ASSOCIATION OF ACTINOBACILLUS-ACTINOMYCETEMCOMITANS LEUKOTOXIN WITH NUCLEIC-ACIDS ON THE BACTERIAL-CELL SURFACE

Actinobacillus actinomycetemcomitans, a periodontopathic gram-negative bacterium, produces a leukotoxin that is a member of the RTX cytotoxi n family. Although genes may function in toxin secretion, the leukotox in is not secreted extracellularly but remains associated with the bac terial cell surface. We report here that this toxin-cell surface assoc iation is mediated by nucleic acids and directly demonstrate that the extracellular secretion of toxin occurs in growing cultures with incre ased ionic strength of medium. All examinations were performed with fr eshly harvested A. actinomycetemcomitans 301-b from anaerobic fructose -limited chemostat cultures. The occurrence of cell surface-localized DNA was shown by directly digesting whole cells with the restriction e ndonuclease EcoRI or HindIII, which yielded many DNA fragments. The ce ll surface DNA constituted about 20% of the total cellular DNA. The le ukotoxin was released from the whole cells by digestion with DNase I a s well as restriction endonucleases. Because the leukotoxin binds ioni cally to DNA, it is dependent on the ionic strength of buffers or medi a. Accordingly, the toxin was released from cells suspended in saline at pH 7.5 in the presence of increasing amounts of MgCl2 (0 to 10 mM) or NaCl (0 to 50 mM). Moreover, a considerable quantity of leukotoxin was detected in the culture supernatant of fructose-limited chemostat cultures when sodium succinate solution was pumped into the steady sta te as an additional salt (30 and then 50 mM). This toxin-DNA associati on was also found in well-characterized strains including not only the leukotoxin-producing ATCC 29522 but also the toxin production-variabl e ATCC 29523 and the non-leukotoxin-producing ATCC 33384 when these st rains were grown in the chemostat culture.