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.     

Spontaneous lambda OR mutations suppress inhibition of bacteriophage growth by nonimmune exclusion phenotype of defective lambda prophage.

Survivor clones with defects in gene functions that participate in the replicative killing of thermally induced Escherichia coli constructs with integrated lambda N through P or cIII through P gene fragments were selected at a frequency of about 10(-6). Among the population of survivors, clones were identified that exhibited normal lambda immunity at 30 degrees C, as shown by their ability to prevent the plating of lambda wild type and to support the plating of a nearly identical heteroimmune bacteriophage lambda imm434. However, when placed at 42 degrees C to inactivate the cIts857 repressor, these survivor isolates excluded the plating of both lambda wild-type and lambda imm434 phages, a phenotype designated nonimmune exclusion (Nie). Spontaneous mutants of lambda wild type were isolated that overcame the Nie phenotype and would plaque at 42 degrees C on cell lawns of these isolates. The acquired lambda se mutations suppressed nonimmune exclusion, prevented lysogenization by interrupting repressor expression from PRM, and made the phage insensitive to replicative inhibition. The se mutations were genetically mapped and sequenced within the rightward lambda operator site.     

Genetic Analysis of tox+ and tox? Bacteriophages of Corynebacterium diphtheriae

A series of mutants derived from the temperate corynebacteriophages beta(tox+), gamma(tox-), and L(tox+) was isolated and characterized. In three-factor crosses between mutant beta phages the relative map order of the genetic markers determining extended host ranges (h and h') and loss of ability to lysogenize (c) was found to be h--c--h'. Recombination between markers was observed in matings between phage beta and the heteroimmune corynebacteriophages gamma and L. In such matings between heteroimmune phages the c markers of phages beta and gamma failed to segregate from the imm markers which determine the specificity of lysogenic immunity in these phages. The factor which directs the synthesis of diphtherial toxin during infection of appropriate corynebacterial hosts by toxinogenic corynebacteriophages is designated tox(+). It was possible to show that the tox(+) determinant of phage beta behaves as a single genetic element which occupies a position between the loci h and imm on the genetic map of this phage. Genetic recombination between mutants of phage beta occurred at very low frequencies in biparental matings performed by mixed infection of Corynebacterium diphtheriae C7(s)(-)(tox-). Considerably higher recombination frequencies were observed when lysogenic bacterial strains carrying one parental phage as prophage were induced by ultraviolet irradiation and then superinfected by the second parental phage. Maximal stimulation of genetic recombination between mutant beta phages was detected when superinfection followed ultraviolet irradiation of the lysogenic cells within a limited period of time. In matings between phages with incomplete genetic homology, the stimulation of recombination by ultraviolet radiation was much less effective.     

Prophage map of converting corynebacteriophage beta.

A prophage map for corynebacteriophage beta consisting of seven markers has been constructed and compared with the vegetative map. The mapping system utilizes heteroimmune double lysogens and capitalizes on the fact that these double lysogens are very unstable and throw off monolysogenic segregants. The prophage map, produced by characterizing the recombinant phage in these monolysogenic segregants, appears to be a cyclic permutation of the vegetative map with the gene for toxin at one end of the prophage map and the gene for phage immunity at the other. This permutation is in accord with the Campbell model for insertion of lambda phage if a site between the toxin and immunity genes in the vegetative map is designated as the phage attachment site. The position of the gene for toxin in the prophage map suggests that converting phages may have originated as specialized transducing phages for this gene.     

Cloning and characterization of the c1 repressor of Pseudomonas aeruginosa bacteriophage D3: a functional analog of phage lambda cI protein.

We cloned the gene (c1) which encodes the repressor of vegetative function of Pseudomonas aeruginosa bacteriophage D3. The cloned gene was shown to inhibit plating of D3 and the induction of D3 lysogens by UV irradiation. The efficiency of plating and prophage induction of the heteroimmune P. aeruginosa phage F116L were not affected by the presence of the cloned c1 gene of D3. When the D3 DNA fragment containing c1 was subcloned into pBR322 and introduced into Escherichia coli, it was shown to specifically inhibit the plating of phage lambda and the induction of the lambda prophage by mitomycin C. The plating of lambda imm434 phage was not affected. Analysis in minicells indicated that these effects correspond to the presence of a plasmid-encoded protein of 36,000 molecular weight. These data suggest the possibility that coliphage lambda and the P. aeruginosa phage D3 evolved from a common ancestor. The conservation of the functional similarities of their repressors may have occurred because of the advantage to these temperate phages of capitalizing on the potential of the evolutionarily conserved RecA protein to monitor the level of damage to the host genome.     

Mutations in bacteriophage lambda repressor that prevent RecA-mediated cleavage.

In this paper, we report on the isolation and sequence analysis of mutations that confer an induction-deficient phenotype to lambda repressor. A total of 16 different mutations, which occur at 13 different sites in the repressor gene, have been characterized. For most of the mutant lysogens, frequencies of spontaneous induction in a recA+ strain were reduced dramatically in comparison with those for a wild-type phage, and these mutant lysogens showed little or no prophage induction after UV irradiation. The immunity properties of cells containing the mutant repressors show that all of the mutants but one exhibit operator-binding properties indistinguishable from wild-type repressor.     

Control of corynebacteriophage reproduction by heteroimmune repression.

Corynebacteriophages beta and gamma are closely related but heteroimmune; hence, gamma reproduces in C7(beta). A series of gamma mutants, designated gamma-bin (beta-inhibited), has been isolated. They reproduce in only 2 to 14% of infected C7(beta) cells, and, as a result, plaque with an efficiency of 10(-4) to 10(-5) on this strain. The proportion of C7(beta) cells in which gamma-bin phage can replicate is increased to 30 to 80% when immunity is lifted by UV induction of C7(beta) or by heat induction of C7(beta-tsr3). The gamma-bin mutants carry out a normal vegetative or lysogenic cycle in strain C7 and thus do not appear to be defective in any essential phage function. Infection of C7(beta) by gamma-bin results in cell killing whether the infection is productive or nonproductive. The data support the hypothesis that inhibition of gamma-bin is due to the direct or indirect action of a beta prophage gene. The simplest hypothesis is that gamma-bin phages have sustained mutations in an operator site and that beta repressor now combines with the mutated operator to inhibit normal replication in a significant proportion of infected cells.     

Effects of mutations in the immunity system of bacteriophage P1.

A mutant of bacteriophage P1 that made an altered c1 repressor is described. The mutant c1 product had two configurations: in lysogens, at high temperatures, it permitted constitutive expression of the normally repressed DNA replication function ban and was insensitive to the action of ant, a product expressed by the virulent mutant P1virs and by the heteroimmune phage P7 (formerly phiamp+) and normally able to overcome c1 repression; in mutant lysogens at low temperatures, the mutant repressor was apparently normal (able to repress ban and sensitive to ant action). Genetic studies of this mutant led to the isolation of a derivative that formed unstable lysogens. These studies suggested that the ban product was normally under c1 control; they further showed that ant overcame c1 repression by inactivating c1 rather than by creating a bypass of repressor activity.     

Inactivation of prophage lambda repressor in vivo.

Jacob &; Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product.In order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants.The kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case.A high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer.In bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo.From the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.     

Salmonella typhimurium metE operator-constitutive mutations

We used a metE-lacZ fusion phage (lambda Elac) to select for mutants with operator-constitutive mutations in the Salmonella typhimurium metE control region. All of the mutations identified were found to lie within a region containing tandemly-repeating 8-bp palindromes with the consensus sequence 5'-AGACGTCT-3', previously proposed to be the binding region for the metJ-encoded repressor. Lysogens carrying mutant lambda Elac phage exhibit high beta-galactosidase levels that are only partially repressible by methionine. Although repression of metE expression by vitamin B12 is not disrupted in metJ+ lysogens, vitamin B12 repression is disrupted in lysogens lacking an active MetJ repressor. These results suggest that there is an interaction between the metJ-encoded repressor and the vitamin B12 repression system mediated by the metH gene product.     

Mutants in the y region of bacteriophage lambda constitutive for repressor synthesis: their isolation and the characterization of the Hyp phenotype

Bacteriophage lambdahyp mutants have been isolated as survivors of Escherichia coli K-12 bacteria lysogenic for lambda Nam7am53cI857. The hyp mutants are characterized by (i) their localization in the y region very close to the imm lambda/imm434 boundary, (ii) polarity on O gene expression, (iii) immediate recovery of lambda immunity at 30 degrees C after prolonged growth of lambda Nam7am53cI857 hyp lysogens at 42 degrees C even in the presence of an active cro gene product, (iv) ability of phage lambda v2v3vs326 but not lambda v1v2v3 to propagate on lambda cI+hyp lysogens, (v) inability to express lambda exonuclease activity after prophage induction, and (vi) inviability at any temperature of phage carrying the hyp mutation. All these properties are referred to collectively as the Hyp phenotype. We show that the Hyp phenotype is due to cII-independent constitutive cI-gene-product synthesis originating in the y region, which results in the synthesis of anti-cro RNA species, and constitutive levels of cro gene product present even in lambda cI+hyp lysogens. A model is presented which is consistent with all the experimental observations.     

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.     

Regulation of toxinogenesis in Corynebacterium diphtheriae. I. Mutations in bacteriophage beta that alter the effects of iron on toxin production

Diphtherial toxin is produced in maximal yields by Corynebacterium diphtheriae (C7(beta tox+) only when iron is present in growth-limiting amounts. Toxin production is markedly decreased under high-iron conditions. We studied the role of the bacteriophage beta genome in this apparent regulation of toxin production by iron. Using a passive immune hemolysis assay to detect toxin antigen production in individual plaques, we identified rare phage mutants that were toxinogenic in high-iron medium. Lysogenic derivatives of C. diphtheriae C7 harboring such phage mutants were constructed. The lysogens were compared with wild-type strain C7(beta) for their ability to produce toxin in deferrated liquid medium containing varying amounts of added iron. Quantitative tests for extracellular toxin were performed by competitive-binding radioimmunoassays. We identified phenotypically distinct mutant strains that produced slightly, moderately, or greatly increased yields of toxin antigen under high-iron conditions. The toxin produced by the mutant lysogens was biologically active and immunochemically indistinguishable from wild-type toxin. Complementation experiments demonstrated that the phage mutation designated tox-201 had a cis-dominant effect on the expression of the toxin structural gene of phage beta. The characteristics of the tox-201 mutation suggest that it defines a regulatory locus of phage beta that is involved in control of toxinogenesis by iron in C. diphtheriae.     

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.     

Chromosomal Recombination in HAEMOPHILUS INFLUENZAE

Haemophilus influenzae cultures doubly lysogenic for defective phage HP1, with a prophage marker sequence +b+/a+c, always contained some free wild-type phage. Single ultraviolet-irradiated cells produced either no wild-type phage or large numbers of them. This suggested that the phage was not released by the original double lysogen but by internal recombinants, i.e., by double lysogens with altered prophage marker sequence such as +++/abc or +b+/++c. Thirty-one wild-type phage-producing clones have been isolated independently from cultures of this double lysogen and identified. They fell in five classes. Two classes, still possessing all three prophage markers, can be explained by Campbell's (1963) prophage recombination model. The other classes had lost one or more markers. They can be explained by interchromosomal double-strand DNA breakage and rejoining. A single-DNA-strand gene conversion model is discussed in view of the fact that genetic transformation involves single-DNA-strand exchanges. A number of potentially interesting mutants has been analyzed of which only the derivatives of rec1 mutant DB117 (obtained from Dr. J. Setlow) were incapable of internal recombination.     

Heat-inducible mutants of corynebacteriophage.

Heat-inducible mutants of temperate cornebacteriophage beta and gamma, called temperature-sensitive repression (tsr) mutants, were isolated and characterized. Lysogens carrying these mutants were induced at 38 degrees C, produced a normal or slightly increased yield of phage, and underwent extensive lysis at this temperature. In some cases mutation to heat inducibility had altered the UV inducibility of the phage, the changes ranging from loss to enhancement of this trait. Complementation tests showed that all five beta-tsr strains had mutated in the same cistron and suggested that these mutations were in the gene responsible for repressor production.     

Genetic characterization of Mu-like bacteriophage D108.

Infection of Escherichia coli by bacteriophage D108 was shown to result in the generation of apparently random chromosomal mutations. Approximately 1% of the cells lysogenized by D108, as with Mu, acquired new auxotrophic mutations. D108-induced mutations were nonreverting and were most probably the result of insertion of the D108 genome into regions of genetic function. D108 and Mu shared many similar properties but were heteroimmune and had different host ranges. Lytic infections of Mu lysogens with D108 and D108 lysogens with Mu resulted in 100-fold increases in release of phage with prophage markers over those due to spontaneous induction. Phenotypic mixing was common, with most phage carrying the prophage immunity being packaged in particles with the host range of the superinfecting phage. A fraction of the superinfecting phage genomes were, however, packaged in particles with the prophage-specified host range. Although 10% of the prophage progeny were D108-Mu genetic hybrids, superinfecting phage-induced release of the prophage with reciprocal phenotypic mixing occurred in recA hosts, in which the frequency of D108-Mu genetic hybrids was reduced 100-fold.     

Influence of Transformability on the Formation of Superinfection Double Lysogens in Haemophilus influenzae

Superinfection of growing (nontransformable) cells of defectively lysogenic strains of Haemophilus influenzae with wild-type or with mutant phage HP1 resulted in a number of double lysogens and a small number of monolysogens with altered prophage. The double lysogens were identified by analysis of their monolysogenic segregants and by examining their deoxyribonucleic acid in certain test crosses. The results indicate that the majority had been formed by insertion of the infecting phage genome within the resident prophage. Superinfection of transformable bacteria gave rise to cells with altered prophages (presumably transformants) and to double lysogens which had gained or lost wild-type prophage loci.     

The antirepressor needed for induction of linear plasmid-prophage N15 belongs to the SOS regulon

The physiological conditions and molecular interactions that control phage production have been studied in only a few families of temperate phages. We investigated the mechanisms that regulate activation of lytic development in lysogens of coliphage N15, a prophage that is not integrated into the host chromosome but exists as a linear plasmid with covalently closed ends. We identified the N15 antirepressor gene, antC, and showed that its product binds to and acts against the main phage repressor, CB. LexA binds to and represses the promoter of antC. Mitomycin C-stimulated N15 induction required RecA-dependent autocleavage of LexA and expression of AntC protein. Thus, a cellular repressor whose activity is regulated by DNA damage controls N15 prophage induction.     

A new pleiotropic bacteriophage P1 mutation, bof, affecting c1 repression activity, the expression of plasmid incompatibility and the expression of certain constitutive prophage genes.

Summary In bacteriophage P1 an amber mutation in a new gene, bof, has been isolated. The bof-1 phage mutant exhibits a pleiotropic phenotype; bof product is non-essential, and acts as a positive modulator. In P1 bac-1 mutants, in which a dnaB analog product, ban, is expressed constitutively, the bof product activates ban expression both in the prophage state and in lytic growth: P1 bof bac prophages have a reduced ban activity and in lytic growth P1 bof bac phages show a lower ban activity than P1 wild type. This effect on ban activity is observed specifically in P1 bac-1 mutants; it is not mediated by the cl repressor of the lytic functions (repressor of the ban operon) since this effect occurs even if the phage carries a heat sensitive c1 repressor. Thus we concluded that the bac mutation put the ban operon under an abnormal, unknown control, modulated by the bof product. P1 bof lysogens show an increased immunity to superinfecting P1 phage and are affected in their inducibility properties; in the presence of the altered c1-100 repressor, bof product is required for maintenance of lysogeny, as shown by the induction of P1 c1-100 bof-1 lysogens at 30°. P1 bof superinfecting phage can be established together with a resident P1 bof prophage in a recA host, unlike P1 wild type which cannot form double lysogens. P1 bof double lysogens are unstable and segregate one or the other prophage. P1 Cm bof and P1 Km bof lysogens show higher levels of antibiotic resistance than the corresponding bof ⁺ lysogens. The bof gene has been mapped, in an interval defined by P1 prophage deletion end points, far from both ban and c1. All bof phenotypes are reversed by single mutations.