Little is known about the molecular mechanisms of conjugation in Gram-positive bacteria. What is known is that these genetic transfers almost certainly do not involve a pillus structure as in the case of the Gram-negative bacteria. Bacillus amyloliquefaciens is a sporeforming bacterium widely used for production of amylases important in manufacturing of ethanol from corn starch. Bacillus thuringiensis is best known for the potent insecticidal crystal toxin that provides the toxicologic basis of insecticides directed against many important insect pests in both agricultural and medical situations. Recently there have been a number of reports demonstrating genetic exchange between members of the species thuringiensis and a number of other Gram-positive bacteria, including Enterococcus faecalis, Bacillus cereus, B. anthracis B. subtilis, and B. megaterium.

In 1988, my laboratory discovered that the transposon, Tn916, from Enterococcus faecalis, transfers into B. thuringiensis and B. amyloliquefaciens by a conjugation-like process. Bacillus subtilis and E. faecalis serve as donors for this transfer. Once inside these industrially important bacilli, Tn916 inserts into random sites in the bacterial chromosome. Even of more interest, however, is the observation that Tn916 can induce relatively high frequency conjugal transfer of plasmid and chromosomal DNA. Thus Tn916 dependent conjugal transfer is currently the major thrust in our laboratory; several aspects of this problem are of interest. Because Tn916 is able to induce relatively high-frequency transfer of foreign plasmid DNA into B. thuringiensis, transposon-induced genetic exchange is being used to explore gene transfer among B. thuringiensis, B. amyloliquefaciens, and other bacterial species. This research will lead to a better understanding of the genetics of these organisms, better methods for the development of genetically engineered products, and a more clear understanding of genetic exchange in Gram-positive bacteria.

More recently, we demonstrated that the presence of tetracycline (the transposon-borne antibiotic resistance) enhances conjugal transfer of Tn916. Moreover, we reported that Tn916 mediated genetic exchange occurs in soils. These experiments show that the process can take place in the environment. To more clearly establish the role of Tn916-like elements in environmental genetic exchange, we are looking at fecal enterococci in animal wastes. We have found that a substantial portion of these microbes are resistant to tetracycline (80%) and that more than half of these containTn916-like elements.

Tn916-mediated conjugal transfer raises some important questions regarding the safety of genetically engineered microbes that are released into the environment. Because of the recently reported enhancement of transfer when one of the cells in the mating contains Tn916, and because such transposons exist in the environment, we are looking at the nature and frequency of genetic exchange between B. thuringiensis and other Gram positive bacteria of pathogenic significance (Streptococcus, Staphylococcus, Bacillus anthracis, Listeria monocytogenes, etc.). Several questions are of interest, including the rate and nature of this transfer, the likelihood of B. thuringiensis acquiring a pathogenic factor from one of these animal and/or human pathogens, and the safety aspects of genetic exchange occurring in the environment. This research will lead to a better understanding of the risks posed by genetic exchange between B. thuringiensis and other potentially pathogenic bacteria that it meets with in the environment when used as an insecticide.

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