction by P22pro-3 lysates is effected by non-defective transducing phages, which transduce by lysogenization; these lysates also contain a majority of defective phages which do not co-operate in mixed infection.

The P22 pro-1 genome is thought to contain an insertion of bacterial DNA longer than the terminal repetition present in P22 wild type, so that at maturation a population of differently defective phages is produced. The exact structure of the P22pro-3 genome is open to conjecture, but it seems clear that the insertion of bacterial DNA is smaller than that in P22pro-1. Both P22pro-1 and P22pro-3 are defective in integration at ataA under non-selective conditions, although both integrate on medium that lacks proline.

     

Isolation and characterization of plaque-forming lambdadnaZ+ transducing bacteriophages.

The Escherichia coli dnaZ gene, a deoxyribonucleic acid (DNA) polymerization gene, is located 1.2 min counterclockwise from purE, at approximately min 10.5 on the E. coli map. From a lysogen with lamdacI857 integrated at a secondary attachment site near purE, transducing phages (lambdadnaS+) that transduced a dnaZts (lambda+) recipient to temperature insensitivity (TS+) were discovered. Three different plaque-forming transducing phages were isolated from seven primary heterogenotes. Genetic tests and heteroduplex mapping were used to determine the length and position of E. coli DNA within the lambda DNA. Complementation tests demonstrated that the deletions in all three strains removed both att P and the int gene, i,e., DNA from both prophage ends. Heteroduplex mapping confirmed this result by demonstrating that all three strains had deletions of lambda DNA that covered the b2 to red region, thereby removing both prophage ends. Specifically, the deletions removed lambda DNA between the points 39.3 to 66.5% of lambda length (measured in percent length from the left and of lambda phage DNA) in all three strains. The three strains are distinct, however, because they had differing lengths of host DNA insertions. These phages must have been formed by an anomalous procedure, because standard lambda transducing phages are deleted for one prophage end only. In lambdagal and lambdabio strains, the deletions of lambda DNA begin at the union of prophage ends (i.e., position 57.3% of lambda length) and extend leftward or rightward, respectively (Davidson and Szybalski, in A, D. Hershey [ed.], The Bacteriophage Lambda, p. 45-82, 1971). Models for formation of the lambdadnaZ+ phages are discussed.     

Construction of φ80 dhis Carrying Salmonella typhimurium Histidine Operon Mutations

Starting with a previously isolated F'his episome and phi80 dhis imm(lambda) cI857 transducing phage, we have constructed recombinant elements bearing previously isolated his mutations from Salmonella typhimurium. These phages were constructed as sources of deoxyribonucleic acid for in vitro biochemical experiments on gene regulation. The manipulation of genes between S. typhimurium and Escherichia coli described here may be useful in studying other S. typhimurium operons.     

The transposable element Tn3 promotes general recombination at the neighboring regions

Transposon Tn3 was inserted into a tRNA operon of the amber suppressor Su+2 on a transducing phage (lambda hcI857nin5pSu+2) by selecting phages with ampicillin resistance and Su- phenotypes. In a strain thus obtained, Tn3 was inserted between the promoter and the first tRNA gene of the operon, which was determined by DNA sequencing. The Su+2 tRNA operon on the transducing phage consisted of two tRNA genes for tRNA(Met) and Su+2 tRNA(2Gln), which was a deletion derivative of the supB-E tRNA operon of E. coli containing seven tRNA genes in the order of promoter-Met-Leu-Gln1-Gln1-Met-Gln2-Gln2. Proliferating the lambda hcI857nin5pSu+2::Tn3 in E. coli cells, a number of phages which had lost Tn3 were isolated, and their tRNA gene compositions as well as the DNA structures of the tRNA operon were analyzed. In many cases the tRNA genes which had been deleted from the original transducing phage were regained from the chromosomal supB-E operon. Thus the loss of Tn3 from the phages was not due to excision of the transposon but due to the replacement of a portion of the tRNA operon, including Tn3, with the host homologous region that did not contain Tn3. This type of replacement takes place rather efficiently as a consequence of Tn3 insertion, owing to the general recombination occurring between homologous tRNA genes of phage and host chromosomes in the presence of either host recA or phage red. No such enhanced recombination in a similar cross between phage and host chromosomes was observed with the Tn3 present in the trans position on an independent plasmid. We conclude that inserting Tn3 in cis promotes general recombination in the neighboring regions. Possible mechanisms for this new type of genetic effect of Tn3 are discussed. During the course of this study, a natural defective mutation (T11) was also detected in one of the duplicated tRNA(2Gln) genes in an E. coli K12 strain we used.     

Isolation of Lambda Transducing Phage with the bio Genes Inserted between Lambda Genes P and Q

Plaque-forming, biotin-transducing phages were constructed with the bio genes inserted between lambda genes P and Q. These phages were isolated for the eventual aim of fusing the lambda Q gene to the bio operon. The following steps were used to construct these phages: A defective temperature-sensitive lysogen was constructed with the bio genes adjacent to and to the left of lambda genes beta NcI857OPQSRA. Heat-resistant survivors were screened for deletions with endpoints in the bio operon and to the right of lambda P and to the left of lambda A. Five of approximately 1,600 heat-resistant survivors had these properties. Two had the gene order bioAB .... lambda QSRA. When these two strains were lysogenized with lambda cI857b221 and heat induced, the desired transducing phages were obtained. We characterized these phages and studied one in detail. Two-thirds of the plaque-forming transducing phages isolated carried the entire bioB gene and only part of the bioA gene, and one-third carried the entire bioA and bioB genes. The phages isolated lost the bio genes upon propagation, indicating that they contain a partial duplication of phage genes. The duplication was shown not to involve the entire lambda Q gene in one of these phages, lambda bioq1b221. A recombinant of this phage, lambda Nam7am53c17b221, failed to form plaques under biotin-derepression conditions. We conclude that if the lambda Q gene was fused to the bio operon in this phage, not enough lambda Q gene product was made to allow phage propagation.     

Evolutionary drift of the argF and argl genes. Coding for isoenzyme forms of ornithine transcarbamylase in E. coli K12.

Considerable genetic drift has occurred during the evolution of the two genes, argF and argl, which individually code for isoenzyme forms of ornithine transcarbamylase in E. coli K12. The use of the experimental protocol described in this work established that between 25-40% of the base pairs in the genes argF and argl have changed since they diverged from a hypothetical ancestral gene. The extent of divergence of the genes was determined by mRNA-DNA hybridization utilizing arginine transducing DNA as a hybridization probe and mRNA prepared in vivo from appropriate bacterial strains and with mRNA synthesized in vitro using template DNA isolated from the specialized transducing phages gammacI857dargl and phi80dargF.     

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.     

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.     

The molecular structure of the transducing particles of Salmonella phage P22. II. Density gradient analysis of DNA

Summary CsCl density gradient analysis showed that the DNA of plaque forming particles ofSalmonella phageP22 is lighter than the host DNA. The DNA of transducing phages exhibits an intermediate density, but close to host DNA. BU labelling of DNA synthesized in the cells after phage infection resulted in a density increase of transducing DNA of about 0.004 gxcm^-3, whereas infectious DNA increased by about 0.045 gxcm^-3. Shearing of isolated DNA molecules from unlabelledP22 lysates demonstrated that transducing DNA consists of two pieces of DNA of different density: 90% stem from the bacterial host whereas 10% are phage DNA and therefore responsible for the BU lable in transducing phages.     

Physical and genetical analysis of bacteriophage T4 generalized transduction

This report describes a comparison of the efficiency of transduction of genes in E. coli by the generalized transducing bacteriophages T4GT7 and P1CM. Both phages are capable of transducing many genetic markers in E. coli although the frequency of transduction for particular genes varies over a wide range. The frequency of transduction for most genes depends on which transducing phage is used as well as on the donor and recipient bacterial strains. Analysis of T4GT7 phage lysates by cesium chloride density gradient centrifugation shows that transducing phage particles contain primarily bacterial DNA and carry little, if any, phage DNA. In this regard transducing phages P1CM and T4GT7 are similar; both phages package either bacterial or phage DNA but not both DNAs into the same particle.     

Transposition of the lac Region to the gal Region of the Escherichia coli Chromosome: Isolation of λlac Transducing Bacteriophages

Defective and plaque-forming lambdalac transducing phages have been isolated from bacterial strains in which the lactose operon has been genetically transposed to the galactose locus.     

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.     

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 att lambda 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' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.