Transduction of bacteriophage lambda by bacteriophage T1.

When bacteriophage T1 was grown on bacteriophage lambda-lysogenic cells, phenotypically mixed particles were formed which had the serum sensitivity, host range, and density of T1 but which gave rise to lambda phage. T1 packaged lambda genomes more efficiently both when the length of the prophage was less than that of wild-type lambda and when the host cell was polylysogenic. Expression of the red genes of lambda or the recE system of Escherichia coli during T1 growth enhanced pickup of lambda by T1, whereas packaging was reduced in recB cells. If donors were singly lysogenic, the expression of transduced lambda genomes as a PFU required lambda-specified excisive recombination, whereas lambda genomes transduced from polylysogens required only lambda- or E. coli-specified general recombination to give a productive infection.     

Transduction of multi-copy plasmid pBR322 by bacteriophage Mu

Summary The temperate bacteriophage Mu transduces the 4363 bp multi-copy plasmid pBR322 at frequencies similar to those of chromosomal markers. Plasmid transducing particles contain DNA molecules of Mu DNA length. Plasmid DNA is transduced as a head-to-tail oligomer that becomes circularized in the recipient cell. The rec system of the donor strain participates in oligomer formation and the rec system of the recipient strain is required for oligomer circularization. Possible mechanisms that may explain the origin of plasmid transducing particles are discussed.     

Transduction of Escherichia coli by bacteriophage P1 in soil.

Transduction of Escherichia coli W3110(R702) and J53(RP4) (10(4) to 10(5) CFU/g of soil) by lysates of temperature-sensitive specialized transducing derivatives of bacteriophage P1 (10(4) to 10(5) PFU/g of soil) (P1 Cm cts, containing the resistance gene for chloramphenicol, or P1 Cm cts::Tn501, containing the resistance genes for chloramphenicol and mercury [Hg]) occurred in soil amended with montmorillonite or kaolinite and adjusted to a -33-kPa water tension. In nonsterile soil, survival of introduced E. coli and the numbers of E. coli transductants resistant to chloramphenicol or Hg were independent of the clay amendment. The numbers of added E. coli increased more when bacteria were added in Luria broth amended with Ca and Mg (LCB) than when they were added in saline, and E. coli transductants were approximately 1 order of magnitude higher in LCB; however, the same proportion of E. coli was transduced with both types of inoculum. In sterile soil, total and transduced E. coli and P1 increased by 3 to 4 logs, which was followed by a plateau when they were inoculated in LCB and a gradual decrease when they were inoculated in saline. Transduction appeared to occur primarily in the first few days after addition of P1 to soil. The transfer of Hg or chloramphenicol resistance from lysogenic to nonlysogenic E. coli by phage P1 occurred in both sterile and nonsterile soils. On the basis of heat-induced lysis and phenotype, as well as hybridization with a DNA probe in some studies, the transductants appeared to be the E. coli that was added. Transduction of indigenous soil bacteria was not unequivocally demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transduction of chromosomal genes between enteric bacteria by bacteriophage P1.

We have used P1 transduction to create intergeneric hybrid strains of enteric bacteria by moving the genA and hut genes between Klebsiella aerogenes, Escherichia coli and Salmonella typhimurium. The use of E. coli as the recipient in such transductions permits the construction of episomes and specialized transducing phage containing non-E. coli material. The effect of host restriction modification and deoxyribonucleic acid homology on the frequency of intergeneric transduction of these loci has been examined.     

Transduction of Escherichia coli by bacteriophage P1 in soil

Transduction of Escherichia coli W3110(R702) and J53(RP4) (10(4) to 10(5) CFU/g of soil) by lysates of temperature-sensitive specialized transducing derivatives of bacteriophage P1 (10(4) to 10(5) PFU/g of soil) (P1 Cm cts, containing the resistance gene for chloramphenicol, or P1 Cm cts::Tn501, containing the resistance genes for chloramphenicol and mercury [Hg]) occurred in soil amended with montmorillonite or kaolinite and adjusted to a -33-kPa water tension. In nonsterile soil, survival of introduced E. coli and the numbers of E. coli transductants resistant to chloramphenicol or Hg were independent of the clay amendment. The numbers of added E. coli increased more when bacteria were added in Luria broth amended with Ca and Mg (LCB) than when they were added in saline, and E. coli transductants were approximately 1 order of magnitude higher in LCB; however, the same proportion of E. coli was transduced with both types of inoculum. In sterile soil, total and transduced E. coli and P1 increased by 3 to 4 logs, which was followed by a plateau when they were inoculated in LCB and a gradual decrease when they were inoculated in saline. Transduction appeared to occur primarily in the first few days after addition of P1 to soil. The transfer of Hg or chloramphenicol resistance from lysogenic to nonlysogenic E. coli by phage P1 occurred in both sterile and nonsterile soils. On the basis of heat-induced lysis and phenotype, as well as hybridization with a DNA probe in some studies, the transductants appeared to be the E. coli that was added. Transduction of indigenous soil bacteria was not unequivocally demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transduction by phiBB-1, a bacteriophage of Borrelia burgdorferi

We previously described a bacteriophage of the Lyme disease agent Borrelia burgdorferi designated phiBB-1. This phage packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genus Borrelia. To demonstrate the ability of phiBB-1 to package and transduce DNA, a kanamycin resistance cassette was inserted into a cloned fragment of phage DNA, and the resulting construct was transformed into B. burgdorferi CA-11.2A cells. The kan cassette recombined into a resident cp32 and was stably maintained. The cp32 containing the kan cassette was packaged by phiBB-1 released from this B. burgdorferi strain. phiBB-1 has been used to transduce this antibiotic resistance marker into naive CA-11.2A cells, as well as two other strains of B. burgdorferi. This is the first direct evidence of a mechanism for lateral gene transfer in B. burgdorferi.     

Inhibition of bacteriophage Mu transposition by Mu repressor and Fis

In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor. Fis can lower the transposition frequency of a mini-Mu 3–80-fold, but only if the Mu repressor is expressed simultaneously. In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site leads to the loss of the Fis effect. As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor. However, the role of Fis and repressor is not only to inactivate the IAS, since a 4bp insertion in the IAS, which changes the spacing of the repressor-binding site, abolishes the enhancing function of the IAS but leaves the repressor-Fis effect intact. A likely target for Fis in this regulation is a strong Fis-binding site, which is located adjacent to the L2 transposase-binding site. However, when this Fis-binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed. Although it is still possible that Fis can function by binding to this non-specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.     

Preferential generalized transduction by bacteriophage Mu

Summary The generalized transduction by bacteriophage Mu was found to be preferential for the 0–1 min segment of the E. coli K12 chromosome. This transduction pattern is obtained with phage lysates grown on all F^-, F⁺ and Hfr tested, and is not marker-specific.Phages grown by both lytic infection and by heat induction of prophages at different locations of the host's chromosome show the same transduction pattern, indicating that generation of transducing DNA does not directly depend on excision events. Conjugation of independently obtained Muc ⁺-lysogenic strains of HfrC with a multiauxotrophic F^- recipient strain lysogenic for a Mucts62 prophage, shows that transfer of the temperature-resistance character (Muc ⁺) is not preferentially linked to the 0–1 min segment. The lysogenizing integrations do therefore not take place within the segment preferentially transduced by the phage.A model¹ for the generation of the transducing DNA is proposed, which assumes that for its replication, Mu DNA is integrated close to the 0–1 min segment of the host chromosome, which is then preferentially replicated and packaged into the phage heads.     

Exclusion of bacteriophage T1 by bacteriophage lambda. II. Synthesis of T1-specific macromolecules under N-mediated excluding conditions.

The results of experiments investigating T1 macromolecular synthesis under N-mediated excluding conditions failed to demonstrate a substantial alteration in the T1 mRNA production in excluding cultures at any stage in the T1 infectious cycle. The number of T1 DNA sequences in the excluding culture was found to be one-third to one-half that found in T1-infected cultures. The most severe reduction in T1-specific macromolecules was seen in protein synthesis. Total incorporation of labeled amino acids was reduced sixfold, and gel experiments confirmed that the T1-specific proteins capable of detection are reduced in excluding cells.     

In vivo mutagenesis of bacteriophage Mu transposase.

We devised a method for isolating mutations in the bacteriophage Mu A gene which encodes the phage transposase. Nine new conditional defective A mutations were isolated. These, as well as eight previously isolated mutations, were mapped with a set of defined deletions which divided the gene into 13 100- to 200-base-pair segments. Phages carrying these mutations were analyzed for their ability to lysogenize and to transpose in nonpermissive hosts. One Aam mutation, Aam7110, known to retain the capacity to support lysogenization of a sup0 host (M. M. Howe, K. J. O'Day, and D. W. Shultz, Virology 93:303-319, 1979) and to map 91 base pairs from the 3' end of the gene (R. M. Harshey and S. D. Cuneo, J. Genet. 65:159-174, 1987) was shown to be able to complement other A mutations for lysogenization, although it was incapable of catalyzing either the replication of Mu DNA or the massive conservative integration required for phage growth. Four Ats mutations which map at different positions in the gene were able to catalyze lysogenization but not phage growth at the nonpermissive temperature. Phages carrying mutations located at different positions in the Mu B gene (which encodes a product necessary for efficient integration and lytic replication) were all able to lysogenize at the same frequency. These results suggest that the ability of Mu to lysogenize is not strictly correlated with its ability to perform massive conservative and replicative transposition.     

DNA-binding proteins specified by bacteriophage T1

Abstract A type A Clostridium perfringens enterotoxin was chially purified by ammonium sulfate precipitation (0 to 15%) and was submitted to polyacrylamide gel electrophoresis (7%). A specific enterotoxin antiserum was obtained by inoculating a rabbit with the polyacrylamide gel strip containing the enterotoxin. This serum gave only one precipitin line with purified enterotoxin and cellular extract in immunodiffusion and immunoelectrophoresis. The titer (1:8) in counter-immunoelectrophoresis was sufficient to detect 0.39 μg/ml enterotoxin by this technique. This serum neutralized the mouse lethality, cytotoxicity and plating efficiency of Vero cells.     

Localization and regulation of bacteriophage Mu promoters.

Mu promoters active during the lytic cycle were located by isolating RNA at various times after induction of Mu prophages, radiolabeling it by capping in vitro, and hybridizing it to Mu DNA fragments on Southern blots. Signals were detected from four new promoters in addition to the previously characterized Pe (early), PcM (repressor), and Pmom (late) promoters. A major signal upstream of C was first observed at 12 min and intensified thereafter with RNA from cts and C amber but not replication-defective prophages; these characteristics indicate that this signal arises from a middle promoter, which we designate Pm. With 20- and 40-min RNA, four additional major signals originated in the C-lys, F-G-I, N-P, and com-mom regions. These signals were missing with RNA from C amber and replication-defective prophages and therefore reflected the activity of late promoters, one of which we presume was Pmom. Uninduced lysogens showed weak signals from five regions, one from the early regulatory region, three between genes B and lys, and one near the late genes K, L, and M. The first of these probably resulted from PcM activity; the others remain to be identified.     

Defective prophages of bacteriophage Mu

Summary A method is described for the isolation of thermoinducible defective Mu lysogens. Four of these defective lysogens were studied more extensively. By marker-rescue experiments it was shown that the strain harbouring the smallest defective prophage contains the immunity gene cts and the genes A and B; the strain with the largest defective prophage still contains all the known essential genes of Mu, A to S (see Fig. 1).After induction at 43° C all the defective lysogens are killed, whereas no lysis occurs.Although in all the thermoinducible defective lysogens the A and B gene products could be demonstrated by complementation, these gene products are not responsible for the killing of the host, suggesting the presence of another unknown early gene product of Mu. The level of complementation of a mutation in gene A is reduced by the presence in the cell of another defective Mu prophage containing the G part of Mu. This effect on A gene complementation is markedly enhanced when the defective prophage, containing the G part, is located on an episome instead of on the chromosome. Complementation of late genes by a defective prophage located on the chromosome, is extremely low or undetectable. A stimulation of complementation by a factor of 10 to 40 was found when the same defective prophage was situated on a F factor. A possible explanation for this episome effect will be discussed.     

Regulation of bacteriophage Mu transposition

Bacteriophage Mu is a transposon and a temperate phage which has become a paradigm for the study of the molecular mechanism of transposition. As a prophage, Mu has also been used to study some aspects of the influence of the host cell growth phase on the regulation of transposition. Through the years several host proteins have been identified which play a key role in the replication of the Mu genome by successive rounds of replicative transposition as well as in the maintenance of the repressed prophage state. In this review we have attempted to summarize all these findings with the purpose of emphasizing the benefit the virus and the host cell can gain from those phage-host interactions.     

Specificity of bacteriophage Mu excision

Summary To study the excision of bacteriophage Mu at the DNA sequence level, the Mu-derived phage placMu3 was transposed to the transcribed but non-translated leader region of a plasmid-borne tetracycline (tet) resistance gene. Revertants (excision products) were then selected by Tet⁺ restoration of Tet⁺ and characterized. Of 21 independent Tet⁺ revertants, 17 contained simple deletions of most or all of placMu3, while the other four contained more complex rearrangements in which one end of placMu3 had been transposed, and most of the prophage had been deleted. The deletion endpoints were found in short direct repeats in each of the complex rearrangements and in 11 of the 17 simple deletion excisants. The results suggest models of slipped mispairing of template and nascent DNA strands facilitated by proteins of the Mu transposition machinery.