Integration defective mutants of bacteriophage P2

Summary Mutants of phage P2 unable by themselves to be integrated as prophages have been isolated. These mutants (int) are complemented by the wild type allele and may then yield stable lysogenic strains carrying an int prophage at location I in Escherichia coli C. These lysogens produce either no phage or little phage, depending on the int mutant used. All int mutants isolated appear to belong to a single complementation group.Exceptional lysogens carrying two or more int prophages may be obtained: they may produce spontaneously even more phage than normal lysogens, and they segregate out defective, singly lysogenic clones at low frequency. These exceptional lysogens carry both prophages in location I, presumably in tandem.Strains carrying two or more int prophages but defective in phage production were also isolated. One of these carries its prophages at two different, not closely linked, chromosomal locations.     

Transduction of R plasmids by bacteriophages P1 and P22

Summary We demonstrated that bacteriophages P1 and P22 were able to form various types of hybrids with six out of seven different R plasmids tested. When the same R plasmid was used for isolation, P1-R hybrids usually carried more resistance markers than P22-R. Several genetical observations suggest that the hybrid prophages carried the resistance markers transposed to the phage genomes without loss of essential phage genes. Upon UV-irradiation the prophages produced phage lysates that transduced the relevant resistance markers at high frequencies by lysogenic conversion. The insertion of the resistance markers was even acquired by the P1 or P22 genomes during one-step growth in R⁺ cells. Some lytically prepared lysates grown on R⁺ cells contained the hybrids at a frequency of 10^-7 to 10^-6/plaqueforming unit. Analysis of P1-transductants for resistance markers of the R plasmids revealed in some cases more specialized transductants than generalized transductants. These results strongly indicate that a precise genetic map of an R plasmid can not be established only on the basis of co-transduction frequencies of the resistance markers of the R plasmid.     

Interactions of Tn7 and temperate phage F116L of Pseudomonas aeruginosa

Summary Tn7 insertions into the genome of F116L, a Pseudomonas aeruginosa generalized transducing phage, were isolated by repeated cycles of transducing phage, were of strains lysogenic for F116cts mutants with selection for trimethoprim resistance (Tp¹). Two non-defective F116Lcts:Tn7 phage were characterized. They have reduced plaquing ability, produced non-lysogenic Tp^r transductants, and have yielded a deletion mutant of the phage genome upon selection for plaque formation in single infection. F116L DNA is circularly permuted and terminally redundant. A circular restriction map of 61.7 kb has been defined, and a cleavage site common to many enzymes has been identified at coordinate 23.3 kb on the map. It is presumed that this site represents the sequence for the initiation of DNA encapsidation by a headful packaging mode. The Tn7 insertion targets and a 13.4 kb deletion define regions of the F116L genome non-essential for either vegetative growth or lysogenization. The restriction map of Tn7 has been determined for five enzymes. Non-lysogenic Tp^r transuctants reveal a Tn7 insertion hot-spot in the P. aeruginosa genome.     

Phage A25-mediated transfer induction of a prophage in Streptococcus pyogenes

Summary The group A streptococcal strain 56188 used as standard donor in transduction with the virulent phage A25 is lysogenic for a phage called P56188. By using specific antiphage sera it is shown that A25 lysates obtained from 56188 contain a fraction of about 10^-4 phenotypically A25 but genotypically P56188 particles. A25-mediated transduction of prophage P56188 is measured by scoring plaques produced by transfer induction on 5004, a lysogenic strain unable to support the growth of A25. Data are obtained suggesting that A25 can also transduce a prophage carried by strain T25₃.Prophage P5004 present in 5004 is found to interfere with the propagation of A25 but does not seem to exert its action by directing extensive degradation of A25 DNA. Lysogenization of SM27 with P5004 leads to dramatically decreased burst sizes of A25, associated with the loss of its ability to plaque on this strain. Furthermore, P5004 lysogens of SM27 yield fewer streptomycin resistant transductants than their parent but gain the ability to serve as donors in A25-mediated transduction. A comparison of the burst size and the yield of transducing particles of A25 on various lysogenic and nonlysogenic hosts suggests that interfering with A25 growth is a widespread property of streptococcal prophages, which might favour processes leading to the formation of transducing A25 particles.     

Analysis of the dnaB function of Escherichia coli K12 and the dnaB-like function of P1 prophage

The dnaB function of Escherichia coli K12 was studied with a series of isogenic strains differing from each other only by a mutation in the dnaB gene. The strains showed different phenotypes depending on the particular dnaB mutation they carry. A clear example is provided by a strain carrying dnaB266 mutation which turned out to be an amber mutation. When the mutation was suppressed by different suppressors, the strains showed different phenotypes. Thus, dnaB proteins which differ from each other by only one amino acid at the mutation site give different phenotypes. Mutation dnaB266 is lethal to the host when not suppressed. Hence the dnaB protein is essential for bacterial growth.Three P1 mutants, P1mcb-4, P1mcb-5 and P1mcb-8, were isolated which converted the temperature-sensitive bacterial growth of dnaB266-supE to resistant growth. Lysogenization with P1mcb allowed growth of dnaB266su⁻ strain which was absolutely defective in the bacterial dnaB function, indicating that the dnaB-like function of P1 prophage can substitute for the bacterial dnaB function. However, lysogenization by P1mcb did not support the growth of λ and λπ phages on dnaB 266su⁻. While P1mcb-4 and P1mcb-5 prophages altered the phenotypes of other dnaB strains to permit the growth of bacterial and λ phage at 32 °C and 42 °C, P1mcb-8 prophage supports the growth of λ phages and bacteria at 42 °C but not λ phage growth on groP-bacteria at 32 °C. The alteration of phenotypes of the P1mcb lysogens varied depending on the dnaB mutations they carried. Mutual interaction between the bacterial dnaB protein and the phage dnaB-like protein which results in different phenotypes of lysogens is suggested.     

Suppression of a thermosensitive dnaA mutation of Escherichia coli by bacteriophage P1 and P7.

Like other plasmids, the P1 and P7 prophages suppress E. coli dnaA(Ts) mutations by integrating into the host chromosome. This conclusion is supported by three lines of evidence: (1) Alkaline sucrose gradients reveal the absence of plasmid DNA in suppressed lysogens; (2) the prophage is linked to host chromosomal markers in conjugation; and (3) auxotrophs whose defect is linked to the prophage are found among suppressed colonies. No phage or bacterial mutation is required for suppression. Integrative suppression by P1 and P7, unlike suppression by F, does not require the host recA⁺ function. Among suppressed P7 lysogens are some that do not produce phage; these contain defective prophages. The genetic extent of the deletions contained by these defective prophages delineates the prophage regions which are not necessary for suppression of dnaA(Ts). The possible mechanisms of integration and deletion formation are discussed.     

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.     

Physical mapping of LP51 and LP52 prophages of lysogenic strains of Bacillus licheniformis

Summary The physical maps of the LP51 and LP52 prophages in lysogenic strains of Bacillus licheniformis were constructed on the basis of data obtained by hybridization of phage DNA probes with Southern blots of restricted DNA of the lysogens. The data were compatible with the Campbell model for chromosomal integration; the attP site was mapped at 58.7–61.8 map units of the genomes of both phages. Identification of prophage-host DNA junction fragments indicated the presence of a unique attB site on the bacterial chromosome; the set of junction fragments in the strain B. licheniformis ATCC 10716 was identical to that of ATCC 11946, but different from ATCC 8187. Both the LP51 and LP52 phages used the same integration sites. Upon reinfection with either phage, the cured strains UM12 and UM18 (i.e. 10716 and 11946 cured of LP52 or LP51, respectively) turned out to be integration deficient. In surface cultures the reinfected bacteria could be maintained in the lysogenic state without, however, integrating the phage genome; when these bacteria were passaged in submerged cultures, several modes of anomalous integration were observed, and the phage segregated into a variety of forms, discernible by virulence and plaque morphology. In liquid cultures of UM12(LP51) or UM12(LP52) lytic forms finally predominated, while most lysogenized UM18 were converted into defective lysogens which contained a defective prophage in a stably integrated form.     

Use of a biotinylated DNA probe to detect bacteria transduced by bacteriophage P1 in soil

Presumptive bacteriophage P1 transductants of Escherichia coli, isolated from soil inoculated with lysates of transducing phage P1 and E. coli, were confirmed to be lysogenic for phage P1 by hybridization with a biotinylated DNA probe prepared from the 1.2-kilobase-pair HindIII 3 fragment of bacteriophage P1. No P1 lysogens of indigenous soil bacteria were detected with the DNA probe. The sensitivity and specificity of the DNA probe were assessed with purified and dot blot DNA, respectively. In addition, two techniques for the lysis and deproteinization of bacteria and bacteriophages on nitrocellulose filters were compared. These studies indicated that biotinylated DNA probes may be an effective alternative to conventional radiolabeled DNA probes for detecting specific gene sequences in bacteria indigenous to or introduced into soil.     

Use of a biotinylated DNA probe to detect bacteria transduced by bacteriophage P1 in soil.

Presumptive bacteriophage P1 transductants of Escherichia coli, isolated from soil inoculated with lysates of transducing phage P1 and E. coli, were confirmed to be lysogenic for phage P1 by hybridization with a biotinylated DNA probe prepared from the 1.2-kilobase-pair HindIII 3 fragment of bacteriophage P1. No P1 lysogens of indigenous soil bacteria were detected with the DNA probe. The sensitivity and specificity of the DNA probe were assessed with purified and dot blot DNA, respectively. In addition, two techniques for the lysis and deproteinization of bacteria and bacteriophages on nitrocellulose filters were compared. These studies indicated that biotinylated DNA probes may be an effective alternative to conventional radiolabeled DNA probes for detecting specific gene sequences in bacteria indigenous to or introduced into soil.     

X-ray sensitivity of Escherichia coli lysogenic for bacteriophage P2.

Summary Strains of Escherichia coli C or K lysogenic for the non-inducible phage P2 show a lower survival following X-ray irradiation as compared to nonlysogenic strains. This difference in X-ray sensitivity is not accompanied by a significant difference in X-ray induced mutability. The capacity of X-irradiated P2 lysogens to multiply any of a number of unirradiated infecting phages is severely impaired. These effects of X-ray treatment can be most simply explained as a consequence of the fact that protein and RNA syntheses are strongly inhibited in P2 lysogens after X-irradiation. All the above events specifically occurring in X-rayed P2 lysogens are dependent on the P2 gene old.     

Initiation of recA+-dependent recombination in Escherichia coli (lambda). II. Specificity in the induction of recombination and strand cutting in undamaged covalent circular bacteriophage 186 and lambda DNA molecules in phage-infected cells.

Covalent circular λ DNA molecules produced in Escherichia coli (λ) host cells by infection with labeled λ bacteriophages are cut following superinfection with λ phages damaged by exposure to psoralen and 360 nm light. This cutting of undamaged covalent circular molecules is referred to as “cutting in trans”, and could be a step in damage-induced recombination (Ross &; Howard-Flanders, 1977). Similar experiments performed with the temperate phage 186, which is not homologous with phage λ, showed cutting in trans and damage-induced recombination to occur in homoimmune crosses with phage 186 also. Double lysogens carrying both λ and 186 prophages were used in a test for specificity in cutting in trans and in damage-induced recombination. The double lysogens were infected with ³H-labeled 186 and ³²P-labeled λ phages. When these doubly infected lysogens containing covalent circular phage DNA molecules of both types were superinfected with psoralen-damaged 186 phages and incubated, the covalent circular 186 DNA was cut, while λ DNA remained intact. Similarly, superinfection with damaged λ phages caused λ, but not 186, DNA to be cut. Evidently, cutting in trans was specific to the covalent circular DNA homologous to the DNA of the damaged phages. Homoimmune phage-prophage genetic crosses were performed in the double lysogenic host infected with genetically marked λ and 186 phages. Damage-induced recombination was observed in this system only between the damaged phage DNA and the homologous prophage, none being detected between other homolog pairs present in the same cell. This result makes it unlikely that the damaged phage DNA induces a general state of enhanced strand cutting and genetic recombination affecting all homolog pairs present in the host cell. The simplest interpretation of the specificity in cutting and in recombination is as follows. When they have been incised, the damaged phage DNA molecules are able to pair directly with their undamaged covalent circular homologs. The latter molecules are cut in a recA ⁺ -dependent reaction by a recombination endonuclease that cuts the intact member of the paired homologs.     

Properties of natural interspecific hybrids of transposable phages of Pseudomonas aeruginosa: specific characteristics of phage PL24 transposition

Properties of natural hybrid transposable phages (TP) of Pseudomonas aeruginosa, including phage PL24 and lysogens for this phage, were studied. PL24 possesses the properties of TP from two previously described groups, B3 and D3112. Its genome, unlike the genome of D3112, contains many sites susceptible to the SalGI restriction endonuclease and possesses no more than 100 nucleotides of bacterial origin located at the left genome end. However, unlike B3, phage PL24 failed to induce auxotrophic mutants upon integration in the bacterial genome. This phage differed from both B3 and D3112 in sensitivity to chloroform treatment. A more detailed examination of a group containing 25 randomly isolated lysogens for phage PL24 revealed previously unknown processes occurring at early stages of bacterial lysogenization. There are at least two different modes of cell lysogenization with phage PL24. In the first case, the emerging lysogens contained a single prophage genome located (in each lysogen) at individual sites. In the second case, polylysogenic bacteria appeared, and, after primary integration of a phage genome, replicative transposition occurred at new sites (often accompanied by the appearance of prophage clusters at these sites). The choice of the mode of lysogenization can be determined both by differences in the physiological state of bacteria and by specific features of phage PL24, which possibly affect the time of repressor accumulation to the concentration sufficient for blocking phage growth or the stability of the lysogenic state.     

Spontaneously Induced Prophages in Lactobacillus gasseri Contribute to Horizontal Gene Transfer

Lactobacillus gasseri is an endogenous species of the human gastrointestinal tract and vagina. With recent advances in microbial taxonomy, phylogenetics, and genomics, L. gasseri is recognized as an important commensal and is increasingly being used in probiotic formulations. L. gasseri strain ADH is lysogenic and harbors two inducible prophages. In this study, prophage ϕadh was found to spontaneously induce in broth cultures to populations of ∼10⁷ PFU/ml by stationary phase. The ϕadh prophage-cured ADH derivative NCK102 was found to harbor a new, second inducible phage, vB_Lga_jlb1 (jlb1). Phage jlb1 was sequenced and found to be highly similar to the closely related phage LgaI, which resides as two tandem prophages in the neotype strain L. gasseri ATCC 33323. The common occurrence of multiple prophages in L. gasseri genomes, their propensity for spontaneous induction, and the high degree of homology among phages within multiple species of Lactobacillus suggest that temperate bacteriophages likely contribute to horizontal gene transfer (HGT) in commensal lactobacilli. In this study, the host ranges of phages ϕadh and jlb1 were determined against 16 L. gasseri strains. The transduction range and the rate of spontaneous transduction were investigated in coculture experiments to ascertain the degree to which prophages can promote HGT among a variety of commensal and probiotic lactobacilli. Both ϕadh and jlb1 particles were confirmed to mediate plasmid transfer. As many as ∼10³ spontaneous transductants/ml were obtained. HGT by transducing phages of commensal lactobacilli may have a significant impact on the evolution of bacteria within the human microbiota.     

Control of the Replication Complex of Bacteriophage P22

A replication complex for the vegetative synthesis of the deoxyribonucleic acid (DNA) of the temperate phage P22 previously has been described. This complex is an association of parental phage DNA, most of the newly synthesized phage DNA made during pulses with (3)H-thymidine, and other cell constituents, and has a sedimentation rate in neutral sucrose gradients of at least 1,000S. The complex is one of the intermediates, intermediate I, in the synthesis and maturation of phage P22 DNA after infection or induction. Evidence supporting the replicative nature of intermediate I is presented. Phage replication is repressed in lysogenic bacteria. On superinfection of P22 lysogens with nonvirulent phage, little association of the input phage DNA with a rapidly sedimenting fraction is demonstrable. However, after induction with ultraviolet light, the superinfecting parental phage DNA quickly acquires the rapid sedimentation rate characteristic of intermediate I; phage DNA synthesis follows; and progeny phages are produced. Infection with a virulent mutant of P22 produces progeny phages in lysogens. Its DNA associates with intermediate I. In mixed infection with the virulent phage, replication of nonvirulent phage P22 is still repressed, even though the virulent replicates normally. The nonvirulent input DNA does not associate with intermediate I. The repressor of the lysogenic cell prevents replication by interfering with the physical association of template material with intermediate I. A phage function is required for association of phage template with the replication machinery.     

Characterization of temperate bacteriophages of Leuconostoc oenos and evidence for two prophage attachment sites in the genome of starter strain PSU-1

The close relatedness between 17 Leuconostoc oenos bacteriophages, induced with mitomycin C from strains isolated in different geographic regions, was inferred from their morphology, DNA homology and protein composition. The genome of all the phages had cohesive end termini and ranged in size from 36.4 to 40.9 kb. According to the restriction patterns obtained by digestion with five enzymes, the phages were divided in six groups. Lysogenization of a spontaneous phage-cured derivative of Leuc. oenos strain PSU-1 was achieved with 16 phages and the analysis of the lysogens showed that the phage DNA integrates in the host chromosome in one or two sites. The att B loci were located on the macrorestriction Asc I and Not I fragments of the recipient strain. A survey of Leuc. oenos strains with a phage DNA probe confirmed the lysogenic nature of several, but not all of the original phage hosts. These results are discussed in the light of evidence for the instability of some lysogenic PSU-1 derivatives.     

Restoration of immunity in lysogens carrying prophage P2, derepressed at high temperature

Summary The possibility that a strain lysogenic for phage P2 could be brought into the so-called antiimmune state in which the synthesis of phage repressor is permanently turned off, was tested in the following way. Two lysogenic strains that could be derepressed at 42°C were prepared. In one, the prophage had, in addition to a temperature-sensitive repressor mutation (c5), amber mutations in the two early genes A and B. In the other, the prophage had an unknown defect that blocked expression of the A and B genes. Both strains could multiply at 42° C as well as or almost as well as a non-lysogen. After the strains had grown for several generations at 42° C, they were returned to 30° and the resynthesis of repressor was followed by measuring the restoration of immunity to super-infection. In both cases, the immunity returned slowly over a period of 2 to 3 h. In a strain made doubly lysogenic for two amA amB c5 prophages, immunity was restored at a more rapid rate, suggesting that the rate of restoration depended mainly on the number of copies of repressor gene present. Attempts to demonstrate channeling towards the lytic pathway in the derepressed lysogens was also negative. The temperature treatment tended instead to increase the frequency of lysogenization of superinfecting P2. Thus, the presence of a system, similar to the cro system described for phage lambda, to regulate repressor synthesis in phage P2 could not be demonstrated.     

Amplification of chloramphenicol resistance transposons carried by phage P1Cm in Escherichia coli.

Summary We have characterized a number of P1Cm phages which contain the resistance genes to chloramphenicol and fusidic acid as IS1-flanked Cm transposons. Restriction cleavage and electron microscopic analysis showed that these Cm transposons were carried as monomers (M) or tandem dimers (D). Lysogens of P1Cm (D) are more resistant to chloramphenicol than those of its P1Cm (M) presumably as a result of an increased gene dosage. Amplification of the Cm transposons to tandem multimers was frequently observed in P1Cm (D) lysogens grown in the presence of high concentrations of chloramphenicol or fusidic acid and was also detected in P1Cm (M) lysogens. The degree of amplification varied in different clones which suggests that cells containing spontaneously amplified Cm transposons were selected by high doses of the antibiotics. The dimeric as well as the amplified Cm transposons carried in P1Cm lysogens grown in the absence of chloramphenicol displayed considerable stability. Mechanisms for the amplification of the IS1-flanked transposons are discussed.     

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.     

Characterization of SE1, a new general transducing phage of Salmonella typhimurium

A transducing phage, SE1, which is able to infect Salmonella typhimurium was isolated from a Salmonella enteritidis strain. SE1 is a temperate phage which is heteroimmune with respect to phages P22, L, KB1 and ES18. It is similar in morphology and size to phages P22, L and KB1 and is serologically related to phages P22 and L but not to KB1. Efficiencies of generalized transduction effected by phage SE1 are similar to those for P22HT (int7), a mutant which mediates a high frequency of chromosomal gene transduction. The lengths of chromosomal DNA transduced by SE1 and P22HT (int7) are similar. Furthermore, the SE1 prophage does not exclude the transducing particles from cells it has lysogenized; consequently it is possible to use both SE1 lysogens and non-lysogenic strains as recipients in SE1-mediated transduction experiments, and obtain similar transduction efficiencies. However, the SE1 prophage gives rise to a lysogenic conversion that decreases the rate of adsorption of SE1 and L phages by about 50%, but does not affect adsorption of P22. Altogether these results suggest that phage SE1 may be a useful tool in the genetic manipulation of S. typhimurium.