Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.     

Interspecies transduction of plasmids among Bacillus anthracis, B. cereus, and B. thuringiensis

Bacteriophage CP-51, a generalized transducing phage for Bacillus anthracis, B. cereus, and B. thuringiensis, mediates transduction of plasmid DNA. B. cereus GP7 harbors the 2.8-megadalton multicopy tetracycline resistance plasmid, pBC16. B. thuringiensis 4D11A carries pC194, the 1.8-megadalton multicopy chloramphenicol resistance plasmid. When phage CP-51 was propagated on these strains, it transferred the plasmid-encoded antibiotic resistances to the nonvirulent Weybridge (Sterne) strain of B. anthracis, to B. cereus 569, and to strains of several B. thuringiensis subspecies. The frequency of transfer was as high as 10(-5) transductants per PFU. Tetracycline-resistant and chloramphenicol-resistant transductants contained newly acquired plasmid DNA having the same molecular weight as that contained in the donor strain. Antibiotic-resistant transductants derived from any of the three species were effective donors of plasmids to recipients from all three species.     

Plasmid transduction by Bacillus anthracis bacteriophage CP54

Possibility of plasmid transduction in Bacillus anthracis vaccine strains Sterne and STI-1 by bacteriophage CP54ant having an increased ability of adsorbtion and a shortened period of latent development in Bacillus anthracis cells has been isolated. The main parameters of plasmid transduction by the bacteriophage have been established for the plasmid pTG141 (TcR). They include the effect of multiplicity of infection, the level of UV-inactivation of bacteriophage, the presence of antiphage serum in the incubation medium. Plasmid transduction by the mutant phage CP54ant was found to be more efficient as compared with the one by the parent phage. The isolated transductants served as donors of the transduced plasmid for Bacillus anthracis and Bacillus thuringiensis strains.     

Transduction in Bacillus cereus by Each of Two Bacteriophages

The ability of phage CP-51 to mediate transduction both homologously and heterologously in some of its hosts was investigated. CP-51 was shown to transduce Bacillus cereus strains 6464, 9139, and T in addition to 569 which was reported earlier from this laboratory. Furthermore, CP-51 grown on B. thuringiensis was shown to transduce some mutants of B. cereus. During this investigation, a second transducing phage for B. cereus 569 was isolated from lysates of phage CP-51 grown on B. cereus 6464. This phage, designated CP-53, is carried by wild-type strain 6464 possibly as prophage. All auxotrophic mutants of B. cereus 569 tested, those requiring tryptophan, histidine, methionine, and leucine, were transduced to prototrophy by CP-53. Electron micrographs of the two phages revealed that CP-51 has a tail core surrounded by a contractile sheath and CP-53 has a long flexible tail without a contractile sheath. CP-53 is stable in the cold, whereas CP-51 is rapidly inactivated at 4 C.     

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis

A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.     

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis.

A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.     

Transduction in Bacillus thuringiensis.

Bacteriophage CP-51, originally reported as a generalized transducing phage for Bacillus cereus and B. anthracis, has been shown to carry out generalized transduction in several strains of B. thuringiensis. A newly isolated phage, CP-54, which has a broader host range than CP-51, also mediates generalized transduction in B. thuringiensis. CP-51 and CP-54 are similar in size and morphology and are related serologically, but they are not identical. CP-54 is more cold labile than CP-51, and, as with CP-51, its stability both at 0 and 15 degrees C is enhanced by the presence of 0.02 M Mg2+. Some examples of cotransduction of linked markers in B. thuringiensis are presented, demonstrating the feasibility of chromosomal mapping in this organism. The rare occurrence of cross-transduction among strains of B. thuringiensis is probably a reflection of nonhomology rather than restriction, since phage itself did not appear to be restricted when grown on a particular host and assayed with other hosts as indicator.     

High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage phiadh

The temperate bacteriophage phiadh mediates plasmid DNA transduction in Lactobacillus gasseri ADH at frequencies in the range of 10 to 10 transductants per PFU. BglII-generated DNA fragments from phage phiadh were cloned into the BclI site of the transducible plasmid vector pGK12 (4.4 kb). Phage phiadh lysates induced from Lactobacillus lysogens harboring pGK12 or the recombinant plasmids were used to transduce strain ADH to chloramphenicol resistance. The transduction frequencies of recombinant plasmids were 10- to 10-fold higher than that of native pGK12. The increase in frequency generally correlated with the extent of DNA-DNA homology between plasmid and phage DNAs. The highest transduction frequency was obtained with plasmid pTRK170 (6.6 kb), a pGK12 derivative containing the 1.4- and 0.8-kb BglII DNA fragments of phiadh. DNA hybridization analysis of pTRK170-transducing phage particles revealed that pTRK170 had integrated into the phiadh genome, suggesting that recombination between homologous sequences present in phage and plasmid DNAs was responsible for the formation of high-frequency transducing phage particles. Plasmid DNA analysis of 13 transductants containing pTRK170 showed that each had acquired intact plasmids, indicating that in the process of transduction a further recombination step was involved in the resolution of plasmid DNA monomers from the recombinant pTRK170::phiadh molecule. In addition to strain ADH, pTRK170 could be transduced via phiadh to eight different L. gasseri strains, including the neotype strain, F. Gasser 63 AM (ATCC 33323).     

Specialized transduction of kanamycin resistance in a Providence strain.

Properties of a transducing system with a phage able to transduce a kanamycin-resistance marker of the T compatibility group plasmid R394 at a frequency of 2 times 10(-2)/plaque-forming unit adsorbed are described. The phage was detected in Providence strain P29 transduced to kanamycin resistance by Providence phage PL25 grown on this strain harbouring the R factor. Four P29 transductants, specially selected at the lowest multiplicities of infection of the high frequency transducing (HFT) phage, were defective lysogens. They plated PL25 with an efficiency of I and only one liberated low-titre phage spontaneously or on u.v. induction. The defect in maturation function could be corrected by introduction of a wild PL25 prophage. The transducing phage was serologically frequency was increased by the simultaneous presence of homologous non-transducing phage. Transductants did not transfer the kanamycin-resistance marker by conjugation, and produced kanamycin-sensitive segregants at a moderate rate. These segregants could be transduced to kanamycin resistance by the HFT phage. Irradiation of HFT lysates by u.v. produced an exponential fall in transduction frequency. It was concluded that the defective phage transduced by lysogenization. Kanamycin-resistant transductants could themselves be transduced by streptomycin resistance by PL25 reared on a streptomycin-resistant mutant. Lysogenic transductants produced by the HFT phage did not always liberate HFT phage on u.v. induction. Possible explantations are considered.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

A specialized host-vector system for the in Vivo cloning of the trp operon of wild-type and mutant strains of Salmonella typhimurium by generalized transduction

Using in vitro methods, a 14.2-kb EcoRI fragment of the Salmonella typhimurium chromosome containing the trp operon plus associated flanking sequences from deletion mutant delta trpDCB763 was cloned into the EcoRI site of plasmid pBR322 in a S. typhimurium host. An in vivo cloning vector was constructed from the recombinant plasmid by the in vitro excision of a SalI fragment that contains the entire trp operon. The derived plasmid (pSTP21) carries a hybrid insert made up of the 5.4-kb EcoRI-SalI upstream flanking sequence and the 3.2-kb SalI-EcoRI downstream flanking sequence. Plasmid pSTP21 has been used as a receptor plasmid to clone a variety of mutant and wild-type trp operons by RecA-dependent in vivo recombination between the insert DNA of the plasmid and the homologous trp flanking sequences of transducing DNA fragments transferred into the cell by bacteriophage P22. The host-vector system developed for the in vivo cloning permits the differentiation of plasmid transductants from chromosomal transductants on the primary selective medium. Expression of the cloned trp operons is regulated normally by tryptophan. A substantial amplification of trp enzymes is attainable upon derepression. The recombinant plasmids are stably inherited in RecA+ and RecA- S. typhimurium hosts. However, conditions of high expression of the trp operon lead to a rapid loss of cellular viability and of plasmid stability.     

Self-transfer and mobilisation capabilities of the pXO2-like plasmid pBT9727 from Bacillus thuringiensis subsp. konkukian 97-27

Recent characterisations of plasmids related to the anthrax virulence plasmids pXO1 and pXO2 in clinical isolates of Bacillus cereus and Bacillus thuringiensis have contributed to the emerging picture of a virulence-associated plasmid pool in the B. cereus sensu lato group. The family of pXO2-like plasmids includes the conjugative plasmid pAW63 from the biopesticide strain B. thuringiensis subsp. kurstaki HD73 and the heretofore cryptic plasmid pBT9727 from the clinical strain B. thuringiensis subsp. konkukian 97-27. Comparative sequence analysis of these three plasmids suggested that they were derived from an ancestral conjugative plasmid, with pAW63 retaining its self-transfer capabilities, and pXO2 having lost them through genetic drift. Such properties had not been investigated in pBT9727, but sequence homologies led us to predict that it may possess self-transfer capabilities. Here, we report that pBT9727 is indeed conjugative, and is able to promote its own transfer as well as that of small mobilisable plasmids.     

Effects of the Recipient Strain and Ultraviolet Irradiation on Transduction Kinetics of the Penicillinase Plasmid of Staphylococcus aureus

When the penicillinase plasmid of Staphylococcus aureus PS 81(P(81))(T(81)) was transferred to its cured derivative of PS 81(N(P))(T(81)), there was a fivefold increase in the transduction frequency of penicillinase plasmid markers after ultraviolet (UV) irradiation of the phage instead of the expected decrease typical for plasmid-borne markers. These results were independent of the transducing phage, the donor, and the method of curing the recipient and were also obtained with a cured derivative of PS 80(PI(80)). With PS 52, a naturally occurring penicillin-sensitive strain, and a cured transductant of PS 52 as the recipients, typical plasmid kinetics were observed. The plasmid location of penicillinase plasmid markers in transductants was confirmed by their instability in ethidium bromide (EB). In a cross between isogenic plasmids (PI(258)penZ cad x PI(258)penI asa ero), transductants were doubly selected for cadmium and erythromycin resistances. There was a twofold increase in transduction frequency after UV irradiation of the transducing phage and an increase in the proportion of recombinant type transductants. CsCl-EB density centrifugation revealed that plasmid deoxyribonucleic acid (DNA) was present in PS 81(P(81))(N(T)) and its cured derivative [PS 81(N(P))(N(T))], but not in PS 52. Sucrose gradient analysis of plasmid DNA showed that the penicillinase plasmid of PS 81(P(81))(N(T)) was larger than the plasmid in its cured derivative. Thus, the cured derivative contains plasmid DNA which appears to recombine with the incoming plasmid, causing the rise in transduction frequency noted after UV irradiation of transducing phage.     

Transduction ability of mutants of phage CP51, virulent for bacteria of the Bacillus cereus group

The virulent phage CP51 used usually to transfer chromosomal and plasmid markers between bacteria of the Bacillus cereus group was treated with N-methyl-N'-nitro-N-nitroguanidine. Mutants with reduced viability and ts-mutants were isolated. Some of the mutants were found to have an increased efficiency of transduction and allow to simplify the process. Transfer frequencies of the plasmid pBC16 by the phages CP51-26 and CP51-4-59 were 5 x 10(-4) per plaque-forming unit and 4-5 x 10(-3) per bacterial cell, respectively. Possibilities of further increasing the transduction efficiency of Bacillus thuringiensis genetic material using phage CP51 mutants are discussed.     

Converting bacteriophage for sporulation and crystal formation in Bacillus thuringiensis.

Bacteriophage TP-13, a converting phage for sporulation and crystal formation in Bacillus thuringiensis, was isolated from soil. The phage converted anoligosporogenic (sporulation frequency, 10(-8), acrystalliferous mutant to spore positive, crystal positive at a high frequency. Each plaque formed by TP-13 in a lawn of sensitive cells contained spores and crystals. These spores were heat stable, and each one was capable of producing a plaque from which TP-13 could be reisolated. Conversion of cells to sporulation and crystal formation was independent of the ho-t used for TP-13 propagation. When converted cells were cured of TP-13, they lost the ability to produce spores and crystals. Incubation of TP-13 with antiserum prepared against purified phage particles prevented conversion. TP-13 has some characteristics similar to those of SP-15 and PBS-1, including large size, morphology, and adsorption specificity of motile cells. TP-13 mediated generalized transduction in several strains of B. thuringiensis at frequencies of 10(-6) to 10(-5). Comparison of cotransduction values indicated that TP-13 transduced considerably larger segments of deoxyribonucleic acid than CP-51 or TP-10, two other transducing phages for B. thuringiensis.     

Regulation of protoxin synthesis in Bacillus thuringiensis.

A derivative of Bacillus thuringiensis subsp. kurstaki (HD-1) formed parasporal inclusions at 25 degrees C, but not at 32 degrees C. This strain differed from the parent only in the loss of a 110-megadalton (Md) plasmid, but plasmid and chromosomal copies of protoxin genes were present in both strains. On the basis of temperature shift experiments, the sensitive period appeared to be during midexponential growth, long before the time of protoxin synthesis at 3 to 4 h after the end of exponential growth. The conditional phenotype could be transferred by cell mating to naturally acrystalliferous Bacillus cereus. In all such cases, a 29-Md protoxin -encoding plasmid was transferred, but this plasmid alone was barely sufficient for protoxin synthesis. Protoxin production increased to detectable levels, but well below those of the parental donor strain, by simultaneous transfer of a 44-Md protoxin -encoding plasmid. Transfer of a 5-Md plasmid with the two larger protoxin -coding plasmids resulted in a protoxin synthesis level approaching that of the donor strain. A role for some of the cryptic plasmids of kurstaki in parasporal body formation was implied. In contrast, a closely related B. thuringiensis strain, HD73 , produced crystals at both 25 and 32 degrees C even when the capacity was transferred on a 50-Md plasmid to B. cereus. The amount of protoxin produced in these B. cereus transcipients , however, was somewhat less than that produced in the parental strain HD73 , implying that catabolic differences, gene dosage, or the presence of a chromosomal gene (or a combination of these) may be necessary for maximum production. A regulatory component of the 29-Md plasmid appeared to be trans-acting and dominant since B. cereus transcipients containing the 29-Md plasmid from kurstaki and the 50-Md plasmid from HD73 produced more protoxin at 25 degrees C than at 30 degrees C. Similar results were obtained when protoxin synthetic capacity was transferred from B. thuringiensis subsp. israelensis to the conditional B. thuringiensis subsp. kurstaki strain.     

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.     

Characteristics of transduction in Erwinia by phage 59

A temperate bacteriophage 59 from polylysogenic strain Erwinia carotovora 268 transduces the following genetic markers: arg+, ilv+, leu+, met+, thr+, thy+, trp+, ura+. The transduction frequencies varied from 1 x 10(-8)- to 1 x 10(-6) and dependent on the multiplicity of infection, UV-irradiation of transducing bacteriophage, the nature of phage lysates. The characteristics of single transductants have been studied.Analysis of the obtained results suggests bacteriophage 59 to perform the generalized transduction.     

Unique regulation of crystal protein production in Bacillus thuringiensis subsp. yunnanensis is mediated by the cry protein-encoding 103-megadalton plasmid.

In sporulating cultures of Bacillus thuringiensis subsp. yunnanensis HD977, two cell types are observed: cells forming only spores and cells forming only crystals. Curing analysis suggested that the crystal proteins are plasmid encoded. Through plasmid transfer experiments, it was established that a 103-MDa plasmid is involved in the crystal production. Conjugal transfer of this plasmid to Cry- recipient cells of Bacillus thuringiensis subsp. kurstaki HD73-26 conferred the ability to produce crystals exclusively on asporogenous cells of the recipient, indicating that the 103-MDa plasmid mediates the unique regulation of Cry protein production. When the dipteran-specific cryIVB gene was introduced into wild-type (Cry+) and Cry- backgrounds of B. thuringiensis subsp. yunnanensis by phage CP51ts45-mediated transduction, similar to all other B. thuringiensis strains, irregular crystals of CryIVB protein were produced by spore-forming cells in both backgrounds. However, the synthesis of the bipyramidal inclusions of B. thuringiensis subsp. yunnanensis was still limited only to asporogenous cells of the transductant. Thus, it appears that the unique property of exclusive crystal formation in asporogenous cells of B. thuringiensis subsp. yunnanensis is associated with the crystal protein gene(s) per se or its cis acting elements. As the crystals in B. thuringiensis subsp. yunnanensis were formed only in asporogenous cells, attempts were made to find out whether crystal formation had any inhibitory effect on sporulation. It was observed that both Cry+ and Cry- strains of B. thuringiensis subsp. yunnanensis (HD977 and HD977-1, respectively) exhibited comparable sporulation efficiencies. In addition, the Cry- B. thuringiensis subsp. kurstaki host (HD73-26) and its Cry+ transconjugant (HD73-26-16), expressing the B. thuringiensis subsp. yunnanensis crystal protein, were also comparable in their sporulation efficiencies, indicating that production of the crystal proteins of B. thuringiensis subsp. yunnanensis does not affect the process of sporulation.     

Design and use of synthetic oligonucleotide probes in the cloning of delta-endotoxin genes from Bacillus thuringiensis.

A detailed protocol is described for the design and use of synthetic oligonucleotide probes for screening DNA libraries from Bacillus thuringiensis var. kurstaki (strain HD191) for copies of the gene (tox) encoding the insecticidal delta-endotoxin. Two homologous tox genes were identified in this organism; one of these was located on a 75-kb plasmid and the other on a second large plasmid or the bacterial chromosome. A tox gene was isolated as a 6.5-kb HindIII fragment of B. thuringiensis plasmid DNA.