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

Abstract

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...

Genomics of the group of organisms

“…The B. cereus large plasmids harbor candidate pore formation genes, such as the TraD/G conjugation proteins, that could form a pore through a membrane complex however their functionality has not been demonstrated experimentally. Sequencing of mobilizable B. cereus group plasmids such as pOX11 [92], pXO12 [92] or pXO16 [74,104] would advance our understanding of plasmid transfer mechanisms in this group of organisms. A number of limited studies have been undertaken that demonstrate that B. cereus group plasmid transfer is not affected by DNase, involves membrane interaction [74] and in some cases employs an Ôaggregation sub-stanceÕ [104], all which suggest a conjugative transfer mechanism.…”

Conjugative transfer, stability and expression of a plasmid encoding acry1Ac gene inBacillus cereusgroup strains

“…2B). Transfer of B. thuringiensis plasmids encoding Cry proteins has been described [12,13], but to our knowledge this is the ¢rst description of B. thuringiensis plasmid transfer to B. mycoides. Although several attempts were made, we were unable to transfer the pHT73-Em R to B. weihenstephanensis and to ¢ve B. cereus strains among which one was an emetic isolate.…”

“…israelensis pXO16 plasmid. Battisti et al [13] observed a limited host range among donors in conjugation experiments, as only 83% of B. thuringiensis strains containing pBC16 were able to transfer this plasmid to a B. cereus strain, and only 50% of the B. thuringiensis strains were able to transfer pBC16 to a B. anthracis strain.…”

Transformation of a cloning vector, pUB110, into Bacillus anthracis

“…Bacillus anthracis, the agent of anthrax, causes a highly infectious disease in domestic animals and humans. The two major virulence factors of B. anthracis are a poly-D-glutamic acid capsule and a tripartite toxin, which are associated with large plasmids, 60 and 110 MDa plasmid, respectively [1][2][3]. Recently, one of these toxin genes was cloned in Escherichia coli [4,5].…”

“…anthracis were not examined at all, because of the lack of a suitable method for phenotypic expression in B. anthracis. The introduction of plasmid DNA into B. anthracis has been achieved by transduction [6] and mobilization [1]. These are rather laborious methods and not widely applicable in recombinant DNA technology.…”