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C B Thorne

Publications and source records attributed to C B Thorne.

At least 19 recordsLinked to original sources
PubMed1993

Cloning and characterization of a gene whose product is a trans-activator of anthrax toxin synthesis.

The 184-kb Bacillus anthracis plasmid pXO1, which is required for virulence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), lef (lethal factor gene), and pag (protective antigen gene). Expression of the three proteins is induced by bicarbonate or serum. Using a pag-lacZ transcriptional construct to measure pag promoter activity, we cloned in Bacillus subtilis a gene (atxA) whose product acts in trans to stimulate anthrax toxin expression. Deletion analysis located atxA on a 2.0-kb fragment between cya and pag. DNA sequencing identified one open reading frame encoding 476 amino acids with a predicted M(r) of 55,673, in good agreement with the value of 53 kDa obtained by in vitro transcription-translation analysis. The cloned atxA gene complemented previously characterized Tn917 insertion mutants UM23 tp29 and UM23 tp32 (J. M. Hornung and C. B. Thorne, Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991, abstr. D-121, p. 98), which are deficient in synthesis of all three toxin proteins. These results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef. beta-Galactosidase synthesis from the pag-lacZ transcriptional fusion construct introduced into an insertion mutant (UM23 tp62) which does not require bicarbonate for toxin synthesis indicated that additional regulatory genes other than atxA play a role in the induction of anthrax toxin gene expression by bicarbonate.

Amino Acid Sequence↗
PubMed1989

Involvement of Tn4430 in transfer of Bacillus anthracis plasmids mediated by Bacillus thuringiensis plasmid pXO12.

The self-transmissible plasmid pXO12 (112.5 kilobases [kb]), originally isolated from strain 4042A of Bacillus thuringiensis subsp. thuringiensis, codes for production of the insecticidal crystal protein (Cry+). The mechanism of pXO12-mediated plasmid transfer was investigated by monitoring the cotransfer of the tetracycline resistance plasmid pBC16 (4.2 kb) and the Bacillus anthracis toxin and capsule plasmids, pXO1 (168 kb) and pXO2 (85.6 kb), respectively. In matings of B. anthracis donors with B. anthracis and Bacillus cereus recipients, the number of Tcr transcipients ranged from 4.8 x 10(4) to 3.9 x 10(6)/ml (frequencies ranged from 1.6 x 10(-4) to 7.1 x 10(-2), and 0.3 to 0.4% of them simultaneously inherited pXO1 or pXO2. Physical analysis of the transferred plasmids suggested that pBC16 was transferred by the process of donation and that the large B. anthracis plasmids were transferred by the process of conduction. The transfer of pXO1 and pXO2 involved the transposition of Tn4430 from pXO12 onto these plasmids. DNA-DNA hybridization experiments demonstrated that Tn4430 was located on a 16.0-kb AvaI fragment of pXO12. Examination of Tra- and Cry- derivatives of pXO12 showed that this fragment also harbored information involved in crystal formation and was adjacent to a restriction fragment containing DNA sequences carrying information required for conjugal transfer.

Bacillus anthracis↗
PubMed1988

Five unique temperate phages from a polylysogenic strain of Bacillus thuringiensis subsp. aizawai.

Five temperate phages were isolated from strain 4042B of Bacillus thuringiensis subsp. aizawai. The phages, which were heteroimmune, could also be distinguished by their host ranges, plaque and particle morphologies, serological specificities, and locations of restriction endonuclease cleavage sites on their chromosomes. Besides maintaining a stable lysogenic relationship with the 4042B host strain, each phage formed a stable lysogen with Bacillus cereus.

Bacillus thuringiensis↗
PubMed1987

Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies.

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.

Bacillus anthracis↗
PubMed1987

Bacillus subtilis (natto) plasmid pLS20 mediates interspecies plasmid transfer.

The 55-kilobase plasmid, pLS20, of Bacillus subtilis (natto) 3335 promotes transfer of the tetracycline resistance plasmid pBC16 from B. subtilis (natto) to the Bacillus species B. anthracis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, and B. thuringiensis. Frequency of pBC16 transfer ranged from 2.3 x 10(-6) to 2.8 x 10(-3). Evidence for a plasmid-encoded conjugationlike mechanism of genetic exchange includes (i) pLS20+ strains, but not pLS20- strains, functioned as donors of pBC16; (ii) plasmid transfer was insensitive to the presence of DNase; and (iii) cell-free filtrates of donor cultures did not convert recipient cells to Tcr. Cotransfer of pLS20 and pBC16 in intraspecies matings and in matings with a restriction-deficient B. subtilis strain indicated that pLS20 was self-transmissible. In addition to mobilizing pBC16, pLS20 mediated transfer of the B. subtilis (natto) plasmid pLS19 and the Staphylococcus aureus plasmid pUB110. The fertility plasmid did not carry a selectable marker. To facilitate direct selection for pLS20 transfer, plasmid derivatives which carried the erythromycin resistance transposon Tn917 were generated. Development of this method of genetic exchange will facilitate the introduction of plasmid DNA into nontransformable species by use of transformable fertile B. subtilis or B. subtilis (natto) strains as intermediates.

Bacillus subtilis↗
PubMed1985

Demonstration of a capsule plasmid in Bacillus anthracis.

Virulent and certain avirulent strains of Bacillus anthracis harbor a plasmid, designated pXO2, which is involved in the synthesis of capsules. Two classes of rough, noncapsulated (Cap-) variants were isolated from the capsule-producing (Cap+) Pasteur vaccine strains ATCC 6602 and ATCC 4229. One class was cured of pXO2, and the other class still carried it. Reversion to Cap+ was demonstrable only in rough variants which had retained pXO2. Proof that pXO2 is involved in capsule synthesis came from experiments in which the plasmid was transferred by CP-51-mediated transduction and by a mating system in which plasmid transfer is mediated by a Bacillus thuringiensis fertility plasmid, pXO12. Cells of Bacillus cereus and a previously noncapsulated (pXO2-) strain of B. anthracis produced capsules after the acquisition of pXO2.

Bacillus anthracis↗
PubMed1985

Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

To facilitate the analysis of genetic determinants carried by large resident plasmids of Bacillus anthracis, a mating system was developed which promotes plasmid transfer among strains of B. anthracis, B. cereus, and B. thuringiensis. Transfer of the selectable tetracycline resistance plasmid pBC16 and other plasmids from B. thuringiensis to B. anthracis and B. cereus recipients occurred during mixed incubation in broth. Two plasmids, pXO11 and pXO12, found in B. thuringiensis were responsible for plasmid mobilization. B. anthracis and B. cereus transcipients inheriting either pXO11 or pXO12 were, in turn, effective donors. Transcipients harboring pXO12 were more efficient donors than those harboring pXO11; transfer frequencies ranged from 10(-4) to 10(-1) and from 10(-8) to 10(-5), respectively. Cell-to-cell contact was necessary for plasmid transfer, and the addition of DNase had no effect. The high frequencies of transfer, along with the fact that cell-free filtrates of donor cultures were ineffective, suggested that transfer was not phage mediated. B. anthracis and B. cereus transcipients which inherited pXO12 also acquired the ability to produce parasporal crystals (Cry+) resembling those produced by B. thuringiensis, indicating that pXO12 carries a gene(s) involved in crystal formation. Transcipients which inherited pXO11 were Cry-. This mating system provides an efficient method for interspecies transfer of a large range of Bacillus plasmids by a conjugation-like process.

Bacillus↗
PubMed1984

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.

Bacillus anthracis↗
PubMed1984

Chromosomal mapping of Bacillus thuringiensis by transduction.

Three groups of linked markers were mapped in Bacillus thuringiensis 4042B by using two-, three-, and four-factor crosses mediated by the temperate bacteriophages TP-13 and TP-18. The order of markers was (trp-11, trp-2)-(leu-1, leu-2)-his-1-(lys-1, lys-2)-cys-1 in the first group; met-1-(argCl, argOl)-met-2-(pyr-1, pyrA2) in the second group; and met-3-pur-1-(nal-1, nal-2)-str-1-(pur-2, pur-4)-pur-3 in the third group. Electron microscopic measurements of head sizes suggested that the volume of the TP-13 phage head is seven times greater than that of the TP-18 phage head. The TP-18 genome was shown by DNA restriction analysis to have a molecular mass of 36 megadaltons. TP-13 was useful for scanning large segments of the B. thuringiensis chromosome, and TP-18 was effective for ordering markers too closely linked for simple resolution with TP-13.

Bacillus thuringiensis↗
PubMed1979

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.

Bacillus thuringiensis↗
PubMed1978

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.

Amino Acids↗
PubMed1974

Cold lability of Bacillus cereus bacteriophage CP-51.

Phage CP-51 was rapidly inactivated when stored at the usual refrigerator temperatures (2 to 4 C) and even more rapidly when exposed to 0 C. The loss in viability resulting from exposure to cold appeared to correlate with the increase in number of phage particles having contracted tails. High concentrations (0.01 M) of Mg(2+), Ca(2+), or Mn(2+) stabilized the phage considerably, but even in the presence of these divalent cations, it was much less stable at 0 C than at 15 C.

Bacillus cereus↗
PubMed1971

Genetic mapping of genes concerned with glutamyl polypeptide production by Bacillus licheniformis and a study of their relationship to the development of competence for transformation.

By transduction with SP-15, we have mapped some of the genetic sites involved in glutamyl polypeptide (Pep) production by Bacillus licheniformis 9945A. Eleven peptide mutations were assigned to group 1 which lies between the gly-42 and his-6 markers. Three other mutations were assigned to group 2 which lies between the ade-2 and str-1 sites. A series of 28 additional Pep(-) mutants did not belong to either group 1 or 2; the chromosomal location of those peptide mutations is unknown. All three mutants of group 2 were highly transformable, but only two of group 1 transformed well under the conditions employed. Knowing the chromosomal markers that were linked to peptide mutations made it possible to investigate the effect peptide mutations might have on the development of competence for transformation. Nontransformable organisms were rendered transformable upon the introduction, by transduction, of Pep(-) mutations from transformable strains. These results supported the conclusion that the ability of cells to develop competence for transformation when grown under appropriate conditions was related to the physiological effects of particular peptide mutations.

Bacillus↗
PubMed1971

Comparison of Bacillus cereus bacteriophages CP-51 and CP-53.

Transducing bacteriophages CP-51 and CP-53 were compared. Unlike CP-51, CP-53 appeared to be a lysogenizing phage. CP-51 gave greater frequencies of co-transduction for linked markers than did CP-53. CP-51 was found to be a larger phage which carried more deoxyribonucleic acid (DNA) than CP-53. CP-51 DNA contained about 43% guanine plus cytosine and in addition contained 5-hydroxymethyluracil in place of thymine. CP-53 DNA contained no unusual bases; its guanine plus cytosine content was 37%.

Adsorption↗
PubMed1970

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.

Animals↗
PubMed1968

Transducing bacteriophage for Bacillus cereus.

A phage, designated CP-51, that carries out generalized transduction in Bacillus cereus 569 was isolated from soil. All auxotrophic mutants tested, those requiring tryptophan, histidine, leucine, isoleucine, methionine, or phenylalanine, were transduced to prototrophy. The phage was extremely unstable when stored at 2 to 4 C, but stability was enhanced by storage at higher temperatures. The optimal temperature of those tested for maintenance of plaque-forming units was 15 C.

Amino Acids↗