The relationships between leukotoxin production, growth rate and the bicarbonate concentration in a toxin-production-variable strain of Actinobacillus actinomycetemcomitans.

Abstract

Actinobacillus actinomycetemcomitans, a Gram-negative periodontopathic bacterium, produces a leukotoxin belonging to the RTX family. The production of leukotoxin varies greatly among different strains of this species. In this paper the effects of growth rate and bicarbonate on the leukotoxin production by a toxin-production-variable strain (301-b) during growth in a chemostat were examined. When the bacterium was grown in anaerobic fructose-limited chemostat cultures (pH 7.0 and 37 degrees C) at dilution rates (D) ranging from 0.04 to 0.20 h-1 in the absence and presence of 10 mM bicarbonate, it produced leukotoxin as a cluster of two polypeptides (M(r) 113,000 and 120,000) and complexed with nucleic acids on the bacterial cell surface. The relationship between leukotoxin production and specific growth rate was analysed by plotting the specific rate of leukotoxin production [qLT, in microgram (mg dry wt)-1 h-1] against D. The plots were approximated to the linear relationships qLT = 2.7D-0.058 and qLT = 9.3D-0.407 without and with bicarbonate, respectively. These relationships suggest that the apparent leukotoxin production is a result of both growth-rate-dependent production and growth-rate-independent decomposition. The cellular leukotoxin level was also followed after the change from chemostat to batch culture in the same fermenter. In batch culture leukotoxin production stopped immediately and the cellular toxin level rapidly decreased, suggesting toxin decomposition. From the slopes of the approximated linear relationships between qLT and D, a theoretical maximum leukotoxin yield (YLT) was estimated as 2.7 and 9.3 micrograms (mg dry wt)-1 in the absence and presence of 10 mM bicarbonate, respectively. The increased YLT value in the cultures containing bicarbonate indicated that the addition stimulated the efficiency of leukotoxin synthesis up to about threefold. Further increases of bicarbonate concentration to between 20 and 40 mM had no effect on the total leukotoxin production, but the amount of extracellular leukotoxin increased with higher bicarbonate concentrations.