Formation of lambda transducing phage in vitro: involvement of DNA gyrase

We found that transducing phages carrying the gal or bio regions of the Escherichia coli genome were formed during in vitro packaging of endogenous lambda DNA. Structural analysis of the transducing phage genomes indicated that they were formed by abnormal excision of lambda prophage. Formation of transducing phages was stimulated by oxolinic acid, an inhibitor of DNA gyrase, implying that DNA gyrase participates in the abnormal excision of lambda prophage. When pBR322 DNA was added to the reaction mixture, transducing phages into which pBR322 had been inserted were produced at a high frequency. This reaction was also stimulated by oxolinic acid. Sequence analyses revealed that pBR322 is inserted into the sites of abnormal excision of the prophage. These results show that transducing phages can be formed by DNA gyrase-dependent illegitimate recombination in an in vitro system and that secondary recombination takes place frequently at the site where the first recombination occurs.     

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 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.     

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.     

Molecular cloning of four tricarboxylic acid cyclic genes of Escherichia coli.

A fragment of DNA (3.1 kilobases [kb]) from a ColE1 Escherichia coli DNA hybrid plasmid containing the bacterial citrate synthase gene (gltA) was subcloned in both orientations into phage lambda vectors by in vitro recombination. The resulting phages were able to transduce gltA and, as prophages, complemented the lesion of a gltA mutant, showing that a functional gltA gene is contained in the 3.1-kb fragment. The segment of E. coli DNA cloned in these lambda gltA phages was extended in vivo by prophage integration and aberrant excision in the gltA region. Plaque-forming derivatives, carrying up to three additional tricarboxylic acid cycle genes, succinate dehydrogenase (sdh), 2-oxoglutarate dehydrogenase (sucA), and dihydrolipoamide succinyltransferase (sucB), were isolated and characterized by their transducing and complementing activities with corresponding mutants, and the order of the genes was confirmed as gltA-sdh-sucA-sucB. Physical maps of a variety of the transducing phages showed that the four tricarboxylic acid cycle genes are contained in a 12.8-kb segment of bacterial DNA. The four gene products, plus a possible succinate dehydrogenase small subunit, were identified in postinfection labeling studies, and the polarities of gene expression were defined as counterclockwise for gltA and clockwise for sdh, sucA, and sucB, relative to the E. coli linkage map.     

Precise mapping of the gpp gene involved in guanosine tetraphosphate synthesis and ilvC-gpp deletion in the region of the Escherichia coli chromosome

Using the set of transducing lambda phages the gpp gene, responsible for pppGpp to ppGpp conversion, was localized between rep and trxA genes on 85 min of the Escherichia coli genetic map. Taking advantage of the Tn10 transposon inserted into the adjacent ilvY locus, we deleted the region of E. coli chromosome covering ilvC, rep and gpp genes. The metabolism of (p)ppGpp in the deletion-containing cells confirms that the product of the gpp gene, guanosine pentaphosphatase, is not the only enzyme, responsible for pppGpp degradation and ppGpp synthesis.     

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.     

Metabolism of arginine-specific messenger ribonucleic acid in Escherichia coli K-12.

Ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization was employed for the determination of the level of messenger RNA (mRNA) transcribed from seven of the nine genes of the arginine regulon of Escherichia coli K-12. The quantity of RNA complexing with each of the separated DNA strands of the argA, argF, argE, and argCBH operons carried on specialized transducing phages was measured. The derepressed:repressed ratio of mRNA formed in vivo was found to vary between about 3 and 4 when measured by hybridization to DNA isolated from specialized transducing phages carrying the argA, argE, argCBH, argF, and argI operons.     

Cosmid DNA packaging in vivo

The packaging of cosmid DNA into phage particles during phage lambda growth is described. Evidence is presented supporting the work of others that cosmid transducing phages contain linear multimers of cosmid DNA in which the number of cosmid copies is that required to make a packagable DNA length (greater than 0.77 of the lambda DNA length). The yield of cosmid transducing phages declines sharply as the number of cosmid copies required to make a packagable DNA length increases. The cosmid DNA replication that produces the packaging substrate shares with lambda rolling-circle replication a dependence on the lambda gam gene product.     

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 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.     

A new cloning system for Bacillus subtilis comprising elements of phage, plasmid and transposon vectors

A new cloning system for Bacillus subtilis was devised which makes use of a combination of Tn917-containing phage SP beta derivatives and Tn917-containing Escherichia coli-B. subtilis shuttle plasmids. This system allows the initial cloning of genes in single copy, via 'prophage transformation', with a selection for complementation of mutational defects in B. subtilis hosts and permits subsequent transfer of the cloned material by homologous recombination to low-copy and high-copy vectors that replicate in both B. subtilis and E. coli. Because cloned sequences are adjacent to pB322-derived DNA in the recombinant phages, inserts can also be 'rescued' directly from the phage DNA after digestion with appropriate restriction enzymes, circularization of the fragments by ligation and transformation of an E. coli recipient. Two genomic libraries of B. subtilis chromosomal Sau3A-generated partial-digest fragments in the size ranges of 5-8 kb and 8-10 kb were constructed and screened for the complementation of mutations aroI906, cysA14, dal-1, glyB133, metC3, purA16, purB33, thrA5, trpC2 and recE4. In all cases, specialized transducing phages carrying inserts that complemented the selected markers were recovered. Inserts complementing the dal-1 and trpC2 mutations could be transferred from recombinant phages to Tn917-containing plasmids by homologous recombination without in vitro subcloning. Another insert complementing the purB33 mutation was rescued directly into E. coli from a recombinant phage DNA.     

Autogenous regulation of RNA polymerase beta subunit synthesis in vitro.

The effects of Escherichia coli RNA polymerase and its subassemblies and subunits on the in vitro synthesis of beta subunit directed by DNA from a lambda transducing phage lambdadrif+-6 were investigated. This phage carries the structural gene (rpoB) for beta subunit as well as the genes for EF (translation elongation factor)-Tu, some ribosomal proteins, and stable RNAs of the E. coli chromosome. Among the RNA polymerase proteins examined, the two oligomers, holoenzyme and alpha2beta complex, repressed the synthesis of only the beta subunit but not of other proteins encoded by the phage DNA. The results indicate that the expression of at least the betabeta' (rpoBC) operon is under autogenous regulation, in which both holoenzyme and alpha2beta complex function as regulatory molecules with repressor activity.     

Isolation of generalized transducing bacteriophages for uropathogenic strains of Escherichia coli

The traditional genetic procedure for random or site-specific mutagenesis in Escherichia coli K-12 involves mutagenesis, isolation of mutants, and transduction of the mutation into a clean genetic background. The transduction step reduces the likelihood of complications due to secondary mutations. Though well established, this protocol is not tenable for many pathogenic E. coli strains, such as uropathogenic strain CFT073, because it is resistant to known K-12 transducing bacteriophages, such as P1. CFT073 mutants generated via a technique such as lambda Red mutagenesis may contain unknown secondary mutations. Here we describe the isolation and characterization of transducing bacteriophages for CFT073. Seventy-seven phage isolates were acquired from effluent water samples collected from a wastewater treatment plant in Madison, WI. The phages were differentiated by a host sensitivity-typing scheme with a panel of E. coli strains from the ECOR collection and clinical uropathogenic isolates. We found 49 unique phage isolates. These were then examined for their ability to transduce antibiotic resistance gene insertions at multiple loci between different mutant strains of CFT073. We identified 4 different phages capable of CFT073 generalized transduction. These phages also plaque on the model uropathogenic E. coli strains 536, UTI89, and NU14. The highest-efficiency transducing phage, ΦEB49, was further characterized by DNA sequence analysis, revealing a double-stranded genome 47,180 bp in length and showing similarity to other sequenced phages. When combined with a technique like lambda Red mutagenesis, the newly characterized transducing phages provide a significant development in the genetic tools available for the study of uropathogenic E. coli.     

Isolation of specialized transducing bacteriophages carrying deletions of the regulatory region of the Escherichia coli K-12 tryptophan operon.

A lysogen of a wild-type strain of Escherichia coli K-12 carrying a heat-inducible lambda-phi80 hybrid prophage was induced to yield transducing phages carrying all of the structural genes of the tryptophan operon. The presence or absence of elements of the trp regulatory region was determined by examining the effects of lambda genes N and cI on trp gene expression. The phages were further characterized by transduction studies and by examining anthranilate synthetase (EC 4.1.3.27) (TRYPE+D) synthesis in the presence of the lambda cI product. A number of phages deleted for the trp promoter were found. Recombination studies between trpOc bacteria and the transducing phages have yielded information that can be used to order the trp end points of some phages and to provide an estimate of the size of the trp promoter region.     

Relative map location of the rep and rho genes of Escherichia coli.

The rep gene of Escherichia coli was mapped between ilvC and rho by three-factor P1 transductional crosses and also by complementation with a set of lambda transducing phages that contain known amounts of bacterial DNA linked to ilvC. The physical distance between ilvC and rep and between rep and rho were calculated with an accuracy of +/- 0.4 kilobase to be 0 less than or equal to ilvC-rep less than or equal to 3.4 kilobases and 2.0 less than or equal to rep-rho less than or equal to 6.0 kilobases. It was shown that rho-15 is Gro+ for phage ST-1. An ilv::Tn10 mutation was located in ilvY.