PLASMID 36, 75–85 (1996) ARTICLE NO. 0035 Mobilization of ‘‘Nonmobilizable’’ Plasmids by the Aggregation-Mediated Conjugation System of Bacillus thuringiensis LARS ANDRUP, 1 OLE JØRGENSEN,* ANDREA WILCKS,LASSE SMIDT, AND GERT B. JENSEN Department of Toxicology and Biology and *Department of Industrial Hygiene, National Institute of Occupational Health, Lersø Parkalle ´ 105, DK-2100 Copenhagen, Denmark Received May 2, 1996; revised August 6, 1996 The aggregation-mediated conjugation system of Bacillus thuringiensis subsp. israelensis (Bti ), encoded by the 200-kb plasmid pXO16, is highly potent in transferring itself and efficient in mobilizing other nonconjugative plasmids. In the present study we have analyzed the native Bacillus cereus plasmid pBC16. This plasmid has previously been shown to harbor a mob gene (ORFb) and a locus functioning as an oriT site in plasmid pLS20-mediated conjugation in Bacillus subtilis. However, in the conjugation system of Bti we found that a derivative of pBC16 deleted for both these loci was mobilizable, although at a reduced frequency. Another derivative of pBC16, containing a deletion spanning the first half of the coding region of the mob gene, was found to be nearly as mobilizable as the intact pBC16, suggesting its dispensability in the transfer process. Other plasmids based on the u-replicating origins, pAMb1, pLS20, ori43, ori44, and ori60, were also consistently mobilized in the conjugation system encoded by Bti plasmid pXO16. Analyzing the conjugation process by the use of scanning electron microscopy revealed the presence of connections between cells in the mating mixtures. These connections did not appear in monocultures of the donor strain or the recipient strain and may be conjugational junctions.  1996 Academic Press, Inc. Bacterial conjugation is a mechanism of ge- (Andrup et al., 1993; Jensen et al., 1995) are netic exchange that requires cell-to-cell con- capable of sustaining DNA transfer in liquid tact and which is not susceptible to DNase media. These gram-positive conjugation sys- present in the mating medium. Conjugation tems differ from the gram-negative systems systems are encoded by large plasmids or by in that there are no pili involved in mating-pair conjugative transposons (Clewell, 1993; formation. Other, yet still poorly understood, Scott, 1993). Several conjugation systems in means of mating-pair formation and establish- gram-positive bacteria have recently been dis- ment of physical contact are employed here. covered and characterized in some detail (for The different conjugation systems in gram- review see (Clewell, 1993)). The best studied positive bacteria vary with respect to host is the pheromone-induced conjugation system range, requirement of solid surfaces, transfer of Enterococcus faecalis (Dunny et al., 1978), frequencies, and ability to mobilize other which along with the conjugation systems of DNA elements. The majority of conjugative Lactococcus lactis (van der Lelie et al., 1991), plasmids reported in E. faecalis, Staphylococ- Lactobacillus plantarum (Reniero et al., cus aureus, Staphylococcus epidermidis, and 1992), and the aggregation-mediated conjuga- various bacilli can mobilize nonconjugative tion system of the mosquito-toxic bacterium plasmids. However, differences in the ability Bacillus thuringiensis subsp. israelensis (Bti ) 2 of conjugative plasmids to mobilize nonconju- gative plasmids were observed. Some noncon- 1 To whom correspondence should be addressed. Fax: jugative plasmids could be mobilized by one /45 39 270107. E-mail: ANDRUP@internet.dk. conjugative plasmid and not by another. The 2 Abbreviations used: Bti, Bacillus thuringiensis subsp. conjugative plasmid pGO1 from S. aureus israelensis; oriT, origin of transfer; oriL, single-strand origin of replication. could not mobilize plasmids pT181 and 75 0147-619X/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.