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.     

Plasmid transformation of Bacillus cereus protoplasts

The process of polyethyleneglycol-induced plasmid transformation of Bacillus cereus protoplasts was studied. Plasmid transfer into Bacillus cereus strains was demonstrated with the frequencies 1.3.10(1)-1.6.10(2) transformants per 1 mkg of plasmid DNA. The plasmids transferred are stably inherited by Bacillus cereus cells causing tetracycline resistance (pBC16) or kanamycin resistance (pUB110 and pBD64). The proposed method can be used for construction of Bacillus cereus strains having the plasmid determined characteristics.     

Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis.

A number of plasmids have been isolated as covalently closed circular DNAs from strains of Bacillus cereus and B. subtilis. From 12 out of 15 strains of B. cereus, plasmids could be isolated. Most of the B. cereus strains contained two or more plasmids. Their molecular weights ranged from 1.6 X 10(6) to 105 X 10(6). Bacteriocin production could be attributed to a 45 X 10(6)-dalton plasmid (pBC7) from B. cereus DSM 336, and tetracycline resistance to a 2.8 X 10(6) plasmid (pBC16) from B. cereus GP7. Two streptomycin-resistant strains of B. subtilis harbored plasmids of 5.2 X 10(6) and 9 X 10(6), respectively, which were, however, not correlated with the antibiotic resistance. The plasmid carrying resistance to tetracycline, pBC16, which was originally isolated from B. cereus, could be subsequently transformed in B. subtilis, where it is stably maintained.     

Comparative study of the symbiotic plasmid DNA in free living bacteria and bacteroids of Rhizobium leguminosarum

Plasmids pIM13, pT127 and pBC16 delta 1, introduced by transformation into Clostridium acetobutylicum N1-4081, were shown to replicate in, and to confer antibiotic resistance upon this new host. Recombinant plasmids were constructed by inserting erythromycin-resistant plasmid pIM13 into the unique ClaI site of pBR322 or by ligating a tetracycline-resistant determinant of plasmid pT127 to HindIII-linearized pIM13. The hybrid plasmids replicated and expressed erythromycin resistance in C. acetobutylicum strain N1-4081 and in Escherichia coli or Bacillus subtilis, indicating that they might be useful as shuttle vectors for transferring genes between these strains. The efficiency and stability of different replicons in C. acetobutylicum were compared.     

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 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.     

Transformation of a Leu− Mutant of Rhizopus niveus with the leuA Gene of Mucor circinelloides

The 2287-bp cryptic plasmid, pMA1, from Microcystis aeruginosa f. aeruginosa Kützing, a unicellular cyanobacterium originally derived from Kasumigaura lake, was completely sequenced and analyzed. The predicted amino acid sequence (253 residues) of an open reading frame had identities of 47%, 48%, and 53% with replication-associated proteins of Bacillus amyloliquefaciens’s plasmid, pFTB14, B. subtilis BAA1’s pBAA1, and B. coagulans’s pBC1, respectively, when conservative amino acid substitutions were included. Such high-level identities were also shown with rep proteins and ori regions in a group of Gram-positive bacterial plasmids such as Lactococci and Staphylococci that are known to replicate via single-stranded intermediates. The pMA1 does hybridize with a plasmid, pUS1-3, derived from another unicellular cyanobacterium, Synechocystis sp. PCC 6803. Novel features of pMA1 are discussed.     

Syntheses and Flavors of Four Possible Optical Isomers of Marmelo Lactones

We have isolated a tetracycline-resistant (Tc^r) Bacillus species (named HE-1) which carries multiple plasmids. HE-1 was identified as Bacillus cereus and found to bear four plasmids. Tetracycline resistance could be attributed to one of four plasmids (designated as pTIT β2 (4.7 kb)) indistinguishable from pBC16, a Tc^r plasmid formerly found in B. cereus [K. Bernhard, H. Schrempf, and W. Goebel, J. Bacteriol., 133, 897 (1978)]. All the other three plasmids (named pTITα (4.0 kb), pTIT β1 (4.7 kb) and pTIT γ (12.4 kb)) were cryptic and did not correlate with bacterial phenotypic traits such as antibiotic resistance or antibiotic and bacteriocin production. B. cereus HE-1 also showed resistance to penicillin, but this seemed very likely to be chromosomally determined in B. cereus. Of interest was the fact that pTITα, pTIT β1, and pTITγ had a noticeable DNA homology among them in blot hybridization. pTIT β2 alone did not shared sequence homology with the other three plasmids.     

Characteristics of Plasmids in Rhizobium huakuii

Rhizobium huakuii nodulates Astragalus sinicus, an important green manuring crop in Southern China, which can be used as forage. The plasmid profiles of 154 R. huakuii strains were examined with the Eckhardt procedure. The plasmid number of the strains varied from one to five, and their molecular weights were estimated from 42 to 600 mDa or more. The plasmids were hybridized with probes nodABC and nifHDK. The results showed that there was one plasmid carrying the nod and nif genes in the strains that harbor two or more plasmids, and the molecular weights of the symbiotic plasmids varied from 117 to 251 mDa. Homology was not observed on plasmids in the strains having only one plasmid; presumably the symbiotic genes are on the chromosome. Plasmid curing was carried out with the Bacillus subtilus sacB to generate derivatives of Rhizobium huakuii strain CH203, which harbors three plasmids, pRHa(97MD), pRHb(168MD), and pRHc(251MD). The largest plasmid (pRHc) carried both nodulation and nitrogen fixation genes. When pRHc was cured, the strain lost its symbiotic ability. The other two plasmids were also related to symbiosis. The derivative cured of pRHb did not nodulate on the host plant, had an altered lipopolysaccharide, and grew much more slowly than the parent strain. Curing of the smallest plasmid (pRHa) resulted in delaying the strain nodulation and made it lose nitrogen fixation ability. Curing of each plasmid in strain CH203 reduced its acid tolerance. Complementation of plasmid-cured strains with appropriate plasmids restored their original phenotypes. Received: 18 December 1996 / Accepted: 28 March 1997     

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.     

Construction and characterization of plasmid vectors for cloning in the entomocidal organism Bacillus sphaericus 1593

A plasmid vector for cloning in Bacillus sphaericus 1593 was constructed in B. subtilis from two parent plasmids, pBC16 and pBD64. When characterized, the 3.9-MDa chimeric plasmid pNN101 was found to consist of the MspI fragment containing the chloramphenicol acetyltransferase (CAT) gene from pBD64 inserted into an MspI site in pBC16. pNN101 was shown to replicate, express, and be stably maintained in B. sphaericus 1593 without affecting the mosquito larvicidal activity of this organism. A derivative of this plasmid, pNN302, was constructed in which a unique HindIII site was introduced into the CAT gene without loss of chloramphenicol resistance.     

Protoplast fusion in Bacillus cereus

Polyethyleneglycol induced fusion of Bacillus cereus protoplasts and its genetic consequences have been investigated. The technique used allows the transfer of a small plasmid pBC16 between Bacillus cereus cells. Fusion has resulted in isolation of hybrid cells having acquired TcR phenotype (harbouring pBC16) with high frequencies (10(-2)-10(-3)). However, the genetic instability of hybrid cells and segregation effects have influenced dramatically the final results of plasmid transfer. Nevertheless, the fusion of protoplasts proves to be useful in construction of BAcillus cereus strains inheriting plasmid determinants.     

Functional analysis of the pBC1 replicon from <Emphasis Type="Italic">Bifidobacterium catenulatum</Emphasis> L48

To determine the minimal replicon of pBC1 (a 2.5-kb cryptic plasmid of Bifidobacterium catenulatum L48) and to check the functionality of its identified open reading frames (ORFs) and surrounding sequences, different segments of pBC1 were amplified by polymerase chain reaction (PCR) and cloned into pBif, a replication probe vector for bifidobacteria. The largest fragment tested in this manner encompassed most of the pBC1 sequence, while the shortest just included the repB gene and its immediate upstream sequences. Derivatives were all shown to allow replication in bifidobacteria. Surprisingly, both the transformation frequency and segregational stability in the absence of antibiotic selection decreased with reducing plasmid length. The relative copy number of the constructs (ranging from around 3 to 23 copies per chromosome equivalent, as compared to 30 copies for the original pBC1) was shown to be strain dependent and to decrease with reducing plasmid length. These results suggest that, although not essential, the copG-like and orfX-like genes of pBC1 play important roles in pBC1 replication. Interruption of repB produced a construct incapable of replicating in bifidobacteria. The analysis of pBC1 will allow its use in the construction of general and specific cloning vectors.     

Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation.

Mobilizations of pBC16 and pAND006, containing the replicon of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3, between strains of B. thuringiensis subsp. israelensis were examined. Transconjugants appeared after a few minutes and reached a maximum frequency after approximately 2 h. Plasmid pBC16 was mobilized at a frequency approximately 200 times that of pAND006. However, pAND006 was consistently transferred, suggesting that the replicon of pTX14-3 is sufficient to sustain mobilization in B. thuringiensis subsp. israelensis. A specific protease-sensitive coaggregation between strains of B. thuringiensis subsp. israelensis was found to be unambiguously correlated with plasmid transfer. Two aggregation phenotypes, Agr+ and Agr-, were identified in this subspecies. Aggregation disappeared when the optical density of the mating mixture at 600 nm exceeded approximately 1, and it did not reappear upon dilution. Aggregation was shown to involve interactions of cells with opposite aggregation phenotypes, and evidence of a proteinaceous molecule on the surface of the Agr- that is cells involved in aggregation formation is presented. Matings and selection for the presence of two antibiotic resistance plasmids followed by identification of the host cell revealed that mobilization was unidirectional, from the Agr+ cell to the Agr- cell. The aggregation phenotype was found to be transferred with high frequency (approximately 100%) in broth matings, and the appearance of Agr- isolates from Agr+ strains suggested that the loci involved in aggregation formation are located on a plasmid. No excreted aggregation-inducing signals were detected in the supernatant or culture filtrate of either the donor, the recipient, or the mating mixture.     

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 transformation in Bacillus subtilis: effects of insertion of Bacillus subtilis DNA into plasmid pC194

Summary We have constructed a hybrid plasmid, pBC1, which consists of plasmid pC194 with an insert of B. subtilis DNA at its HindIII restriction site. This plasmid is stably maintained in B. subtilis. In contrast with pC194, monomeric ccc forms of pBC1 are active in transformation. Transformations with these monomeric molecules of pBC1 have a stringent requirement for recombination proficieny., as defined by recE in the recipient cell. The extent of dependence of the transforming activity of oligomeric pBC1 DNA on the recombination proficiency of the recipient cell decreases with increasing oligomer size. A model of DNA proccssing during plasmid transformation of B. subtilis is presented.     

Structural organization of pBC1, a cryptic plasmid from Bacillus coagulans.

The complete nucleotide sequence of the Bacillus coagulans plasmid pBC1 was determined. The sequence revealed an open reading frame encoding a polypeptide of 259 amino acids. This open reading frame shows sequence similarity to genes coding for replication-associated proteins in a group of gram-positive bacterial plasmids known to replicate via single-stranded intermediates. A region required for replication in cis, when the intact replicon is supplied in trans, was identified as well.     

Transfer of plasmids and chromosomal genes amongst subspecies ofBacillus thuringiensis

Summary The plasmids pBC16 and pC194 fromBacilus thuringiensis subsp.israelensis strains A084-16-194 were transferred to 25 subspecies ofB. thuringiensis by a conjugation-like process using broth mating technique. The frequencies of transfer varied considerably between different mating pairs, ranging from 1.1×10^–9 to 9.8×10^–5. Additionally, chromosomal transfer could also be demonstrated in tenB. thuringiensis subspecies with very low frequencies (4.3×10^–9 to 3.7×10^–7). The intersubspecies matings within a group of eight subspecies strains gave higher frequencies of transfer than the matings between the subspecies. Furthermore, the results indicated that the capability to transfer plasmids among these various subspecies did not depend on the presence of large plasmid.     

Inhibition of a conjugation-like gene transfer process in Bacillus thuringiensis subsp. israelensis by the anti-s-layer protein antibody

Extraction of the S-layer protein by treatment with 6 m urea revealed a high-molecularweight protein in the extracts obtained from Bacillus thuringiensis subsp. israelensis (B.t.i) strain 4Q2. This protein band was found to be absent in partially cured (4Q2-72) and completely cured (c4Q2-72) strains. The antibody toward this S-layer protein was prepared and used to locate its antigenic protein on B.t.i cells by using indirect immunofluorescence. Immunodiffusion reactions and Western blot analysis confirmed the specificity of the anti-S-layer protein antibody. It was found that the antibody against 4Q2 S-layer protein, inhibited plasmid transfer via a conjugationlike process between, B.t.i. strains 4Q2-16 and c4Q2-72. That is, the frequency of transfer of plasmid pBC16 was reduced from 9.7×10^-6 in the absence of the antibody to less than 1.0×10^-8 in the presence of the antibody. The antibody was also found to reduce the frequency of pBC16 plasmid transfer via a conjugation-like process between B.t.i. strains A084-16-194 and c4Q2-72 from 2.2×10^-5 in the absence of the antibody to 1.2×10^-6 in the presence of the antibody.