Downloaded from www.microbiologyresearch.org by IP: 54.162.190.106 On: Thu, 11 Feb 2016 14:41:47 Printed in Great Britain Microbiology (1 998), 144, 1263-1 270 Characterization of plasmid pAW63, a second self-transmissible plasmid in Bacillus thuringiensis subsp. kurstaki HD73 Andrea Wilcks,' Nicole Jayaswal,' Didier Lereclus2 and Lars Andrupl Author for correspondence: Andrea Wilcks. Tel: +45 39165248. Fax: +45 39165201. e-mail: aw@ami.dk National Institute of Occupational Health, Lers0 Parka116 105, DK-2100 Copenhagen, Denmark Unite de Biochimie Microbienne, lnstitut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France Bacillus thuringiensis subspecies kurstaki HD73, toxic for lepidopteran larvae, contains two large self-transmissible plasmids of approximately 75 kb, pHT73 and pAW63. The conjugative plasmid pHT73 has been studied extensively and has been shown to harbour the toxin gene crylAc, the transposon Tn4430 and several insertion sequences. In this study it was demonstrated that the minor plasmid pAW63 is also self-transmissible and about 10-30 times more efficient in mobilizing plasmid pBC16. To facilitate direct selection for pAW63 transfer, the plasmid was tagged with the tetracycline resistance transposon Tn5401 and in intraspecies matings it was found that after 2 h, all recipients had acquired a copy of the plasmid. Mating experiments demonstrated that pAW63 could be transferred to Bacillus thuringiensis subsp. israelensis, Bacillus cereus, Bacillus licheniformis, Bacillus subtilis and Bacillus sphaericus, and that the conjugative functions were expressed in these hosts. Hybridization studies showed that the replicons of pAW63 and pHT73 were distinct from one another. Sequences homologous to transposon Tn4430 and several insertion sequences were, however, shown to reside on both plasmids. Keywords: Bacillus thuringiensis, HD73, self-transmissible plasmid, conjugation, mobilization INTRODUCTION Bacterial conjugation is a mechanism of genetic ex- change that requires cell-to-cell contact and which is not susceptible to DNase present in the mating medium. Conjugation systems are encoded by large plasmids or by conjugative transposons (Clewell, 1993 ; Scott, 1993), which besides being capable of transferring themselves are able to co-transfer smaller mobilizable plasmids. Conjugation is a well-known process in Gram-negative bacteria with the F plasmid from Escherichia coli being particularly well studied. The cell-to-cell contact in Gram-negative bacteria is established by the sex pilus, which retracts and brings donor and recipient cells into close physical contact (Firth et al., 1996). In Gram-positive bacteria, DNA transfer by conjugation has been found in an increasing number of species. In these bacteria cell-to-cell contact is not mediated by pili, but other, still scarcely characterized surface structures Abbreviations: B.t., Bacillus thuringiensis; Cm, chloramphenicol ; Em, erythromycin; Nal, nalidixic acid; Sm, streptomycin; Tc, tetracycline. are involved. Recently, several Gram-positive systems capable of sustaining DNA transfer in liquid media have been discovered and characterized in some detail (for review, see Clewell, 1993). The best studied is the pheromone-induced conjugation system of Entero- coccus faecalis (Dunny et al., 1978), which along with the conjugation systems of Lactococcus lactis (van der Lelie et al., 1991), Lactobacillus plantarum (Reniero et al., 1992) and the mosquito-toxic bacterium Bacillus thuringiensis (B.t.) subsp. israelensis (Andrup et al., 1993 ; Jensen et al., 1995), mediates plasmid transfer via the formation of large aggregates in liquid medium. In 1982, Gonzalez & Carlton reported the conjugative transfer of B.t. plasmids in mixed culture which led to the discovery that the insecticidal crystal protein genes were located on large conjugative or mobilizable plasmids in several subspecies (Gonzalez et al., 1982). One of the subspecies analysed was B.t. kurstaki strain HD73. B.t. kurstaki flagella serotype 3ab comprises both strain H D l (crystal serotype K-1) and HD73 (crystal serotype K-73) (Krywienczyk et al., 1978). Strain HD73 contains six plasmids ranging from 7.5 to 77 kb (Gonza- lez et al., 1981). The genetic basis of the insecticidal 0002-221 1 0 1998 SGM 1263