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, pXOll and pXO12, found in B. thuringiensis were responsible for plasmid mobilization. B. anthracis and B. cereus transcipients inheriting either pXOll 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 pXOll were Cry-. This mating system provides an efficient method for interspecies transfer of a large range of Bacillus plasmids by a conjugation-like process. A previous report from this laboratory (15) demonstrated the utility of the generalized transducing bacteriophage CP-51 in transferring plasmids among the three species Bacillus anthracis, B. cereus, and B. thuringiensis. However, the size of plasmids that can be transferred by CP-51 is limited by the size of the phage and the corresponding amount of DNA it can package (ca. 50 megadaltons). Therefore, with the hope of being able to transfer the large plasmids of B. anthracis to assess more adequately their biological significance, we decided to investigate whether the B. thuringiensis mating system described by Gonzalez et al. (5, 6) could be applied to B. anthracis. Our strategy was to look for transfer of the selectable tetracycline resistance plasmid pBC16 (2) and then to examine tetracycline-resistant transcipients for the acquisition of additional plasmids. We have found that certain plasmids which promote their own transfer from B. thuringiensis are also effective in promoting the transfer of a variety of plasmids among the three Bacillus species. Evidence is presented that each of two plasmids, pXO11 and pXO12, found in B. thuringiensis subsp. thuringiensis is capable of bringing about its own transfer as well as that of other plasmids. Plasmid analyses confirmed the transfer of a variety of plasmids from B. thuringiensis subsp. thuringiensis to B. anthracis and B. cereus. Transcipients of the latter two organisms that inherited either pXOll or pXO12 were, in turn, effective donors. The mating system is thus a useful and efficient means of...

The self-transmissible plasmid pXO12 (112.5 kilobases [kbj), originally isolated from strain 4042A of BaciUus 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, pXOl (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 104 to 3.9 x 106/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 Cryderivatives 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.Recent reports from our laboratory have concerned the identification and characterization of six self-transmissible plasmids from five subspecies of Bacillus thuringiensis (1,19). One of these plasmids, designated pXO12 (112.5 kilobases [kb]), was isolated from strain 4042A of B. thuringiensis subsp. thuringiensis. Plasmid pXO12 is capable of mediating its own transfer as well as that of a large range of Bacillus plasmids among strains of Bacillus anthracis, Bacillus cereus, and B. thuringiensis. In addition to conjugal transfer functions, pXO12 also encodes production of the insecticidal toxin known as the 8-endotoxin or the parasporal crystal (Cry').The widespread occurrence of large self-transmissible plasmids among B. thuringiensis strains suggests that conjugation may be an important means of plasmid dissemination in naturally occurring Bacillus populations (4). In the laboratory, this mating system has provided us with an efficient method of shuttling a wide range of plasmids among B. thuringiensis, B. cereus and B. anthracis. For example, the utility of this conjugation system in the genetic analysis of the B. anthracis toxin and capsule plasmids, pXO1 and pXO2, respectively, has been well documented (1,6,19,24).Prior to the development of this mating system, the most reliable system available for transferring plasmids among these three Bacillus species was transduction (21). Therefore, the importance of this system as an additional means of genetic exchange among these organisms warrants further genetic and physical analysis of these conjugative plasmids. We have been interested in determining the mechanism of pXO12-mediated transfer of both lar...