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

Abstract

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 ("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.Conjugation, the direct transfer of genes from one bacterial cell to another, is often mediated by large conjugative plasmids. These large plasmids are transferred at high frequency, and occasionally chromosomal genes and smaller nonconjugative plasmids are cotransferred by the transfer apparatus of the conjugative plasmids. In gram-negative bacteria many small nonconjugative plasmids can be transferred (mobilized) if an additional self-transmissible plasmid is present in the host (13, 46, 47). Recent reports have described similar mobilizations of small plasmids in gram-positive bacteria (2,5,26,34,40). The first studied and best understood of the conjugative plasmids is the F plasmid, which can replicate in Escherichia coli and certain closely related enteric bacteria. The mechanism of F plasmid-mediated conjugation involves physical contact of the cells and transfer of plasmid DNA brought about by the sex pilus (23). Studies of the gram-positive bacterium Enterococcus (formerly Streptococcus) faecalis suggest a mechanism quite different from that in the better-studied gram-negative systems. Pili do not play any role in conjugation between strains of this organism, but pheromone-induced aggregation between cells has been shown to be a major characteristic (11,15).

The genetic basis of the aggregation system in Bacillus thuringiensis subsp. israelensis is located on the large conjugative plasmid pXO16

“…The aggregation phenotypes are characterized by the appearance of macroscopic aggregates when cells of exponentially growing strains of Agr ϩ and Agr Ϫ bacteria are mixed in broth. The mobilization of small plasmids was found to be unidirectional, from Agr ϩ cells to Agr Ϫ cells (1). In addition, the Agr ϩ phenotype was transferred at a high frequency (Ϸ100%) to Agr Ϫ cells during broth matings.…”

Phage-Borne Factors and Host LexA Regulate the Lytic Switch in Phage GIL01

“…The natural host of phage GIL01 is the insect pathogen B. thuringiensis subspecies israelensis strain NB31 (2). The permissive plasmid-free strain GBJ002 (31), derived from strain 4Q2 and related to NB31 (32), was used to propagate GIL01.…”

The sex pheromone system of Enterococcus faecalis

“…These include Bacillus thuringiensis (Andrup et al, 1993), Lactobacillus plantarum (Reniero et al, 1992), Lactococcus lactis The clumping phenomenon is clearly visible. Aggregation substance was detected by reacting the cells/clumps with a polyclonal antiserum directed against pAD1-encoded aggregation substance and decorating the bound antibodies with a commercial preparation of goat (anti-rabbit) antibodies conjugated to gold particles (10 nm diameter).…”