Defective Transducing Phage Research Articles

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.

Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome.

A series of defective lambda transducing phage carrying genes from the lip-leuS region of the Escherichia coli chromosome (min 14 on the current linkage map) has been isolated. The phage defined the gene order as lac---lip-dacA-rodA-pbpA-leuS---gal. These included the structural genes for penicillin-binding protein 2 (pbpA) and penicillin-binding protein 5 (dacA) as well as a previously unidentified cell shape gene that we have called rodA. rodA mutants were spherical and very similar to pbpA mutants but were distinguishable from them in that they had no defects in the activity of penicillin-binding protein 2. The separation into two groups of spherical mutants with mutations that mapped close to lip was confirmed by complementation analysis. The genes dacA, rodA, and pbpA lie within a 12-kilobase region, and represent a cluster of genes involved in cell shape determination and peptidoglycan synthesis. A restriction map of the lip-leuS region was established, and restriction fragments were cloned from defective transducing phage into appropriate lambda vectors to generate plaque-forming phage that carried genes from this region. Analysis of the proteins synthesized from lambda transducing phage in ultraviolet light-irradiated cells of E. coli resulted in the identification of the leuS, pbpA, dacA, and lip gene products, but the product of the rodA gene was not identified. The nine proteins that were synthesized from the lip-leuS region accounted for 57% of its coding capacity. Phage derivatives were constructed that allowed about 50-fold amplification of the levels of penicillin-binding proteins 2 and 5 in the cytoplasmic membrane.

Regulation of phospholipid biosynthesis in Escherichia coli. Cloning of the structural gene for the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

The structural gene for the Escherichia coli biosynthetic sn-glycerol-3-phosphate (glycerol-P) dehydrogenase gpsA, was transferred from a defective transducing phage (lambda dcysE, gpsA) into the Eco RI site of plasmid pMB9 by recombinant DNA techniques. The recombinant plasmids suppressed the glycerol-P requirement of gpsA- mutants and strains bearing one such plasmid, pDC2, overproduced the glycerol-P dehydrogenase about 60-fold. The glycerol-P dehydrogenase from a strain bearing the pDC2 was purified 200-fold to homogeneity. This is contrasted to the 12,000-fold purification required to purify the enzyme from a wild type strain (Edgar, J. R., and Bell, R. M. (1978) J. Biol. Chem. 253, 6348-6353). The homogeneous enzyme purified from a strain bearing the pDC2 plasmide was strongly inhibited by glycerol-P (Ki of 2.5 microM). The introduction of the pDC2 plasmid into glycerol-P auxotrophs containing a Km-defective glycerol-P acyltranferase, defined by the plsB locus, caused a 60-fold overproduction of the glycerol-P requirement. This strongly suggests that the intracellular level of glycerol-P is stringently regulated in vivo by a mechanism involving feedback inhibition of the glycerol-P dehydrogenase by glycerol-P.

Construction, propagation and expression of simian virus 40 recombinant genomes containing the Escherichia coli gene for thymidine kinase and a Saccharomyces cerevisae gene for tyrosine transfer RNA

Recombinant simian virus 40 (SV40) virus genomes have been constructed in vitro by joining SVGT1, the segment of SV40 DNA between map co-ordinates 0.15 and 0.73 (clockwise), to either the Escherichia coli gene for thymidine kinase ( Ecotdk), or one of the Saccharomyces cerevisae genes for tRNA Tyr ( ScetyrG); the resulting recombinants were propagated in CV-1 monkey cells at 41 °C using tsA58 as a helper. The sEcotdk gene was obtained first as an approximately 20 kb ‡ DNA segment in a defective transducing phage, φ80 dtdk5, then as overlapping 1.85 and 2.35 kb segments in pMB9- Ecotdk, prior to insertion into SVGT1. The ScetyrG segment introduced into SVGT1 was a 1.25 kb DNA fragment contained in λgtl- ScetyrG, a recombinant that had been cloned previously by Olson et al. (1979). After infection of CV-1 monkey cells with the SVGT1- Ecotdk or SVGT1- ScetyrG hybrid viruses, RNA complementary to the exogenous DNA was produced. With SVGT1- Ecotdk the RNA homologous to the Ecotdk segment was heterogeneous, ranging in size from 1 to > 8 kb in length. At least 30 to 40% of this RNA was covalently joined to SV40-specific RNA. No E. coli thymidine kinase enzyme activity could be detected in the infected cells. By contrast, infection with SVTG 1- ScetyrG resulted in the formation of a transfer RNA-sized RNA, complementary to the tRNA Tyr coding sequence of the ScetyrG segment, as well as a population of heterogenous large RNAs. Since the formation of the tRNA-sized RNA occurred after infection with recombinants having the ScetyrG segments in either of the two alternative orientations in SVGT1, it is possible that transcription of the tyrG sequence is initiated within the cloned segment. ‡ Abbreviation used: kb, kilobase (10 3 bases)

Identification of transfer RNA suppressors in Escherichia coli: I. Amber suppressor su+2, an anticodon mutant of tRNA 2Gln

In order to isolate the gene for amber suppressor su +2 ( SupE) in Escherichia coli, a non-defective su +2-transducing phage lambda was isolated in three steps: first, deletion derivatives of F′ su +2 gal (λ) were selected, linking su +2 to the right-hand prophage attachment site, att λ PB′; second, these F′-factors were relysogenized by λ and defective transducing phages, λd su +2, were produced by induction; and third, non-defective λp su +2 transducing phages were produced by recombination of λd su +2 isolates with λ. Upon infection by λp su +2, the production of transferRNAs accepting glutamine and methionine was markedly stimulated. Fingerprint analysis of these tRNAs revealed that they consisted of normal tRNA 2 Gln, mutant tRNA 2 Gln and tRNA m Met. The mutant tRNA 2 Gln carried a singlebase alteration from G to A at the 3′-end of the anticodon. The production of tRNA 1 Gln was not stimulated by the infection of λp su +2. We conclude that the wild-type allele of su +2 ( SupE) is the structural gene for tRNA 2 Gln, and the su +2 amber suppressor was derived by a single base mutation, changing the anticodon from CUG to CUA, in one of the multi-copy genes for tRNA 2 Gln. The fact that λp su +2 also induces the production of tRNA m Met suggests that this tRNA is encoded in the same chromosomal region of E. coli as is tRNA 2 Gln.

The separation of phage promoter from bacterial [formula omitted] promoter for β-galactosidase expression in transducing phage λp [formula omitted

The replication defective transducing phage λp lac5 O29 P 3 carries a portion of the E. coli lac operon in the b 2 region of the lambda phage. This lac operon segment contains the lac promoter, the lac operator, and the β-galactosidase z gene, but does not contain the lac repressor i gene. The z gene can be expressed from both the inserted lac promoter and the phage promoter. When E. coli strain 594 ( z −, i +) or JC6256 (Δ lac ) is infected by λp lac5 O29 P 3 in the absence of additional cyclic AMP, β-galactosidase synthesis is shown to be expressed from the phage promoter. When 594 ( λ +) or JC6256 ( λ +) is infected by λp lac5 O29 P 3 in the presence of additional cyclic AMP and IPTG, β-galactosidase synthesis is shown to be expressed from the inserted lac promoter. The ability to separate the phage promoter from the inserted lac promoter for β-galactosidase expression will simplify the interpretation whenever λp lac 5 is used.

Specialized transducing phage lambda carrying the genes for coupling factor of oxidative phosphorylation of Escherichia coli: increased synthesis of coupling factor on induction of prophage lambda asn.

Studies were made of the synthesis of the coupling factor complex (F1--F0) of oxidative phosphorylation after prophage induction of a set of Escherichia coli strains lysogenic for defective transducing phage lambda asn, lambda uncA, or lambda bglC. The transducing phages had been isolated from a strain of E. coli carrying prophage lambda cI857 S7 within the bglB gene located near the unc gene cluster [Miki, T., Hiraga, S., Nagata, T. & Yura, T. (1978) Proc. Natl. Acad. Sci. USA 75, 5099--5103]. When lysogenic cells carrying lambda asn and lambda cI857 S7 were induced at high temperature, synthesis of the F1-ATPase portion of the complex increased to severalfold that of the noninduced cells. In contrast, no increase was observed upon thermoinduction of cells carrying lambda uncA or lambda bglC. The number of membrane sites that could bind purified F1-ATPase also increased significantly upon induction by lambda asn but not by lambda uncA or lambda bglC. In addition, F1-depleted membranes prepared from lambda asn-induced bacteria required more dicyclohexylcarbodiimide to seal the proton pathway than did those from noninduced bacteria. These results strongly suggest that lambda asn carries a set of bacterial genes coding for all the F1 polypeptides (the alpha, beta, gamma, delta, and probably the epsilon subunits) and at least some of the genes involved in formation of F0 polypeptides. Although lambda uncA carries the structural gene (uncA) for the alpha subunit of F1-ATPase, it apparently does not carry the whole set of F1--F0 genes.

Isolation of specialized transducing bacteriophages carrying the structural genes of the hexuronate system in Escherichia coli K-12: exu region

In Escherichia coli HfrH 58, isolated by Shimada et al., a heat-inducible phage has been integrated in a secondary attachment site. We have characterized tha nature of the lambda integration. The exuR regulatory gene is inactivated by prophage integration. Genetic and biochemical analysis indicated a gene order: uxaA-uxaC-exuT-(exuR')-lambdaNRAJ (exuR''). By induction of HfrH 58, one class of deletions extending into the exu region was obtained. Analysis of these deletions confirms the exu region topography and the regulatory mechanism of the hexuronate system previously described. It has been possible to regenerate a functional exuR gene by prophage exision. Various lambda transducing particles plaque-forming and defective transducing phages carrying the left part or the right part of the exu region, have been derived from the secondary site lysogen HfrH 58. A phage carrying the entire exuR region was constructed by a cross between these two types of phage. The construction and characterization of these exu transducing phages are presented.

Transposition of a DNA sequence determining kanamycin resistance into the single-stranded genome of bacteriophage fd

Derivatives of bacteriophages fd which transduce kanamycin resistance were selected after growth of the phage in an E. coli strain that carried transpoon 5 (Tn5). Different clones of transducing phage and their DNAs were characterized by gel electrophoresis, electron microscopy, and by their ability to multiply in the absence of helper phage. Integration of the intact transposon into the full size phage genome was correlated with an increase in size of the phage particle from 0.95 mu to 1.7 mu, and with the appearance in the phage DNA of the stem loop structure characteristic for single-stranded Tn5 DNA. In non-defective phages the site of insertion was mapped by heteroduplex analysis within the intergenic region of the phage genome. Defective transducing phages were characterized as an insertion of Tn5 into a phage gene, and/or as a partial deletion or duplication of phage and transposon DNA. The size of the transducing phage from different defective clones varied from 0.6 mu to 3.0 mu and was directly proportional to the DNA content. These results demonstrate that filamentous bacteriophage are highly capable to replicate and package very different amounts of foreign DNA.

Secondary-site attachment of coliphage lambda near the thr operon

Phage λ has been integrated at a secondary attachment site near the thr oporon of Escherichia coli. The integration caused a pleiotropic effect since both thrA and thrB enzyme activities were lost. Lysogenic, thr + revertants regained thrA and thrB activities although the enzymes were synthetized constitutively at low levels. Four classes of prophage deletion strains were isolated with deletions extending into trpR and the thr operon. Genetic and biochemical analyses indicated a gene order: trpR .. λ .. thrA .. B .. C. Defective transducing phage carrying thr A, B,C were also isolated.

Dual control of arabinose genes on transducing phage λd ara

A λ-φ80 hybrid derivative of the defective transducing phage φ80d ara (Gottesman & Beckwith, 1969) has most of its late genes replaced by Escherichia coli DNA containing an intact arabinose operon. In a cell lysogenic for this phage the arabinose operon on the phage responds normally to its regulatory gene araC and to the level of l-arabinose in the growth medium. However, as a consequence of transcriptional read-through from phage late genes to the arabinose operon, arabinose enzymes are also synthesized under the control of phage gene Q during growth of the phage in the absence of l-arabinose. We also show that the gene araC has the same transcriptional orientation as the phage late genes and the arabinose operon genes B, A and D. Since monomers of phage lysozyme and l-arabinose isomerase are produced in comparable quantities under phage late control, it follows that if there is transcription termination at the end of the gene araC it is no more than 90% efficient.

Autonomous replication of a defective transducing phage in Pseudomonas putida

The mandelate gene cluster for four enzymes has been transduced from Pseudomonas putida strain PRS1 (ATCC 12633) to the camphor-growing, mandelate-deleted strain PUG2 (ATCC 17452). Whereas crosses within strain PRS1 using phage pf16h2 yield principally phage-sensitive transductants, the interstrain crosses of PRS1 and PUG2 yield mostly immune ones, unstable for the mandelate and immune characters. The mandelate cluster is segregated at an approximate frequency of 10 −3 per bacterium per division during the cellular life cycle of the transductants. The immune heterogenotic transductants do not produce active vegetative phages on induction, but upon UV irradiation and superinfection with viable nontransducing phages they produce lysates which transduce at frequencies of 10 −5 to 10 −6 instead of the usual 3 × 10 −8. The release of transducing phages is dependent on the multiplicity of the superinfecting phages and on the dose of UV irradiation. Phage-sensitive transductants of PUG2 also occur, and these also produce high-frequency transducing lysates without producing viable phages. We conclude that the transducing particles consist of different amounts of the phage genome, with or without an immunity-controlling region, and lack some essential phage function. These defective phages have been termed pf dm in analogy to λ dg or Pl dl. In CsCl gradient the infective particles in high-frequency transducing lysates have a characteristic density of 1.519 g/cm 3, whereas three of the pf dm elements tested vary in density from 1.526 to 1.534 g/cm 3, suggesting compositional heterogeneity of the transducing particles. Phage-sensitive transductants of PUG strains can be cured of the defective pf dm particles by ultraviolet irradiation. They contain a satellite DNA band in addition to the bacterial DNA in a CsCl density gradient. These cells overproduce mandelate enzymes, suggesting that pf dm particles replicate autonomously in the phage-sensitive PUG cells.

Isolation and characterization of non-defective transducing elements of bacteriophage phi-80.

Abstract Several nondefective, tryptophan-transducing derivatives of bacteriophage φ80 (φ80 pt) have been isolated which carry various segments of the tryptophan operon. Tryp+ transductants arising from infection by φ80 pt phages are distinguished from those resulting from defective transducing phages by having rough edges and lytic halos. The most frequently encountered type of φ80 pt is that carrying all the tryptophan genes except that specifying anthranilate synthetase. This suggests that in most events leading to transducing phage formation, a specific region in the tryptophan operon interacts with a homologous prophage region. Mutations in the tryptophan operon including both the localized ones and those of the T1r trypdel type can be rapidly mapped employing transduction frequency tests with the various φ80 pt phages. No correlation was found between buoyant densities of the φ80 pt phages and the amount of tryptophan genes incorporated into their genomes. This suggests that during formation of transducing phage unequal exchange of bacterial and phage genetic material occurs.

Specialized transduction of tryptophan markers in Escherichia coli K12 by bacteriophage ∅80

A UV-inducible temperate phage ∅80, capable of lysogenizing on E. coli K12, has been isolated. Some of its properties have been investigated. Prophage ∅80 is closely linked to the try marker on the chromosome of strain K12. Specialized transduction of try + genes from tryptophan positive ( try +) to tryptophan negative cells ( try −) by ∅80 has been demonstrated. No markers except those in the try region have been transduced by this phage. The frequency of transduction resembles that of other transduction systems such as λ. Some try + transductants have been found to be heterogenotes. Heterogenotic cultures produce high frequency transducing (HFT) lysates, which contain both active phage ∅80 and defective transducing phage ∅80 dt carrying try genes. Each ∅80 dt strain carries the set of genes of the tryptophan operon, replacing a part of the phage genome which includes the immunity-related locus.

On the Mechanism of Unrepressed Galactosidase Synthesis Controlled by a Transducing Phage

Phage PI dl, a defective transducing phage derived from PI, carries a lac operon derived from a previous host (Luria et al., 1960). With PI dl phage carrying i−z+ alleles of the repressor and the β-D-galactosidase genes, peculiar regulatory effects are observed (Revel et al., 1961a, b, c). Notable among these are the constitutive synthesis of galactosidase following infection of i+z− cells (escape synthesis) and a similar constitutive synthesis following ultraviolet induction of bacteria of the genetic constitution i+z−(Pl dl i−z+) (derepressed synthesis). Constitutive enzyme production, at lower levels, is observed also with PI dl i+z+ phage in analogous situations. When these findings were first reported, two interpretations were considered: (a) a topological effect, by which a repressor would not be fully effective on the operator site when this was part of a vegetative phage element; or (b) a multiplicity effect, that is, a failure of repression when the ratio...