Genetic characterization of a phi80 transducing bacteriophage carrying the histidine operon of Salmonella typhimurium.

A phi 80 transducing phage, phi 80imm lambdadhis, carrying the Salmonella his-gnd region, was characterized by immunity studies, tonB deletion analysis, and marker rescue analysis. Phi 80imm lambdadhis retains the phage immunity region of the phi 80-lambda hybrid phage from which it was derived. Bacterial genes replace most late phage genes. Deletion analysis shows the prophage gene order to be immlambda-his-gnd and indicates the orientation of the his operon to be hisOGDCBHAFIE-gnd. The structure of phi 80imm lambdadhis is remarkably similar to two independently isolated phi 80 phages that carry the his-gnd region of Escherichia coli and that, like phi80imm lambdahis, were derived by directed gene transposition to the tonB locus. A derivative of phi 80imm lambdadhis that is phi 80 immune is also reported.     

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.     

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.     

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.     

Isolation and characterization of lambda transducing bacteriophages for the su1+ (supD minus) amber suppressor of Escherichia coli.

Specialized lambda transducing phages for the sul+ (supD-) amber suppressor in Escherichia coli K-12 have been isolated, using a secondary site lambda-cI857 lysogen in which we have shown the prophage to be closely linked to sul+.sul+ transducing particles were detected frequently, at 10-5 per plaque-forming unit, in lysates prepared from the secondary-site lysogens. High-frequency transducing lysates were obtained from several independently isolated sul+ transductants and were analyzed by CsCl equilibrium density gradient centrifugation. The transducing phages are defective; marker rescue analysis indicates that the lambda-N gene is not present. In lambda-cI857DELTANdSul+, a bio-type transducing phage, the genes specifying recombination and excision functions have been replaced by bacterial deoxyribonucleic acid.     

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.     

Isolation and Characterization of φ80 Transducing Bacteriophage for a Ribonucleic Acid Polymerase Gene

A new method for isolating specialized transducing phages is described. It was used to isolate a group of phi80 transducing phages which carry various bacterial markers from the metB region of the Escherichia coli chromosome. Some of the phages selected for transduction of the supA36 marker were also shown to carry rif, a locus known to specify the beta subunit of ribonucleic acid polymerase. Expression of the prophage rif(r) gene in lysogens was demonstrated by its ability to confer rifampin resistance on part of the cellular ribonucleic acid polymerase pool.     

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.     

A FhuA mutant of Escherichia coli is infected by phage T1-independent of TonB

Infection of Escherichia coli K-12 by phages T1 and phi 80 requires the FhuA outer membrane protein and the TonB protein. Mutations in the N-terminal globular domain close to the predicted channel in the beta-barrel of FhuA were created. The FhuA Delta 107-111 N104K K110D L111P mutant and the FhuA(L(109)DPNGLK(110)) insertion mutant were sensitive to phage T1, but nearly resistant to phage phi 80. FhuA Delta 107-111 N104K K110D L111P mediated phage T1 infection in a tonB mutant without formation of TonB-independent phage T1 host-range mutants. The FhuA mutants showed no altered sensitivity to phage T5. Although the phages share overlapping binding sites in FhuA, the structural alterations elicited by the mutations resulted in very different phage sensitivities. In the FhuA deletion mutant, the TonB requirement for phage T1 infection was partially bypassed.     

Genome of Enterobacteriophage Lula/phi80 and Insights into Its Ability To Spread in the Laboratory Environment

The novel temperate bacteriophage Lula, contaminating laboratory Escherichia coli strains, turned out to be the well-known lambdoid phage phi80. Our previous studies revealed that two characteristics of Lula/phi80 facilitate its spread in the laboratory environment: cryptic lysogen productivity and stealthy infectivity. To understand the genetics/genomics behind these traits, we sequenced and annotated the Lula/phi80 genome, encountering an E. coli-toxic gene revealed as a gap in the sequencing contig and analyzing a few genes in more detail. Lula/phi80''s genome layout copies that of lambda, yet homology with other lambdoid phages is mostly limited to the capsid genes. Lula/phi80''s DNA is resistant to cutting with several restriction enzymes, suggesting DNA modification, but deletion of the phage''s damL gene, coding for DNA adenine methylase, did not make DNA cuttable. The damL mutation of Lula/phi80 also did not change the phage titer in lysogen cultures, whereas the host dam mutation did increase it almost 100-fold. Since the high phage titer in cultures of Lula/phi80 lysogens is apparently in response to endogenous DNA damage, we deleted the only Lula/phi80 SOS-controlled gene, dinL. We found that dinL mutant lysogens release fewer phage in response to endogenous DNA damage but are unchanged in their response to external DNA damage. The toxic gene of Lula/phi80, gamL, encodes an inhibitor of the host ATP-dependent exonucleases, RecBCD and SbcCD. Its own antidote, agt, apparently encoding a modifier protein, was found nearby. Interestingly, Lula/phi80 lysogens are recD and sbcCD phenocopies, so GamL and Agt are part of lysogenic conversion.     

Incorporation of phi80 phage into unusual chromosome sites and isolation of a specialized transducing bacteriophage]

The mutant strain KS713 of Escherichia coli K-12 deleted for the normal insertion site and secondary preferable one was obtained. The insertion frequency of phage phi80 into the double deletion strain is reduced about 30-fold with respect to integration into the strain H47 with deletion of the primary phi80 attachment site and about 500-fold relative to integration into wild type Escherichia coli. Analysis of the rare abnormal lysogens of KS 713 strain indicates that there are secondary sites on the chromosome, which are utilized for prophage attachment if insertion at preferable secondary att80-II site is eliminated too. The insertion of phi80 phage into the bfe locus was obtained by the appropriate selection technique. Induced prophage excision from the bfe site was rather efficient and lysates contained phi80 phage particles that could specificically transduce the argH+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harbouring both the wild-type and the mutant argH genes were isolated. These heterogenotes were used for producing high-frequency transducing lysates.     

Isolation of Transducing Bacteriophages for the Histidine and Isoleucine-Valine Operons in Escherichia coli K-12

In vitro studies have been of great value in elucidating the mechanism of the regulation of several bacterial operons. To obtain a deoxyribonucleic acid preparation enriched for the histidine (his) and for the isoleucine-valine (ilv) operons, we have isolated bacteriophages carrying the his and the ilv regions of the Escherichia coli chromosome. Transposition of the his operon to a site close to the att80 region of the E. coli chromosome has been carried out selecting for integration of a temperature-sensitive F'his(+) in the tonB locus. This transposed strain has been lysogenized with phi80i(lambda). Upon induction of the lysogen, His(+) transductants have been isolated, which, on further induction give rise to HFT (high frequency of transduction) lysates. Preliminary characterization of the transducing phage is reported. The ilv operon, carried on an F' particle, has been fused to an episome carrying the att80 region. The fused episome has been lysogenized with phi80i lambdat68. Upon induction of the lysogen, Ilv(+) transductants have been isolated which on further induction give rise to HFT lysates.     

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.     

Revised restriction maps of Bacillus subtilis bacteriophage phi 105 DNA.

Physical mapping of Bacillus subtilis temperate phage phi 105 DNA was carried out by using restriction endonucleases EcoRI, SmaI, and KpnI, and a new revised EcoRI cleavage map is presented. In addition, the EcoRI cleavage maps of six specialized transducing phages carrying sporulation genes of B. subtilis were revised.     

Evidence that Bacillus subtilis Bacteriophage SPO2 is Temperate and Heteroimmune to Bacteriophage φ105

Some characteristics of Bacillus subtilis phage SPO2 which show that it is a temperate phage are presented. Wild-type SPO2 forms turbid plaques, similar to those of other temperate phages. SPO2 lysogenic strains which are resistant to SPO2 can be isolated; these strains remain stable lysogens despite the fact that they can no longer adsorb SPO2. SPO2 lysogenic strains can be grown for many generations in SPO2 antiserum and remain lysogenic. Phage SPO2 plates on phi105 lysogens and phage phi105 plates on SPO2 lysogens; this indicates that SPO2 and phi105 are heteroimmune. Phage phi105 plates on an SPO2-resistant strain; this indicates that SPO2 and phi105 adsorb to different receptor sites on the bacterial surface.     

Isolation of Plaque-Forming, Galactose-Transducing Strains of Phage Lambda

Plaque-forming, galactose-transducing lambda strains have been isolated from lysogens in which bacterial genes have been removed from between the galactose operon and the prophage by deletion mutation.—A second class has been isolated starting with a lysogenic strain which carries a deletion of the genes to the right of the galactose operon and part of the prophage. This strain was lysogenized with a second lambda phage to yield a lysogen from which galactose-transducing, plaque-forming phages were obtained. These plaque-forming phages were found to be genetically unstable, due to a duplication of part of the lambda chromosome. The genetic instability of these partial diploid strains is due to homologous genetic recombindation between the two identical copies of the phage DNA comprising the duplication. The galactose operon and the duplication of phage DNA carried by these strains is located between the phage lambda P and Q genes.     

Factors that affect transduction in microorganisms]

Data from literature concerning general and specialized transduction in microorganisms are given in the paper. The process of exogenic DNA penetration to the cells of bacteria and participation of protein products of separate phage genes in this process are described. The so-called E-proteins in a set with DNA penetrate through a cell membrane. In phage P22 they are protein products of phage genes 7, 16, 20. In P22 mutants with an altered transducing frequencies (HFT and LFT) the due functions are also coded by the phage genes. It is shown that the process of DNA packing in phages P22, phi 80, lambda and others is genetically determined. The gene transfer frequency depends on UV radiation and the very nature of transducing phages itself. In virulent phages the UV radiation up to inactivation level 95-99% evokes a decrease of their "killer" ability, which is accompanied by an increase of survivability of the formed transductants and, as a result, by enhancement of the transduction transfer frequency. An important role of the transduction analysis for fine mapping of a genome of microorganisms and its significance for practice are shown. A mathematical analysis of the data on cotransduction of linkage markers is presented as such that may be used when determining the value of transduced fragment of a chromosome.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. VI. Mapping of F14 sequences homologous to phi 80dmetBJF and phi 80dargECBH bacteriophages

The positions of the metBJF and the argECBH sequences on F14 have been mapped by studying heteroduplexes of F14 with φ80dmet and φ80darg transducing phage DNAs. The structures of the DNAs of the transducing phage φ80d-metB isolated by Konrad (1969), of two φ80dmetB phages isolated by Press et al. (1971), and of some derived φ80darg phages, have been determined. They all have complex structures. In addition to the bacterial chromosome sequences corresponding to the met and arg genes, they contain certain F sequences, which have been recognized as active in F-related recombination events. Plausible models for the integration and excision events leading to the formation of the phage DNA molecules are proposed.     

Characterization of Serratia entomophila Bacteriophages and the Phage-Resistant Mutant Strain BC4B

Successful large-scale fermentations of the bacterium Serratia entomophila for use in biological control of the soil-dwelling insect Costelytra zealandica has required the development of a phage-resistant mutant, BC4B. We report our investigations into S. entomophila phages and the nature of the phage resistance mechanism of strain BC4B. The parental strain of BC4B, A1MO2, was found to contain two previously unidentified prophages, (phi)9A and (phi)9B, which were UV inducible and also released spontaneously in large numbers. BC4B was shown to be completely cured of (phi)9A. Single lysogens of (phi)9A and (phi)9B were not homoimmune to any other S. entomophila phages. However, on the basis of DNA-DNA homology, all S. entomophila phages except (phi)CW3 were shown to have significant regions of homology and also packaged their DNA via pac-like mechanisms. The failure of phage particles to adsorb was identified as the basis of phage resistance in BC4B. In addition, it was demonstrated that all known S. entomophila phages are naturally temperature sensitive.     

Isolation and characterization of specialized lambda transducing bacteriophage carrying the metBJF methionine gene cluster.

Secondary attachment site lysogens of Deltaatt(lambda)Deltappc-argECBH strains of Escherichia coli with lambdacI857 integrated into the bfe gene (88 min) were isolated. Of 20 such lysogens examined, 2 produce lysates with transducing phage containing the metBJF gene cluster (87 min). Reintroduction of the ppc-argECBH chromosome segment (which lies between the bfe and met genes) into these strains virtually abolishes the production of met transducing phage. All of the phage examined have lost essential genes from the left arm of the lambda chromosome. Approximately 85% of the phage appear to have the same genetic composition, containing the metBJF gene cluster, but not the closely linked gene cytR, and having lost phage genes G and J. Analytical CsCl density gradient centrifugation of five representatives of this major class of phage shows four of them to have identical densities (lighter than lambda), while the fifth cannot be resolved from lambda. The four apparently identical phage were isolated from three separate lysates, which suggests the existence of preferred sites for illegitimate recombination on the bacterial and phage chromosomes. Three specialized transducing phage that carry cytR in addition to metB, metJ, and metF have also been studied. Each of these viruses has a different amount of phage deoxyribonucleic acid. Two of them have less deoxyribonucleic acid than lambda, whereas the third has about the same amount. The metB, metF, and cytR genes of the transducing phage have been shown to function in vivo. The phage-borne metB and metF genes are subject to metJ-mediated repression.