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 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.     

Replication defective RP4 plasmids recovered after chromosomal integration

pHH6000 is a composite replicon made by the in vitro ligation of the IncP plasmid RP4 to a fragment of bacteriophage lambda capable of autonomous replication. Derivatives were selected in which it had integrated into the Escherichia coli chromosome by homologous recombination with the resident lambda prophage, and plasmids were subsequently regenerated from the integrated molecules. Although of the same molecular size as pHH6000, all had altered properties: those recovered from the chromosome of cells simultaneously carrying a distinguishable autonomous IncP plasmid showed a 100- to 1000-fold reduction in their ability to become established in a lambda lysogen; those regenerated from cells with no autonomous IncP plasmid were no longer RP4 replicons, now being dependent on replication functions encoded by the lambda DNA they carry and therefore unable to form a plasmid in a lambda lysogen. This second class of plasmids still exhibited normal RP4 incompatibility and stability even though neither property is encoded by the lambda replicator DNA. It was concluded that expression of RP4 incompatibility and partitioning control do not require an intact RP4 replicon. The data also suggest that the presence in the chromosome of a normal RP4 molecule may be deleterious to the host, although the manner in which the integrated molecules were obtained allows other explanations. The composite plasmids replicating from cloned lambda genes should be useful in analysis of the regulated distribution of RP4 molecules at cell division.     

Isolation of an aroF-lac plasmid by recombination in vivo.

An aroF-lac operon fusion was transferred from a lambda aroF-lac prophage onto a plasmid carrying a 'Mu cts trp-lac fusion by recombination in vivo.     

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.     

Functional analysis of hybrid plasmids carrying genes for lambda site-specific recombination

A number of hybrid plasmids, carrying lambda genes involved in site-specific integrative recombination, have been constructed in vitro. Analysis of protein synthesis in Escherichia coli minicells has shown that Int protein is synthesized only when int gene is expressed constitutively. The plasmids RSF2124::lambda-CD, RSF2124::lambda-Cint-c57, and pInt lambda were able to integrate into the chromosome of E.coli at the attB. The integration of hybrid plasmids into the genome of bacteria has also been shown for polA1 strains restricting the autonomous replication of ColE1 type plasmids. Genetic markers of hybrid plasmids are maintained in polA1 bacteria for at least 50 generations under nonselective conditions. The Southern blotting experiments using [32P]pBR322 DNA and EcoRI fragments of E. coli polA1 chromosome carrying integrated plasmid pInt lambda demonstrated that in this strain hybrid plasmids can be observed only when integrated into the attB of the chromosome according to Campbell's model of integration. In the cells, where autonomous replication of plasmids is possible, they can be observed both in extrachromosomal and integrated states. The integration of the ColE1 replication origin into the chromosome of bacteria is not lethal for the cells. Only attP and the int gene of lambda are necessary for the integration of hybrid plasmids under conditions of effective int gene expression. If the level of Int protein synthesis is high enough, the prophage excision can be observed in the absence of Xis product. The six-fold decrease of Int protein concentration in the cell (in case of pInt lambda 2 as compared to pInt lambda 1) is critical both for integration and excision.     

Isolation of a Specialized Lambda Transducing Bacteriophage Carrying the Beta Subunit Gene for Escherichia coli Ribonucleic Acid Polymerase

A heat-inducible lysis-defective lambda prophage has been integrated directly into the E. coli chromosome at a site (bfe) very closely linked to the ribonucleic acid polymerase mutation rif(d), a dominant rifampin resistance allele. This unusual lysogen has facilitated the isolation of specialized transducing phages conferring rifampin resistance to sensitive cells, and carrying at least the beta subunit gene of RNA polymerase in intact form.     

Isolation and analysis of aroFo mutants by using an aroF-lac operon fusion

The lac structural genes were fused to the regulatory region of the aroF-tyrA operon so that the expression of beta-galactosidase was regulated by the tyrR+ gene product. Transducing phage carrying the aroF-lac fusion were isolated, and a lambda aroF-lac lysogen was used to select for aroFo mutants. A plasmid vector was constructed onto which the aroFo mutations were transferred by recombination in vivo.     

Isolation of Plaque-Forming, Galactose-Transducing Strains of Phage Lambda

Plaque-forming, galactose-transducing lambda strains have been isolated from lysogens in which bacterial genes have been removed from between the galactose operon and the prophage by deletion mutation.—A second class has been isolated starting with a lysogenic strain which carries a deletion of the genes to the right of the galactose operon and part of the prophage. This strain was lysogenized with a second lambda phage to yield a lysogen from which galactose-transducing, plaque-forming phages were obtained. These plaque-forming phages were found to be genetically unstable, due to a duplication of part of the lambda chromosome. The genetic instability of these partial diploid strains is due to homologous genetic recombindation between the two identical copies of the phage DNA comprising the duplication. The galactose operon and the duplication of phage DNA carried by these strains is located between the phage lambda P and Q genes.     

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.     

Prophage lambda induction caused by mini-F plasmid genes

Summary When bacterial cells harboring a temperaturesensitive replication plasmid, which carries the particular ccd segment of a mini-F plasmid, are transferred to 42°C, cell division is inhibited after incubation for an appropriate time. The inhibition occurs, when the copy number of the plasmid decreases to become critically low, about one per cell (Ogura and Hiraga 1983 b). In phage lysogens carrying this type of plasmid, the prophage is induced in a small portion of the cell population under the same conditions, in addition to the inhibition of cell division in most of cells. The prophage induction, but not the inhibition of normal cell division, depends on normal recA function. Both induction of prophage and inhibition of cell division are suppressed by the simultaneous presence of a replication proficient plasmid carrying the ccdA gene. We discuss molecular mechanisms of the ccd function that couples host cell division to plasmid proliferation and induces the prophage. Additionally, we propose a hypothesis that the ccd mechanism of F plasmid contributes to indirect induction of prophage by an F plasmid damaged by UV-irradiation and then introduced into a lysogen via conjugation.Abbreviations kb kilobase pairs. m.o.i., multiplicity of infection     

Phasmids: hybrids between ColE1 plasmids and E. coli bacteriophage lambda

Plasmids carrying cloned lambda att sites may be integrated into the bacteriophage genome by the site-specific recombination mechanism of lambda. The cross, referred to as "lifting" the plasmid, requires mixed infection of an Escherichia coli strain carrying the plasmid with two appropriately constructed "lifting" lambda phages. One phage donates a short left arm and the other donates a short right arm. These two short arms are of insufficient length to produce a viable phage genome and yield no recombinants when crossed on standard bacteria. However, viable recombinants are obtained when the genome length is extended by integration of one or more plasmids. We call these recombinants phasmids. They contain multiple att sites introduced at the ends of the integrated plasmids, and in the presence of integrase, recombination between these att sites can be exploited to effect release of the plasmid components. These novel genetic elements can be used in a variety of ways as vectors in genetic manipulation experiments. Sequences cloned in phasmids may be studied as a component of either a plasmid and or of a phage, and easily interconverted between the two states.     

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.     

Behavior of λ Bacteriophage in a Recombination Deficient Strain of Escherichia coli

The behavior of lambda phage in the Rec(-) strain JC-1569 is compared with that in the Rec(+) strain JC-1557. No difference deemed significant was noted in the adsorption rate, latent period, burst size, frequency of lysogenization, and frequency of vegetative phage recombination. The location of the prophage and its mode of insertion in the Rec(-) lysogen of wild-type lambda (lambda(+)) were inferred to be normal from the results of conjugational crosses. Spontaneous and ultraviolet (UV) irradiation induction of lambda(+) were markedly reduced in the Rec(-) lysogen. On the other hand, thermal induction of a mutant lambda (lambdacI857) lysogen of the Rec(-) strain was not reduced and was only slightly affected by UV irradiation. Phage subject to inhibition by lambda immunity failed to multiply in UV-irradiated cells of the Rec(-) lambda(+) lysogen, whereas those not inhibited by this immunity did multiply. It was concluded that the failure of UV to induce lambda(+) in the Rec(-) lysogen was not due to damage to the prophage, but rather to the inability of the irradiated cells to respond by lifting immunity. Preliminary evidence indicates that a single mutation confers recombination deficiency and the inability to lift immunity after UV irradiation. Possible relationships between recombination and the lifting of immunity are enumerated.     

Isolation of a λdv Plasmid Carrying the Bacterial gal Operon

A lambdadvgal plasmid carrying genes for controlled plasmid replication from phage lambda and the bacterial gal operon was isolated as a deletion mutant of phage lambdagalq4, which carries the gal operon between lambda genes P and Q. The plasmid DNA was found in cell extracts as covalently closed circular molecules. The plasmid was characterized by using genetic crosses, digestion with the specific endonuclease EcoRI, sucrose gradient centrifugation, and electron microscopy. In one clone analyzed, the plasmid was a complete dimer (O(lambda)P(lambda)galO(lambda)P(lambda)gal); in a subclone derived from it, the plasmid was a partial dimer with only one copy of gal (O(lambda)P(lambda)O(lambda)P(lambda)gal). The partial dimer may be a recombination product of the complete dimer, since test crosses show that the gal and lambda sequences in the plasmid can be separated by recombination. Analyses of the EcoRI digests of plasmid DNAs indicated one cleavage site per lambda gene sequence and none in the gal operon. A lambdadvgal monomer was approximately 6.7 x 10(6) daltons and the lambda gene and gal components were 3.9 x 10(6) and 2.8 x 10(6) daltons, respectively. The lambdadvgal plasmid can be introduced into a new bacterial host by transfection at an efficiency of 10(-6) per DNA molecule.     

Rapid construction of adenoviral vectors by lambda phage genetics

Continued improvements of adenoviral vectors require the investigation of novel genome configurations. Since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid DNA, it is possible to separate genome construction from virus production. In this way failure to generate a virus is not associated with an inability to generate the desired genome. We have developed a novel lambda-based system that allows rapid modification of the viral genome by double homologous recombination in Escherichia coli. The recombination reaction and newly generated genome may reside in a recombination-deficient bacterial host for enhanced plasmid stability. Furthermore, the process is independent of any restriction endonucleases. The strategy relies on four main steps: (i) homologous recombination between an adenovirus cosmid and a donor plasmid (the donor plasmid carries the desired modification[s] and flanking regions of homology to direct its recombination into the viral genome); (ii) in vivo packaging of the recombinant adenoviral cosmids during a productive lambda infection; (iii) transducing a recombination-deficient E. coli lambda lysogen with the generated lysate (the lysogen inhibits the helper phage used to package the recombinant andenoviral cosmid from productively infecting and destroying the host bacteria); (iv) effectively selecting for the desired double-recombinant cosmid. Approximately 10,000 double-recombinant cosmids are recovered per reaction with essentially all of them being the correct double-recombinant molecule. This system was used to generate quickly and efficiently adenoviral genomes deficient in the E1/E3 and E1/E3/E4 regions. The basis of this technology allows any region of the viral genome to be readily modified for investigation of novel configurations.     

Structure of cryptic lambda prophages

When Escherichia coli cells lysogenic for bacteriophage lambda are induced with ultraviolet light, cells carrying cryptic lambda prophages are occasionally found among the apparently cured survivors. The lambda variant crypticogen (lambda crg) carries an insertion of the transposable element IS2, which increases the frequency of cryptic lysogens to about 50% of cured cells: 43 of these cryptic prophages have been characterized. They all contain substitutions that replace the early segment of the prophage genome (from the IS2 to near the cos site) with a duplicate copy of a large segment of the host chromosome. The right end of the substitution always results from recombination between the nin-QSR-cos region of the prophage and the homologous incomplete lambdoid prophage Qsr' at 12.5 minutes in the E. coli chromosome. The left end of the substitution is usually a crossover that recombines the IS2 element in the prophage with an E. coli IS2 at 8.5 minutes, near the lac gene, or with a second IS2 located counterclockwise from leu at 2 minutes, generating duplications of at least 200,000 bases. Five cryptic lysogens derived from cells lysogenic for a reference strain of lambda (which lacks the IS2 present in lambda crg) have been characterized. They contain substitutions whose right termini are generated by a crossover with the Qsr' prophage. The left termini of these substitutions are formed either by a crossover between the lambda exo gene and a short exo-homologous segment of Qsr' (2/5), or by a crossover between sequences to the left of attL and an unmapped distant region of the host chromosome (3/5). The large duplications carried by these cryptic lysogens are stable, unlike tandem duplications, and so may significantly influence the cell's evolutionary potential.     

Phasmids. Properties and the use in genetic engineering. I. Construction of the phasmid vector

A phasmid vector molecule designated pMYF11 has been constructed. The vector combines some useful features of plasmid and phage vector molecules. lambda pMYF11 is a hybrid of lambda 47.1 vector and pBR322 plasmid. CI- marker of pMYF11 is replaced with cI+ marker by recombination between the plasmid and prophage 434. The phasmid molecule can be used as a replacement vector for BamHI, HindIII, SalGI endonucleases. The maximum size of fragments to be cloned is 21 kilobase pairs. Positive selection for hybrid molecules is possible because of the Spi phenotype expression after replacement of the central HindIII or BamHI DNA fragment with foreign DNA. A library of Escherichia coli genes is constructed with the help of lambda pMYF11 as a vector molecule. A hybrid phage harboring genes of the proline operon is detected by means of complementation.     

Isolation and characterization of lambda pleu bacteriophages.

In the Escherichia coli lysogen HfrH73 described by Shimada et al. (1973), none of the enzymes coded for by the leucine operon is synthesized due to an insertion of phage lambda into cistron leuA. The orientation of lambda in the chromosome is ara leuDCB lambda JAN leuA. After heat induction of the lysogen, plaque-forming transducing phages of two types are formed at low frequency. One type (e.g., lambda pleu9) transduces leuD, leuC, and leuB strains to prototrophy. The other type (e.g., lambda pleu 13) transduces leuA strains to prototrophy. lambda pleu 13 forms lysogens at low frequency (about 0.2%) by integration into the leucine operon. These lysogens are unstable, segregating phage-sensitive clones at high frequency (about 1%). Phages carrying different portions of the leucine operon were formed by aberrant excision after heat induction of strain CV437 (leuA371 lambda pleu13). A phage carrying the entire leucine operon (lambda K2) was constructed by a cross between lambda pleu9 and lambda pleu13. An analysis of leucine-forming enzyme levels in strains lysogenized with lambdaK2 indicated that leuO and leuP are present and functional in lambda K2. leu-specific messenger ribonucleic acid from E. coli hybridizes to the heavy (r) strand of lambdaK2. The leucine operon of lambda G4 pleuABCD (an S7 derivative of lambda K2) exists intact on a 7.3 x 10(6)-dalton fragment (lambdaG4EcoRI-B) generated by cleavage with endonuclease EcoRI. Heteroduplexes formed between lambda G4 and lambda show a 5.4 x 10(6)-dalton piece of bacterial deoxyribonucleic acid (DNA) replacing a 4.5 x 10(6)-dalton piece of lambda DNA starting at 0.46 fractional unit on the map of lambda. Fragment lambda G4EcoRI-B has about 0.6 x 10(6) daltons of lambda DNA from the b2 region at one end and about 1.4 x 10(6) daltons of lambda DNA from the int region at the other end.