Host mutants resistant to phage lambda killing

Host mutants have been isolated which are resistant to killing by the kil protein of an induced λ prophage. In general, such host mutants are defective in adsorption and production of lambdoid phages, and show altered sensitivities to deoxycholate, colicins, and antibiotics. Most of the mutants are cold sensitive and form filaments at the restrictive temperature. These properties seem to implicate the cell envelope in the kil phenomenon. The cold-sensitive host mutants cannot be cured of their prophage; furthermore, growth of the mutants at different temperatures shows a marked negative correlation with the degree of repressor activity of the temperature-sensitive cI857 repressor. These observations suggest that some phage product, perhaps kil protein, is required for cell survival in these mutants.     

Similarity of vegetative map and prophage map of bacteriophage Mu-1

The vegetative map of bacteriophage Mu was extended by the localization of three more amber mutations belonging to different complementation groups, and the position of the c gene on the vegetative map was approximately determined. The data confirm the previous finding that the vegetative map is linear (Wijffelman et al., 1972).Defective Mu lysogens were isolated from an Escherichia coli K12 strain, which has an insertion of Mu in the trp operon, by selecting for TonB colonies. All these defective lysogens were no longer immune to Mu infection, suggesting that the gene controlling immunity is located near to the end of the prophage proximal to tonB. By analyzing the remaining prophage genes in the defective lysogens, it was possible to determine the order of 16 amber mutations belonging to different complementation groups. The results show that the position of the genes on both the vegetative map and the prophage map is the same.The prophage map of Mu was determined by using the same method in another 9 independently isolated insertion mutants. In 7 of these mutants the gene order in the prophage is the same as found for the above-mentioned strain. The other two contain the prophage genes in the reversed order. All defective lysogens derived from these two insertion mutants by selection for TonB colonies were immune. The results show that Mu is integrated in a unique way and that both orientations may occur. The similarity between prophage map and vegetative map and the consequence for the mechanism of the integration process are discussed.     

Superinfection immunity and prophage repression in phage P1 and P7 III. Induction by virulent mutants

Two types of virulent mutants (virB and virC) have been isolated for phage Pl; both induce a Pl prophage to replicate after superinfection of a lysogen. The genetic lesion of P1virB (and P7virB) is located in the immunity-specific region of the phage, and these mutants induce only a homoimmune prophage. The virB mutants appear to depend for prophage induction on titration of the phage-specific c4 immunity repressor in the lysogen, since DNA synthesis of the superinfecting phage is needed for this induction process. The P1virC mutation, which is located at the left end of the phage map, induces both homoimmune and heteroimmune prophages. The virC mutant causes prophage induction in the absence of phage DNA replication and may induce because of constitutive synthesis of a vegetative repressor (c1) antagonist.     

Suppressor-sensitive mutants of coliphage phi80

About fifty suppressor sensitive (sus) mutants of phage phi80 were isolated after hydroxylamine treatment, and these were classified into fourteen cistrons. In vitro and in vivo complementation experiments revealed that at least five cistrons were concerned with head formation and that at least six cistrons were concerned with tail formation in phi80. Some presumed "early" mutants were also found. Defective lysogens were isolated from a phi80-lysogenic nonpermissive strain using the colicin plate method (Gratia, 1966). In these strains deletions which affected the colicin B receptor gene extended for varying distances into prophage phi80. Marker rescue experiments were carried out with these deletion lysogens by infecting various sus mutants, and the gene order in prophage phi80 was determined. Clustering of all head genes and also of tail genes, as in phage lambda, were demonstrated by the results of the prophage deletion mapping as well as the two-factor crosses performed among the sus mutants. Moreover, the gross gene arrangement of phi80 was also similar to that of phage lambda: namely, the cluster of head genes was found to be located at one end of the vegetative map of phi80, being followed by that of tail genes, and presumed "early" genes are located at the other end of the map.     

Studies on polylysogens containing lambda N-cI- prophages. II. Role of high multiplicities in lysogen formation by lambda N-cI- phage

Results of the experiments presented in this paper show that lambda N-cI- phage can lysogenize a nonpermissive host Escherichia coli when it infects at very high multiplicities (around 100), and lambda N-cI-cII- and lambda cIII-N-cI- lysogenize poorly at similar high multiplicities. The latter two phages lysogenize with appreciable frequency when either lambda N-cI- or lambda int-cN-cI-cII- is used as helper. The phages, lambda N-cI-, lambda N-cI-cII-, and lambda cIII-N-cI- can lysogenize also at relatively low m.o.i. of 20 in presence of the above lambda int-c helper, and the lambda int-cN-cI-cII- phage alone forms converted lysogens at an m.o.i. as low as 12. All these results suggest that the establishment of prophage integration by lambda N-cI- is positively regulated, like lambda N+cI+ phage, by the cII/cIII-promoted expression of the int gene of lambda, and under the N- condition, high multiplicities are needed to provide optimum levels of cII and cIII products, especially the latter.     

Focus assay and defectiveness of avian myeloblastosis virus.

A lysogen for P1d91tet, a defective prophage produced by recombination between P1 and an R factor, is not immune to superinfection by phage P1. However, marker rescue experiments demonstrate that the wild type form of the P1 repressor gene (c1⁺), whose action is necessary for the maintenance of a P1 prophage, is present in d91tet. Furthermore, the c1⁺ gene is active in a P1d91tet lysogen, since a single lysogen for a recombinant P1d91tet plasmid into which a c1ts mutation has been introduced is thermo-inducible. It is therefore concluded that superinfection immunity of a prophage is controlled, at least in part, by a different part of the P1 genome from that needed for prophage repression (gene c1).Marker rescue experiments using the d91tet lysogen allow us to determine the relative order of the genes near the “right-hand” end of the vegetative P1 map, including gene c1.     

Characterization of clear mutants belonging to the Z gene of bacteriophage P2

Gene Z mutants of phage P2 were isolated previously as clear plaque-forming mutants that could complement mutants of the C gene. Gene Z mutants are unable to lysogenize most strains of Escherichia coli C, although stable double lysogens, carrying both a Z⁺ and a Z mutant prophage, are easily obtained. When the Z⁺ prophage is removed from such a double lysogen (for example, by transduction), the resultant single lysogen produces many nonlysogenic segregants and cannot be maintained. These results suggest that the product of the Z gene is necessary for the maintenance of P2 lysogens.Additional mutations (suZ) that suppress the Z mutant phenotype have also been isolated. Such mutations are located near, or within, the Z gene itself and are dominant, since double lysogens carrying both a Z mutant and a suppressed Z mutant prophage are stable. Models for the function of the Z gene are discussed.     

Functions of two new genes in Salmonella phage P22 assembly.

The capsid of bacteriophage P22 is composed of at least eight protein species, six of which had been identified as the products of known P22 genes. The two smallest proteins, pX of 18,000 daltons, and pa of 15,000 daltons, were not the products of any of the known P22 genes. We have isolated and characterized phage carrying amber mutations in the two genes which code for pX and pα (designated gene 7 and gene 4, respectively). The products of these genes are essential for the formation of viable phage. The gene 7 protein is not needed for particle assembly, but rather for particle infectivity. Restrictive cells infected with a mutant in gene 7 accumulate structures with the sedimentation properties and electron microscopic appearance of wild-type phage. Although the defective 7^? particles adsorb to cells, they are unable to recombine with a defective prophage inside the cell or express phage-specific protein synthesis. The 7^? phenotype is similar to the phenotypes of amber mutants in genes 16 and 20 [Botstein et al., J. Mol. Biol.80, 669–695 (1973)]. Genes 7, 16, and 20 map contiguously. The gene 4 product seems to be necessary for the stabilization of newly DNA-filled phage heads. Restrictive cells infected with a mutant of gene 4 accumulate structures with the sedimentation properties and electron microscopic appearance of the “empty heads” that occur in smaller relative amounts in wild-type lysates. These 4^? empty heads are derived from unstably packaged heads inside the cell. The product of gene 4 is not required for cutting phage DNA into mature length pieces. The 4^? phenotype is similar to the 10^? and 26^? phenotypes [Lenk et al., Virology68, 182–199 (1975)]. Genes, 4, 10, and 26 map contiguously. All the proteins found in P22 phage and phage precursor particles have been matched with their genes.     

Damage and mutagenesis of bacteriophage lambda induced by high pH.

Bacteriophage lambda-Escherichia coli complexes exhibited remarkable sensitivity to alkaline pH 10.0 at 37 degrees C. The decline in plaque forming units after alkali treatment was more pronounced in complexes with some of the radiation repair defective mutants of E. coli K-12, i.e. uvrArecA, recA, rer and lexA mutants as compared to those of uvrA, recB and wild-type strains. The red gene of lambda phage and recA gene of E. coli seem to have a complementary effect on the alkali induced lesions. Alkaline treatment to lysogenic lambda phage was also found to be mutagenic. An enhanced level of mutagenesis was observed when treated phage particles were allowed to adsorb on treated wild-type bacteria. Moreover, the alkali treatment to lysogen (lambda cI857-E. coli) resulted in prophage induction in nutrient broth even at 32 degrees C. Thus on the basis of these results the role of error prone SOS repair systems in the repair of alkali induced lesions in lysogenic bacteriophage lambda has been suggested.     

The genomic RNA of mengovirus: I. Location of the poly(C) tract

Strains of λ phage containing a mutated cII, cIII, or cy gene in addition to a cro^? mutation were constructed. Each single mutant and the double mutants were analyzed for growth behavior and for expression of specific early and late λ proteins. λc1857cro 16 is unable to grow well at 30° and at 43°. This strain is incapable of turning off early genes (of the b region, of the P_L and P_R operons, and the cU and int genes) and is defective in the expression of late functions. The clear mutations permit phage growth at 30°. They relieve the inhibitory effect conferred by the cro^? mutation on phage growth at 43° to different extents. Our results also indicate that the cII and cIII proteins do not necessarily act at y in the regulation of late functions.     

Genome analysis of an inducible prophage and prophage remnants integrated in the Streptococcus pyogenes strain SF370

The mitomycin C inducible prophage SF370.1 from the highly pathogenic M1 serotype Streptococcus pyogenes isolate SF370 showed a 41-kb-long genome whose genetic organization resembled that of SF11-like pac-site Siphoviridae. Its closest relative was prophage NIH1.1 from an M3 serotype S. pyogenes strain, followed by S. pneumoniae phage MM1 and Lactobacillus phage phig1e, Listeria phage A118, and Bacillus phage SPP1 in a gradient of relatedness. Sequence similarity with the previously described prophages SF370.2 and SF370.3 from the same polylysogenic SF370 strain were mainly limited to the tail fiber genes. As in these two other prophages, SF370.1 encoded likely lysogenic conversion genes between the phage lysin and the right attachment site. The genes encoded the pyrogenic exotoxin C of S. pyogenes and a protein sharing sequence similarity with both DNases and mitogenic factors. The screening of the SF370 genome revealed further prophage-like elements. A 13-kb-long phage remnant SF370.4 encoded lysogeny and DNA replication genes. A closely related prophage remnant was identified in S. pyogenes strain Manfredo at a corresponding genome position. The two prophages differed by internal indels and gene replacements. Four phage-like integrases were detected; three were still accompanied by likely repressor genes. All prophage elements were integrated into coding sequences. The phage sequences complemented the coding sequences in all cases. The DNA repair genes mutL and mutS were separated by the prophage remnant SF370.4; prophage SF370.1 and S. pneumoniae phage MM1 integrated into homologous chromosomal locations. The prophage sequences were interpreted with a hypothesis that predicts elements of cooperation and an arms race between phage and host genomes.     

Role of gene 8 product in morphogenesis of bacteriophage T3

The product of gene 8 (gp8) of T3 phage is one of the minor head proteins located at the phage head-tail junction. To determine the role of gp8, an amber (8-) and four temperature-sensitive mutants (ts8) were characterized by sedimentation analysis, polyacrylamide gel electrophoresis, and extract complementation. Neither DNA-containing particles nor empty particles were formed in cells infected with 8-. In addition, prohead assembly was greatly reduced. Prohead assembly was also blocked in cells infected with all ts8 mutants at 42 degrees and with some ts8 even at 37 degrees. Proheads containing gpts8 were converted to empty heads when cell lysates were treated with chloroform. The protein compositions of proheads showed that the minor head proteins, gp8, gp15, and gp16, were lost from proheads formed in cells infected with ts8, but these minor proteins were present in proheads formed in cells infected with double mutants of ts8 and 5- or 19-, which are defective in DNA synthesis or DNA maturation, respectively. In vitro complementation experiments suggested that a ts mutation in gene 8 affected not only DNA packaging but also subsequent assembly steps. From these results, it is concluded that gp8 plays multiple roles in T3 phage morphogenesis, including prohead assembly, prohead stabilization, DNA packaging, and subsequent events.     

Control of gene activation of P2 prophage by satellite phage P4

In the preceding paper we report that satellite phage P4 can derepress P2 lysogens (Six and Lindqvist, 1978). We now show that derepression leads to the expression of the early as well as the late gene regions of the P2 prophage. In the absence of transactivation, P2 prophage late genes will be expressed only if the early P2 gene A function is available. This was demonstrated with P4 mutants defective in gene α. Such mutants are proficient in derepression but were found to be transactivation deficient, hence restricted to derepression as a means of turning on P2 prophage late genes. Thus two modes of P4-induced late gene expression can be distinguished: transactivation and derepression. Derepression precedes transactivation by about 30 min, but the extra time needed for transactivation appears not to be due to a requirement for early P2 gene products.     

A phage P1 virulent mutation at a new map location

A newly isolated Pl virulent mutation, vir11, is not suppressed by reb mutations that suppress P1virB mutations. The location of P1virll on the vegetative phage map is at the left-most end, beyond all other known markers. Thus, in the circular prophage, virII may be closely linked to the cl repressor gene.     

Mutants of E. coli in which bacteriophage P4 cannot activate the late genes of its helper, bacteriophage P2.

Mutants of Escherichia coli strain C have been isolated which fail to support normal growth of satellite phage P4 even though its helper P2 is present as a prophage. The host mutations prevent P4-directed activation of the late genes of the P2 helper. In contrast, activation of P2 late genes during P2 infection is unimpaired. These results indicate that P2 and P4 activate the late genes of P2 by different mechanisms. P4-directed transactivation is temperature-sensitive in two of the four mutants. Mutants of P4 which overcome the defect in the host can be isolated only on these thermosensitive host mutants. Growth of coliphages λ, φ80, P1, T4, and φX174 is defective on one or more of these bacterial mutants. Growth of T7 is not affected.     

Superinfection immunity and prophage repression in phage P1. IV. The c1 repressor bypass function and the role of c4 repressor in immunity

Previous work indicated that both genes c1 and c4 of phage P1 and P1 are necessary for maintenance of lysogeny and suggested the c4 but not c1 is required for the specificity of superinfection immunity. We now show that c4^? P1 lysogens do not express superinfection immunity. In addition, we have isolated mutants in a c1 repressor bypass function (Reb) which permits P1 to grow in the presence of c1⁺ product. Reb mutants were isolated by selecting for suppression of the virB or c4 phenotype, so synthesis of reb product is negatively controlled by both c4 repressor and by the site at which virB mutants occur. Because some P1reb mutants are suppressed by nonsense suppressors, reb must code for a protein. However, P1reb mutants cannot be complemented by P1reb⁺ phage in the same cell, which indicates that the reb protein acts predominantly in “cis”. The reb⁺ product is not needed for prophage replication but is necessary for normal vegetative phage growth by c1⁺ phage. Possible modes of action of reb are discussed.     

Morphogenetic structures present in lysates of amber mutants of bacteriophage Mu

The Mu phage particle is structurally similar to that of the T-even phages, consisting of an icosahedral head and contractile tail. This study continues an analysis of the morphogenesis of the Mu phage particle by defining the structural defects resulting from mutations in specific Mu genes. Defective lysates produced by induction of 55 amber mutants, representing 24 essential genes, were examined in the electron microscope and categorized into eight classes based on the observed phage-related structures. (1) Mutations in genes lys, F and G, and some H mutations, did not cause a visible alteration in particle structure. (2) Mutants defective in genes A, B, and C produced no detectable phage structures, consistent with their lack of production of late RNA. (3) Extracts defective in genes L, M, Y, N, P, Q, V, W, and R contained only head structures, and these appeared normal. (4) K-defective mutants accumulated free heads as well as free tails which were longer than normal and variable in length. (5) Tails which appeared normal were the only structures found in T- and some I-defective extracts. (6) Free tails and empty heads accumulated in D-, E-, and some I- and H-defective extracts. These heads were as much as 16% smaller than normal heads. The heads found in some I amber lysates had a protruding neck-like structure and unusually thick shells suggestive of a scaffolding-like structure. (7) Defects in gene J resulted in the accumulation of unattached tails and full heads. (8) Previous analysis of lysates produced by inversion-defective gin mutants fixed in the G(+) orientation demonstrated that S and U mutants produced particles lacking tail fibers (F.J. Grundy and M.M. Howe (1984), Virology 134, 296-317). In these experiments with Gin+ phages S and U mutants produced apparently normal phage particles. Presumably the tail fiber defects were masked by the production of S' and U' proteins by G(-) phages in the population.