Molecular characterization of new group A streptococcal bacteriophages containing the gene for streptococcal erythrogenic toxin A (speA)

Summary Bacteriophage T12 is the prototype phage carrying the streptococcal erythrogenic toxin A (speA) gene. To examine more closely the phages involved in lysogenic conversion, we examined 300 group A streptococcal strains, and identified and isolated two new phages that carry the speA gene. The molecular sizes of these phage genomes were between 32 and 40 kb, similar to that of phage T12 (35 kb). However, as ascertained by restriction analysis, the physical maps of the new phage genomes were different from phage T12 and from each other. Hybridization analysis also showed that all of these phages were only partially related to one another and the speA gene was always located close to the phage attachment site. Additionally, colony hybridization showed that whereas phage T12 or one of its close relatives is the most common phage associated with the group A streptococci, phage 49 has a much stronger association with the speA gene. A defective phage was also found following pulsed field gel electrophoresis of total phage DNA. This phage appears to be a resident of strain T25₃c and is found only following induction of a T25₃c lysogen. Restriction enzyme analysis of the isolated defective phage DNA suggests that it is the source of the submolar amounts of DNA previously found in association with phage T12 digestion patterns. Additionally, the defective phage may serve as the site of integration of the speA gene-carrying phages described above.     

The kinetics of the requirement for X gene product in bacteriophage P 22

Summary The kinetic study of the requirement for X gene product showed that the average burst size of the P22 phage depended on the length of the permissive interval in which the X function was expressed. Results of the temperature shift experiments with the clear plaque recombinants tsX c ₂ ⁵ and ts 25.1 c ₂ ⁵ gave a complicated pattern of the phage yield response.It is concluded that X gene product, besides the control function in the initiation of the phage development, is involved directly or indirectly in the control of late functions and is required throughout the entire period of the phage development.     

Superinfection exclusion and changes in cellular transport processes in phage infected Salmonella typhimurium.

Summary The changes induced by bacteriophage P22 in the cellular transport process(es) of the host Salmonella typhimurium (Taneja et al., 1975; Khandekar et al., 1975; Bandyopadhyay and Chakravorty, 1976) involve interactions between the superinfection exclusion system of the resident prophage and the C immunity region of the superinfecting phage. The sieA gene of the prophage interferes with the changes in the cellular transport process induced by the superinfecting phage. However, if the superinfecting phage carries active C ₁ and C ₂ genes of the superinfecting phage seem to be expressed in the sie A⁺ lysogen.     

Identification of the E. coli groNB(nusB) gene product

Summary The E. coli groNB(nusB) gene product has been previously shown to be necessary for bacteriophage N protein function. The product of the groNB gene has been identified on SDS polyacrylamide gels after infection of UV-irradiated E. coli cells with various groNB ⁺ transducing phage derivatives. It is a polypeptide with an apparent molecular weight of 14,000 daltons. Transducing phage carrying either a deletion or an amber mutation in the groNB gene fail to synthesize the 14,000-M_r polypeptide chain upon infection of a sup ⁺ host. However, am ⁺ revertants of the groNBam phage do induce the synthesis of the polypeptide.     

Specific binding affinity for DNA of the L phage (Salmonella typhimurium) in extracts of Escherichia coli

The repressor gene c _II of the L phage was cloned into plasmid pHC624 and expressed in E. coli. Two separate binding affinities for L phage DNA were identified during fractionation of protein extract of that strain. The activity that salts out in low concentration of ammonium sulphate belonged to the repressor, the activity that salts out in high concentrations of (NH₄)₂SO₄ was proved to be of E. coli origin. Binding sites for the two proteins are located on different fragments of the L phage genome.     

Identity of a gene responsible for suppression of aminoacyl-tRNA synthetase mutations with rpsT, the structural gene for ribosomal protein S20

Summary Specialized transducing lines of phage carrying segments between thr and car from the E. coli chromosome have been isolated. With help of these phages it has been shown that the gene sup _S20 (Böck et al., 1974) corresponds to rpsT, the structural gene for ribosomal protein S20.     

Cloned structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12

Summary pTU 100 is a hybrid plasmid constructed by cloning a 7.5 Kb EcoRI fragment (carrying the wildtype ompA gene) onto pSC 101 (Henning et al., 1979). This plasmid confers sensitivity to phages Tull^* and K3h1 when present in an ompA host strain, due to the expression of the phage receptor protein II^* from the plasmid ompA ⁺ gene. Plasmid mutants have been isolated that have become resistant to one or both of these phages. Restriction endonuclease analysis and DNA-sequencing studies in these plasmids demonstrate that a BamHI site and two PvuII sites are located within the ompA gene. BamHI cuts the gene at a site corresponding to residue 227 within a total of 325 amino acid residues.Neither the wildtype ompA gene nor the BamHI fragment encoding the NH₂-terminal part of the protein (residues 1–227) could be transferred to a high copy number plasmid, presumably due to lethal overproduction of the protein or its NH₂-terminal fragment. However, the NH₂-terminal fragment derived from one of the ompA mutants of pTU100 could be transferred to the high copy number plasmid pBR322, and was expressed in the presence of the amber suppressors supD or supF. Under these conditions two new envelope proteins with apparent molecular weights of 30,000 and 24,000 were synthesized, and the cells became sensitive to phage TuII^*, indicating the presence of phage receptor activity in the outer membrane. The major, 24,000 dalton protein has the molecular weight expected of a protein comprising residues 1–227 of protein II^*. DNA-sequencing studies demonstrated that no termination codons are present in the DNA region immediately downstream from the BamHI site at residue 227 in this hybrid plasmid, and it is therefore likely that the 24,000-dalton protein arises from the posttranslational proteolytic cleavage of a larger polypeptide. The 30,000-dalton protein is a likely candidate for such a larger polypeptide. These results also demonstrate that the 98 CO₂H-terminal residues of wildtype protein II^* (resisdues 228–325) are not required either for the activity of the protein as a phage receptor or for its incorporation into the outer membrane.     

Ant-mediated inactivation of Salmonella phage L-specified repression at OR of prophage L.

Summary Ant product of phage P22 inactivates repression of prophage L at the right-hand operator o_R and allows for transactivation of prophage gene 12. The transactivation efficiency observed with a series of phage and prophage recombinants, using single superinfection of a lysogenic bacterium, is about the same as that recently observed at o_L of prophage L. This finding is in contrast to the failure to demonstrate derepression at o_R of prophage L in an experimental system employing double superinfection (Prell, 1978a). The reasons for the differing results are discussed and it is shown that derepression by the ant product in trans at o_R of the prophage is not modified to any significant degree by the immunity specificity (L or P22) of the prophage or of the superinfecting phage.     

Genetic map of coliphage 186 from a novel use of marker rescue frequencies

Summary A genetic map of phage 186 has been constructed, using the frequency of marker rescue from 186 mutant prophages for genes to the left of att, and int promoted recombination for genes to its right. At the left end of the genome lie 7 genes involved in the formation of the phage head, followed to the right by the lysis gene P, a gene (O) of unknown function, and a group of 11 genes involved in the formation of the phage tail. Gene B, the late control gene, lies to the right of this group but to the left of the phage attachment site. To the right of the att site lie the non-essential genes (cI and cII) involved in lysogen formation and the gene (A) required for 186 DNA synthesis.     

Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa

Pf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf 4 i nhibition t oxin and Rorf0727 be named PfiA for Pf iT a ntitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5′-AATTC N₅GTTAA -3′) overlapping the -35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements.     

Control of the production of orthophosphate repressible extracellular enzymes in Neurospora crassa

Summary N-1, a plasmid isolated from a strain ofShigella flexneri in Japan more than 10 years ago, mediates the phage inhibition phenotype which has recently been found to be characteristic of plasmids of the H₂ incompatibility group. Using the criteria of phage inhibition, surface exclusion and incompatibility, the N-1 plasmid is shown to be closely related to H₂ plasmids isolated from non-typhoid salmonella and distantly related to H₁ plasmids isolated fromSalmonella typhi. Plasmids of other incompatibility groups did not show the H₂ type of phage inhibition.     

Isolation and characterization of a temperature-sensitive amber suppressor mutant of Escherichia coli K12

Summary By mutagenizing an E. coli strain carrying an amber suppressor supD ^- (or su _I ⁺), we isolated a mutant whose amber suppressor activity was now temperature-sensitive. The mutant suppressor gene was named sup-126, which was found to be cotransduced with the his gene by phage P1vir at the frequency of ca. 20%. At 30° C it suppresses many amber mutations of E. coli, phage T4, and phage . At 42° C, however, it can suppress none of over 30 amber mutations tested so far. The sup-126 mutation is unambiguous and stable enough to be useful for making production of an amber protein temperature-sensitive.     

A new class of clear mutants from coliphage 434 hy not complementing CII and C3 mutants for lysogenization

Summary Clear mutants which differ from regular C _I , C _II , CIIIand y mutants have been isolated from phage 434 hy. These mutants resemble C _I mutants in plaque and spot phenotype but efficiently complement C _I mutants for lysogenization. Like C _II mutants, they do not complement authentic C _II mutants for lysogenization but in contrast to C _II mutants they also fail to complement C _III mutants. They map between the lambda-434 non-homology region and Co ₁ (aC _II mutant). On account of this map position adjacent to C _II the mutants of the new type are called C _IIa . They arise from phage 434 hy with a frequency comparable to that of C _I and C _II mutants. Such mutants are also obtained from phage lambda but apparently not from phage b₅. C _IIa mutants would not fit into a picture of three independently acting cistrons C _I , C_II, and C _III . The hypothesis is presented that C _IIa and C _II mutants are in the same structural gene. Two possibilities are discussed that would account for the complementation patterns: 1. C _IIa mutants may block the expression of gene C _III in cis position; or 2. the products of genes C _II and C _III function through an oligomeric complex they form.     

Gene 49 endonuclease VII is not essential for multiplicity reactivation of bacteriophage T4

Summary Endonuclease VII, the product of phage T4 gene 49, has been shown previously to resolve Holliday structures in vitro. Two different processes, genetic recombination and multiplicity reactivation are presumed to have Holliday structure intermediates. Other workers have shown that genetic recombination is reduced in a gene 49 mutant infection. However, in the present study, multiplicity reactivation of UV-irradiated ts or amber mutant phage defective in gene 49 was nearly identical to that of UV-irradiated wild-type phage T4. Thus endonuclease VII is not thought to be essential for multiplicity reactivation of phage T4.     

Circular structure of the genome of phage ?H in a lysogenic Halobacterium halobium

Summary Phage H is a temperate phage, i.e., it can establish lysogeny in the archaebacterium Halobacterium halobium. H-lysogens are immune to phage infection and phage production is spontaneously induced at a rate of about 10^-7. In the prophage state. H DNA exists as a covalently closed circle of 57 kb.The frequent occurrence of clones carrying the phage genome but unable to produce phage is another proof of the high variability of DNA in H. halobium. In one such strain, R₁-3, the phage genome has undergone a structural change which may have abolished an essential phage gene.     

Gene N regulator function of phage λimm21: Evidence that a site of N action differs from a site of N recognition

We report the isolation and characterization of a new mutation in the hybrid phage λimm21. Both genetic and physiological studies demonstrate that this new mutation, N_21?1, is similar to N mutations of phage λ. As in the case of the N gene of λ (Ni_λ), the N_21?1 mutation maps immediately to the left of the cI gene and has a pleiotropic effect on the expression of phage functions. Although these studies strongly suggest that phage 21 has an N function, they do not definitely locate the N_21?1 mutation within the N₂₁ structural gene.Reported here are studies demonstrating that N₂₁ acts in trans, similar to N_λ, to stimulate the expression of phage functions. N products show an immunity specificity; N₂₁ being only active on phage carrying the immunity region of phage 21, while the n_λ is only active on phage carrying the immunity region of λ or phage 434. However, one site of action for N_λ can be rescued from phage 21. We propose that the specificity of an N function is determined by its sites of recognition and that these sites may be different from the sites of N action.