Apparent Mutagenic Effect of Induction of Lambda Prophage Inserted Between lysA and thyA

Induction of a heat-inducible abnormal lambda prophage inserted between lysA and thyA in Escherichia coli resulted in a number of auxotrophic mutants in the surviving cured-cell populations. These mutants could not be accounted for by deletions arising on formation of lambda hybrid particles carrying regions adjacent to the insertion site. The properties of these mutants, which were almost all spontaneously revertable, have been described and mapped by F′ episome complementation. Tentatively, it was suggested that induction of the lambda lysogen leads to a mutagenic state.     

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.     

The cloning, DNA sequence, and overexpression of the gene araE coding for arabinose-proton symport in Escherichia coli K12

A lambda placMu1 insertion was made into araE, the gene for arabinose-proton symport in Escherichia coli. A phage containing an araE'-'lacZ fusion was recovered from the lysogen and its restriction map compared with that of the 61-min region of the E. coli genome to establish the gene order thyA araE orf lysR lysA galR; araE was transcribed toward orf. A 4.8-kilobase SalI-EcoRI DNA fragment containing araE was subcloned from the phage lambda d(lysA+ galR+ araE+) into the plasmid vector pBR322. From this plasmid a 2.8-kilobase HincII-PvuII DNA fragment including araE was sequenced and also subcloned into the expression vector pAD284. The araE gene was 1416-base pairs long, encoding a hydrophobic protein of 472 amino acids with a calculated Mr of 51,683. The amino acid sequence was homologous with the xylose-proton symporter of E. coli and the glucose transporters from a human hepatoma HepG2 cell line, human erythrocytes, and rat brain. The overexpressed araE gene product was identified in Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell membranes as a protein of apparent Mr 35,000 +/- 1,150. Arabinose protected this protein against reaction with N-ethylmaleimide.     

lambda-Transducing phages carrying transfer genes isolated from an abnormal prophage insertion into the traY gene of F

A set of lambda-transducing phages carrying transfer (tra) genes has been isolated from an abnormal lysogen in which a lambda prophage was inserted into the traY gene of Flac. These have been characterized genetically for complementation of Flac tra and finP point mutants and for the presence of oriT. Studies of tra gene expression during lambda repression showed that tra genes on the transducing phages were expressed from the lambda PL promoter as well as from the transfer promoters when these were present. The molecular weights of the traM (14,000) and traJ (23,500) proteins were measured after infection of ultraviolet-irradiated cells with one of the phages, ED lambda 102, and overproduction of the traJ protein upon induction of an ED lambda 102 lysogen was demonstrated. A proportion of this traJ protein was located in the inner membrane and cytoplasmic fractions of the cell, the majority being in the outer membrane. Physical analysis of the DNA carried by the lambda tra phages by determination of the phage buoyant densities in CsCl, by restriction enzyme digestion and by electron microscope heteroduplex analysis, was used to define the DNA segments encoding the tra functions. Correlation of the physical and genetical data improved the positioning of the tra genes within the transfer region. These results were combined with new restriction enzyme cleavage data to construct an improved map of this region.     

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.     

Carbohydrate metabolism and transport in Bacillus subtilis. A study of ctr mutations

Summary Indirect ultraviolet induction of prophage occurs when lysogenic E. coli K12 cells are mated with ultraviolet-irradiated donor strains carrying a transmissible episome such as F lac ⁺. Indirect induction occurs in wild type, uvrA, or recB recipient lysogens, but not in recA lysogens. When nonpermissive lysogens carrying prophages susO or susP are similarly mated, the defective prophages are induced and indirect curing takes place.Although indirect induction is independent of the capacity of the lysogen for repair by pyrimidine dimer excision, indirect curing (and hence indirect induction) is subject to photoreactivation when the recipient lysogen is exposed to visible light after mating. This confirms that the structure initiating indirect ultraviolet induction is a damaged transferred episome consisting of one DNA strand containing ultraviolet photoproducts and a newly synthesized discontinuous DNA strand such that pyrimidine dimers remain in single-stranded regions.F^- lac ⁺ recombinants are formed in either nonlysogenic or lysogenic Lac^- cells receiving damaged F lac ⁺ episomes from ultraviolet irradiated F lac ⁺ donors. prophage induction occurs more frequently in zygotes that form Lac⁺ recombinants than in zygotes that remain Lac^-. In contrast, cells receiving intact (undamaged) episomes are converted to F lac ⁺ secondary donors, but are rarely induced or cured.     

Isolation of P22 Specialized Transducing Phage following F''-Episome Fusion

A P22 specialized transducing phage has been constructed which carries the structural gene for aspartate transcarbamylase (ATCase). This gene (pyrB) was first brought close to the P22 attachment site by fusing an F' pyrB⁺ episome to an F' prolac episome which carries a P22 prophage attachment site. A prophage was added to these fused F' episomes and the lysogen was UV-induced. The specialized transducing phage was isolated from the resulting lysate. The phage also carries argI, the structure gene for ornithine transcarbamylase.     

Operator-promoter functions in the threonine operon of Escherichia coli.

The prophage curing properties of secondary-site lysogens of coliphage lambda have been studied. The site of integration in the original lysogen (L79) is within the ooerator-promoter region of the thr operon. As a result, expression of the thr enzymes is reduced, and the strain is a leaky threonine auxotroph. Heat pulse curing of strain L79 and a thr+ lysogenic revertant (L79-20) showed that heat pulse curing of both lysogens was int and xis dependent and occurred by correct excisions of the prophage. The heat pulse curing restored strain L79 to prototrophy whereas strain L79-20 synthesized the thr enzymes constitutively and at high levels. This indicates that the reversion mutation in strain L79-20 occurred outside of the prophage and within the operator-promoter region of the thr operon. In contrast, spontaneous curing of both lysogens occurred by both correct and incorrect excisions. Spontaneously cured derivatives of strain L79-20 gave rise to three classes of regulatory mutants affecting operator and promoter functions to the thr operon.     

Superinfection exclusion by heteroimmune corynebacteriophages.

Superinfection of Corynebacterium diphtheriae C7(beta) by heteroimmune phage gamma is productive, whereas superinfection by gamma-bin mutants is for the most part nonproductive. Exclusion of gamma-bin phage occurred after its DNA had penetrated and was partially expressed in the heteroimmune lysogen. All of the infected cells were killed, and lysis was observed. The beta inhibitor causing exclusion was produced during the prophage state and appeared to be distinct from immune repressor. The ability of gamma-bin phage to superinfect C7(beta) productively could be restored by recombination with beta phage, indicating that both beta and gamma phages contain either indentical or similar alleles of the bin gene. The bin gene was mapped by vegetative and prophage crosses and found to be located in the region of the phage genome concerned with regulation. Both beta and gamma wild-type phages induced the resident prophage in a significant fraction of superinfeted heteroimmune lysogens. This, coupled with the fact that induction of C7(beta) abolished exclusion, suggests that the bin gene product acts as antirepressor, i.e., it reduces the level of heteroimmune repressor either directly or indirectly. The gamma-bin mutants either failed to produce antirepressor or did so with reduced efficiency. Antirepressor activity was negatively controlled by homoimmune repressor. The isolation of beta mutants that appeared bin-like suggests that beta and gamma phages contain homologous systems of exclusion and antiexclusion. Exclusion of gamm-bin by beta phage in gram-positive C. diphtheriae exhibited striking parallels to the sieB exclusion described for phages P22 and lambda in gram-negative organisms. The extended similarities of these coryngephages to lambda bacteriophage is noted.     

Further characterization of a non-essential mutator gene in Escherichia coli K-12.

The properties of mutR, a mutator closely linked to thyA, have been further characterized. We have found that the mutator gene is carried on a specialized transducing phage (lambdapcI857 thyA) generated by the excision of lambdacI857 integrated at a secondary attachment site between lysA and thyA. We present three lines of evidence indicating that mutR is a nonessential gene. (i) Deletions of the mutator can be found amoung survivors of heat induction of lambdacI857 when the phage is integrated between lysA and thyA. (ii) Mutations in mutR can be induced with the frameshift mutagen ICR-191. (iii) An amber mutant in mutR has been found. Viable strains could be made by combining the mutator with polB, polA polR, ligts7, and uvrA mutations. The mutator was still able to increase the spontaneous mutation frequency in these genetic backgrounds. When the reversion patterns of a series of well-characterized trpA mutations were analyzed, the results suggested that mutR is more efficient at causing transitions than transversion mutations.     

Mapping of insertion mutations in gnd of Escherichia coli with deletions defining the ends of the gene.

A genetic map was prepared for gnd, the gene of Escherichia coli which encodes the metabolically regulated 6-phosphogluconate dehydrogenase. Direct selection methods were used for the isolation of mutants with deletions that define the respective ends of gnd. These selections depended on the availability of a defective lysogen in which gnd was present on a lambda h80 dgnd his prophage located at the att phi 80 region of the chromosome. Mutants with deletions entering gnd from the his-distal end were selected as Gnd- TonB- mutants. Mutants with his-proximal gnd deletions were selected as Gnd-, temperature-resistant mutants of a specially prepared stable lysogen. Gnd- mutants were also isolated after mutagenesis with bacteriophage Mu cts61, and genetic tests were used to determine which mutants carry a Mu cts61 prophage in gnd. The deletion mutations were mapped against each other and against the insertion mutations through the use of F' merodiploid strains. The insertion mutations mapped at seven distinct sites in gnd; three mapped under the deletions defining the his-proximal portion of the gene and three mapped with the his-distal deletions.     

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.     

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.     

Deletions induced by gamma rays in the genome of Escherichia coli.

An Escherichia coli lysogen was constructed with a lambda phage bearing a lacZ gene surrounded by about 100 x 10(3) base-pairs of dispensable DNA. The lacZ mutants induced by gamma rays in this lysogen were more than 10% large deletions, ranging in size from 0.6 x 10(-3) to 70 x 10(3) base-pairs. These deletions were centered, not on lacZ, but on a ColE1 origin of DNA replication located 1.2 x 10(3) bases downstream from lacZ. This suggested that this origin of replication was involved in the process by which the deletions were formed. In agreement with this hypothesis, a lysogen of the same phage without the ColE1 origin showed a very much lower percentage of radiation-induced deletions, as did a second lysogen of a lambda phage without any known plasmid origin of replication. Indirect evidence is presented for radiation-induced deletions centered on the lambda origin of DNA replication in a lysogen. It is suggested that high percentages of large deletions may occur among radiation-induced mutations in mammalian cells because deletions centered on some of the thousands of origins of replication in these genomes do not kill the cells.     

Reconstitution of an operon from overlapping fragments: use of the lambda SV2 integrative cloning system

We have used the lambda SV2 system [Howard and Gottesman. In Gluzman (Ed.), Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 211-216; in Inouye, M. (Ed.) Experimental Manipulations of Gene Expression. Academic Press, New York, 1983, pp. 137-153] to reconstitute the Salmonella typhimurium his operon from overlapping fragments. lambda SV2 can be propagated as an autonomously replicating plasmid or as a prophage integrated in the Escherichia coli chromosome at the lambda attachment site; our reconstitution was accomplished in the integrated state. We first inserted a portion of the his operon into lambda SV2 and integrated the resulting plasmid by site-specific recombination into the E. coli chromosome. This was achieved by brief induction of a resident prophage. The lysogen was then transformed with DNA from a lambda SV2 clone carrying the remainder of the his operon on an overlapping DNA fragment. The second plasmid was forced to integrate into the first by homologous recombination. When this recombination occurs at the his overlap, a lysogen carrying two lambda SV2 prophages is produced. One prophage carries the entire his operon and the other carries the his overlap region. The latter is removed by site-specific recombination, permitting further contiguous sequences to be sequentially added to the remaining prophage. This method should be applicable for the reconstitution and maintenance of large genes or gene clusters in the E. coli genome.     

Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor

Wiesmeyer, Herbert (Vanderbilt University, Nashville, Tenn.). Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor. J. Bacteriol. 91:89-94. 1966.-The concentration of lambda repressor molecules within a lambda lysogenic cell was estimated from the multiplicity of superinfecting homologous phage necessary to permit replication and release of plaque-forming units. A multiplicity of 20 superinfecting phage was found sufficient to permit replication to occur in the normal lambda lysogen. The phage released after lysis of the superinfected lysogen was composed of both prophage and superinfecting phage types. Superinfection of the lysogen at lower multiplicities resulted in the lysis of only a small percentage of infected cells and is thought to represent a possible heterogeneity of repressor concentration in the lysogenic population. Viability of the superinfecting particle was found to be unnecessary for titration of the repressor. The repressor concentration in three lysogens of the nonultraviolet-inducible mutant of lambda, lambda(ind-), was found to be greater than 20 regardless of the host bacterium. However, the number of cells yielding phage after superinfection was found to vary with the particular host. The specificity of the lambda repressor was shown to be limited to homologous phage, as determined following heterologous superinfection experiments with phages T6r, 82c, 434c, 434hy, and 424. In all instances except that of superinfection with phage 434hy, only heterologous phage replication occurred. Superinfection by phage 434hy resulted in the release of both prophage and superinfecting phage types. The latter type represented approximately 80% of the total phage released.     

Mechanism of non-tandem integration of prophage phi 80 into the chromosome of the wild-type Escherichia coli

We studied the ability of lambda, phi 80 and their hybrid lambda att80 to lysogenize homoimmune monolysogens and examined the prophage locations on the chromosome of the resulting polylysogens. We observed an effective integration of phi 80 and lambda att80, in contrast to lambda, into the host chromosome, exclusively, at the attachment sites that were not occupied by the resident prophage (nontandem). Besides, the lambda att80 (int+) prophage was observed to ensure effective nontandem integration of a homoimmune int mutant DNA. Hence, we inferred that the expression of the int gene in the phi 80 prophage is constitutive, cI-independent and results in nontandem integration of the homoimmune prophage. The validity of this inference has been supported experimentally: (i) the only lysogen that was found to contain a phi 80 tandem was highly unstable (spontaneous segregation of monolysogens occurred 6-7 times more frequently than with the lambda tandem); (ii) an int inactivating mutation stabilized the phi 80 tandem; as a result, the int mutant has the frequency of tandem integration as high as that of lambda, while no nontandem integration was observed. A hypothesis is proposed which accounts for the instability of the phi 80 tandems and explains the relation between this phenomenon and the prophage ability to integrate into secondary attachment sites in the presence of the primary (normal) one.     

Gene Regulation in N Mutants of Bacteriophage λ

Mutants (N(-)nin) of bacteriophage lambda in which the N gene product is not required for growth on wild-type Escherichia coli do not plate on recA bacterial mutants. Secondary mutants, selected for growth on recA, lie within the immunity region to the right of gene cI and appear identical to the cro mutants of Eisen et al. In an N(+) phage, a cro mutation causes enhanced and prolonged production of lambda exonuclease. N(-)cro phages make no detectable exonuclease, but show an increased rate of specific excision from lysogens and are excluded by P2 prophage. These properties, together with the ability to plate on recA, suggest that N(-)cro phages express genes to the left of N at a rate that is very low but higher than that for N(-)cro(+) phages. N(-)nin phages can integrate at the normal site on the bacterial chromosome, but specific excision from lysogens is immeasurably low.     

Ultraviolet light-induced mutagenesis in the Escherichia coli chromosome. Sequences of mutants in the cI gene of a lambda lysogen

DNA sequences were determined for 56 mutations induced by ultraviolet light in the lambda cI gene of an Escherichia coli uvr+ lysogen, which should reflect those occurring in the E. coli chromosome. The spectrum of mutagenesis was similar to that found in the cI gene of irradiated phase assayed in uvr- host cells, except that the fraction of transversions is about 35% in prophage and about 15% in phage. The cause of this difference is not known. Of 17 frameshifts in phage and prophage, six have an accompanying base substitution. These double mutational events are consistent with a model in which a photoproduct in a template can cause a DNA polymerase to insert a wrong base and destabilize the next few bases added, thus leading to a frameshift by a slippage mechanism.     

Excision of bacteriophage lambda from a site in the arabinose B gene.

A lambda lysogen with the prophage inserted into the arabinose B gene of Escherichia coli strain K-12 has been prepared. Induction of the phage from this lysogen yields viable phage at a frequency 4 X 10(-6) that found for induction of lysogens with phage inserted at the normal attachment site. Over 30% of the phage particles induced from the insertion in ara are arabinose-transducing phage. The excision end points of 62 independently isolated, nondefective araC-transducing phage containing less than the entire araC gene were genetically determined and were found to be randomly distributed through the araC gene. The amount of arabinose deoxyribonucleic acid contained on four selected transducing phage was determined by electron microscopy of deoxyribonucleic acid heteroduplexes, providing a physical map of the araC gene. The efficiency with which these phage transduce araC and araB point mutations was found to be approximately proportional to the homology length available for recombination.