Isolation of Specialized Transducing Bacteriophages for Gluconate 6-Phosphate Dehydrogenase (gnd) of Escherichia coli

Specialized transducing phages for gluconate 6-phosphate dehydrogenase (gnd), a constitutive enzyme in Escherichia coli, have been isolated using a method previously described for other genes. The gnd-his region, carried on an F' episome, was first transposed to tonB. Rare phages carrying gnd were selected, by transduction, from phi80 lysogens of these strains; one phage also carried his (phi80gndhis). From the transductants, high-frequency transducing lysates were obtained; low multiplicity of infection then yielded defective lysogens. tonB deletion analysis of the phi80dgndhis lysogen shows the order of genes in the prophage to be imm80...hisOGD...gnd; according to a marker rescue experiment most phage late genes have been replaced by bacterial deoxyribonucleic acid. A heat-inducible, lysis-defective lambda-phi80 hybrid derivative of phi80dgndhis has been prepared.     

Derivation of an F-Merogenote and a φ80 High-Frequency Transducing Phage Carrying the Histidine Operon of Salmonella

An F' factor, FS400, carrying the his operon, the gnd gene, and the rfb gene cluster of Salmonella typhimurium was isolated. FS400 was introduced into an Escherichia coli strain having a lengthy deletion of the his gene region. From this strain, Hfr derivatives were isolated which had the F' factor integrated in the tonB locus near the attachment site of phi80. One of the Hfr strains was lysogenized with a heat-inducible, h mutant of phi80, and from this strain a high-frequency transducing phage carrying the his genes and the gnd gene of Salmonella was isolated.     

Molecular structure of hybrid phage phi80hy43

The phage hybrid phi80hy43 derived from a vegetative cross phi80cIhlambda x lambdacIc17 was constructed for discrimination phi80mono- and polylysogens. Molecular structure of this hybrid was established using heteroduplex analysis and restriction endonuclease EcoRI. It is found that the hybrid phi80hy43 represents a phage phi80 containing a foreign piece of DNA between genes cI and 0. The length of this piece of DNA comprises 0.7%, corresponding to the length of cy-cII region of th phage lambda. So it is believed that the hybrid phi80hy43 was formed due to insertion of the lambdacy region with the mutation c17 into phi80hlambda phage genome.     

Integration of specialized transducing bacteriophage lambda cI857 St68 h80 dgnd his by an unusual pathway promotes formation of deletions and generates a new translocatable element.

Molecular and genetic studies have revealed that several illegitimate recombinational events are associated with integration of the specialized transducing bacteriophage lambda cI57 St68 h80 dgnd his into either the Escherichia coli chromosome or into a plasmid. Most Gnd+ His+ transductants did not carry the prophage at att phi-80, and 10% were not immune to lambda, i.e., "nonlysogenic." Integration of the phage was independent of the phage Int and Red gene products and of the host's general recombination (Rec) system. In further studies, bacterial strains were selected which carried the phage integrated into an R-factor, pSC50. Restriction endonuclease analysis of plasmid deoxyribonucleic acid (DNA) purified from these strains showed that formation of the hybrid plasmids resulted from recombination between a single region of pSC50 and one of several sites within the lambda-phi 80 portion of the phage. Furthermore the his-gnd region of the phage, present in the chromosome of one nonlysogenic transductant, was shown to be able to translocate to pSC50. Concomitant deletion of phage DNA sequences or pSC50 DNA was frequently observed in conjunction with these integration or translocation events. In supplemental studies, a 22- to 24-megadalton segment of the his-gnd region of the chromosome of a prototrophic recA E. coli strain was shown to translocate to pSC50. One terminus of this translocatable segment was near gnd and was the same as a terminus of the his-gnd segment of the phage which translocated from the chromosome of the nonlysogenic transductant. These data suggest that integration of lambda cI857 St 68 h80 dgnd his may be directed by a recombinationally active sequence on another replicon and that the resulting cointegrate structure is subject to the formation of deletions which extend from the recombinationally active sequence. Translocation of the his-gnd portion of the phage probably requires prior replicon fusion, whereas the his-gnd region of the normal E. coli chromosome may comprise a discrete, transposable element.     

Organization of the early region of bacteriophage phi 80. Genes and proteins

An EcoRI segment containing the early region of bacteriophage phi 80 DNA that controls immunity and lytic growth was identified as a segment whose presence on a plasmid prevented growth of infecting phi 80cI phage. The nucleotide sequence of the segment (EcoRI-F) and adjacent regions was determined. Based on the positions of amber mutations and the sizes of some gene products, the reading frames for five genes were identified. From the relative locations of these genes in the genome, the properties of some isolated gene products, and the analysis of the structures of predicted proteins, the following phi 80 to lambda analogies are deduced: genes cI and cII to their lambda namesakes; gene 30 to cro; gene 15 to O; and gene 14 to P. An amber mutation by which gene 16 was defined is a nonsense mutation in the frame for gene 15 protein, excluding the presence of gene 16. An amber mutation in gene 14 or 15 inhibits phage DNA synthesis, as is the case with their lambda analogues, gene O or P. Some characteristics of proteins from the early region predicted from their primary structures and their possible functions are discussed.     

Restrictase BamHI, HindII, EcoRI and SmaI mapping of hybrid lambda-phi 80 phages with recombination in the structural gene region

Hybrids lambda H lambda T80 with recombination in the region of structural genes have lambda head and phi 80 tail genes. In this paper the molecular structure of 5 independently isolated hybrids was established using restriction endonucleases. It has been shown that all of them have a recombinant head or tail. A deletion of 4,8% lambda was demonstrated in the immunity region of phi 80vir phage. Co-ordinates of restriction sites for BamHI, HindIII, EcoRI and SmaI restriction endonucleases on phi 80 DNA were calculated.     

Genetics of bacteriophage phi 80--a review

The genetic maps of bacteriophage lambda and lambdoid phage phi 80 are compared. The gene organization of phi 80 is very similar to that of lambda, as shown by isolation and characterization of many am, ts and c (clear) mutants of the phage. In general, the essential genes located in the same position on the genetic map of the phages lambda and phi 80 fulfill the same functions. These include the gene clusters coding for the head and tail proteins, genes for DNA synthesis, and the genes controlling lysogeny and late gene expression. The specific regulatory features of phi 80 in relation to the N function of lambda are discussed, but they require further clarification. The two phages differ in immunity specificity, host range, conversion property and temperature sensitivity.     

New transducing phage with RNA polymerase beta- and beta'-subunit genes derived from a hybrid phage lambda att80: isolation, genetic analysis and physical mapping]

A hybrid lambda att 80 phage with the genetic structure lambda (A-J) phi 80 (att-int-xis) imm lambda..cI857s7 is shown to be a convenient vector for creating transducing phages. On the one hand, the restriction analysis indicates that it has 3 restriction sites for EcoRI in comparison with 5 and 9 sites for parental phages lambda and phi 80 respectively. On the other hand, its buoyant density is less than that of phage lambda and under centrifugation it is easier separated from the phage transducing particles. When lambda att 80 prophage was excluded from the bfe locus of Escherichia coli, transducing phages with genes of two RNA polymerase beta-subunits (rpoB and rpoC) were isolated. To identify the latter, a convenient genetic test was worked out. A physical map of lambda att 80 drifd 35 transducing phage, carrying rpoB and rpoC genes has been constructed using endonucleases EcoRI and HindIII. A comparison of this map and the corresponding maps of transducing phages lambda drifd 18 and lambda drifd 47, studied earlier, led to the discovery of two integration sites of phage lambda within the locus bfe spaced apart by about 1800 nucleotide pairs. At all the sites both phages (lambda and lambda att 80) have integrated in the locus bfe in the counter clockwise order.     

Mapping of the c-region of phage phi80

A set of c-mutants of the phage phi80 is isolated. These mutants fit into three genes. According to plaque morphology and frequency of lysogenization of mutants, the genes were named cI, cII and cIII as it was previously done for phage lambda. Their order, determinated by mutant phage crosses, is cIII-sus326-cI-cII-sus250. Sus326 is a mutation in the gene 15, so it is probably an analogue of the N gene of the phage lambda. Thermoinducible mutants of the phage phi80 cts11 and cts12 correspond to the mutant types cItsB and cItsA of the phage lambda and they complement each other. Thus, it is supposed that phi80 phage repressor molecules consist of few protein subunits.     

Mapping of genes 14 and 16 of phage phi 80

This study was performed to obtain more detailed information on the early region of phage phi 80. For this purpose, a selective method for isolation of early phage ts mutants was developed. 32 ts mutants of phi 80 obtained and early sus mutants isolated by Sato were characterized by complementation tests and deletion mapping. The results of this study differ from those of Sato in two aspects. Firstly, it was shown that sus mutations 250, 258 and 326 of phi 80 define one gene 16, and that gene 15 previously determined by Sato via sus 326 mutation does not exist. Secondly, we found that the order of the phi 80 early genes 14 and 16 is cI-16-14, contrary to the Sato data (cI-14-16).     

Preparation of plasmids from lambdoid phages and studies on their incompatibilities.

Various lambdoid phages, including those carrying the immunity region of 434, 21, and φ80, were found to give rise to fragments of DNA that can be perpetuated in the plasmid state. The plasmids, imm434dv, imm21dv, and φ80dv, were similar to the already known plasmid λdv in size, genetic constitution, oligomeric state, copy number, and stability. Cells carrying two kinds of dv plasmids were constructed by transformation. It was shown that a pair of plasmids is compatible in a cell if they originate from phages differing in immunity region. On the other hand, a pair of plasmids is incompatible if they are derived from phages carrying the same immunity region. These observations were taken to imply that the incompatibility of a pair of plasmids is determined by the “immunity region” of the plasmid genome that contains an autorepressor gene and a promoter-operator. The region that carries initiator genes and a site for initiation of plasmid replication is not primarily important for determination of the incompatibility. Plasmids λdv and imm434dv, which are very closely related to each other, behaved in an intermediate fashion.     

Lambda H-lambda T 80 hybrid study of the DNA structural gene region in lambda and phi 80 phages

Hybrids lambda H lambda T80 are formed due to recombination of the phage lambda att80 and phi 80 prophage partially deleted in the region of structural genes. Genetic structure of 22 independently isolated lambda H lambda T80 hybrids was determined by the restriction method and it was shown that recombination took place in the genes A, C, D and H. The frequencies of hybrid formation diminish from 1.10(-3) to 4.10(-5) for this gene order, which suggests that the polar divergence of nucleotide sequencies in the region of structural genes exists. It was found that formation of hybrids with recombination in the region of "weak" homology (gene H) was possible only when the region of "strong" homology was present in the deleted phi 80 prophage to initiate recombination.     

Isolation and characterization of the specialized transducing bacteriophages phi80dargF and lambdah80cI857 dargF: specific cleavage of arginine transducing deoxyribonucleic acid by the endonucleases EcoRI and SmaR.

The directed transposition of argF to the tonB locus of the Escherichia coli chromosome and the subsequent isolation of the specialized transducing phage phi80dargF is described. The structure of this phage has been has been determined. A hybrid lambdah80cI857dargF phage has been constructed. Deoxyribonucleic acid isolated from these and their parent bacteriophages has been specifically cleaved by the endonucleases EcoRI and SmaR; the unique deoxyribonucleic acid fragments thus obtained have been resolved and analyzed by electrophoresis in agarose gel.     

HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli genome, and location of the tonB gene.

The HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli chromosome are presented. Analysis of phage DNAs carrying tonB mutations has allowed identification of a 1,730-base pair HindII fragment containing at least part of the tonB gene. This fragment is 4,020 base pairs from the end of trpA, with the total distance from attphi80 to trpA being 6,550 +/- 800 base pairs. Properties of hybrid plasmids containing insertions of various tonB+ restriction fragments suggest that tonB lies completely within the 1,730-base pair fragment. In addition, apparent fusions of beta-galactoside to proteins within the tonB region suggest that the entire region codes for more than one polypeptide.     

Excision and duplication of su3+-transducing fragments carried by bacteriophage phi 80. I. Novel structure of phi 80sus2psu3+ DNA molecule.

DNA molecules of phi 80sus2psu3+ and phi 80dsu3+ isolated by Andoh and Ozeki (1968) were studied by the electron microscope heteroduplex method. The phi 80sus2psu3+ and phi 80dsu3+ DNA lengths were found to be 108.7 and 103.3% of the phi 80 DNA, respectively. The phi 80sus2psu3+/phi 80 heteroduplex shows an insertion loop of 8.7% of the phi 80 DNA which migrates from 7.7 to 9.7%, as measured relative to the left (0%) and right (100%) termini of the mature phi 80 DNA molecule. The region of loop migration occupies the central region of the phi 80 head gene cluster. The presence of su3+-containing Escherichia coli DNA of 6.7% phi 80 unit flanked by two homologous regions of phage DNA of 2.0% of phi 80 unit gives rise to a movable insertion loop. In phi 80dsu3+, from which phi 80sus2psu3+ was derived, 50.5% of the phi 80 DNA at the left arm was replaced by E. coli DNA containing the su3+ gene, equivalent to about 53.8% phi 80 unit in length. The phi 80sus2psu3+/phi 80dsu3+ heteroduplex appears as a double-stranded molecule that bifurcates into two clearly visible single-stranded regions, rejoins, bifurcates, and rejoins again. The middle double-stranded stretches of 6.7% phi 80 unit correspond to the E. coli DNA inserted in phi 80sus2psu3+. Therefore the transducing fragment carried by phi 80sus2psu3+ originates from the inside region of the transducing fragment of defective phage phi 80dsu3+ by at least two illegitimate recombination events.     

Lysogenic conversion caused by phage phi 80. III. The mapping of the conversion gene and additional characterization of the phenomenon

The cor gene specifies lysogenic conversion caused by the lambdoid phage phi 80. The cor gene product inhibits tonA function in infected and lysogenic cells. The cells harboring pBR322 plasmid with the cloned cor gene of phi 80 became resistant to the phages T1 and phi 80 (TonA phenotype). The cor gene was mapped between 24 and 13 genes on the phi 80 phage genetic map. It is not essential for phage lytic growth. Its presence in lysogens leads up to accumulation of tonA mutants in a cell population.     

Construction of φ80 dhis Carrying Salmonella typhimurium Histidine Operon Mutations

Starting with a previously isolated F'his episome and phi80 dhis imm(lambda) cI857 transducing phage, we have constructed recombinant elements bearing previously isolated his mutations from Salmonella typhimurium. These phages were constructed as sources of deoxyribonucleic acid for in vitro biochemical experiments on gene regulation. The manipulation of genes between S. typhimurium and Escherichia coli described here may be useful in studying other S. typhimurium operons.     

Nucleotide sequence of the immunity region of Bacillus subtilis bacteriophage phi 105: identification of the repressor gene and its mRNA and protein products

A gene involved in the regulation of lysogeny in the temperate Bacillus subtilis phage phi 105 has been identified and isolated. A plasmid, pDC4, was constructed that contains a 740-bp HindIII-PvuII fragment that is derived from the phi 105 immunity region and is capable of rendering B. subtilis immune to infection by phi 105. Three different hybrid plasmids that contain the 740-bp fragment, pAG101 [Cully and Garro, J. Virol. 34 (1980) 789-791], pDC1 and pDC2, were found to synthesize a common 18-kDal polypeptide in B. subtilis minicells and Escherichia coli maxicells. The nucleotide (nt) sequence of this region revealed three open reading frames (ORFs) that predict proteins with Mrs of 16521, 7332, and 5516. In vivo synthesized phi 105 prophage RNA was mapped by primer extension and shown to be transcribed from the DNA strand coding for the Mr 16521 protein. The 5' end of the phi 105 lysogen RNA was mapped to a region that contains conserved sequences for RNA polymerase recognition.