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 and under different culture conditions. A toxin-production-variable strain, 301-b, stably produces significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures, but does not do so in the presence of excess fructose. This communication describes the cloning and sequencing of the leukotoxin promoter region from 301-b, showing that this strain has a promoter region similar to that from strain 652, a moderately toxic strain. Northern blot analysis using a leukotoxin gene probe demonstrated that change in toxin production in response to the level of external fructose was due to alteration in the transcriptional level of the leukotoxin gene. Pulsing of fructose into the fructose-limited chemostat culture remarkably reduced the intracellular cAMP level from 40 pmol (mg dry wt cells) N1 to 31 pmol (mg dry wt cells) N1 , which was restored when the culture was returned to fructose-limited conditions. Further, it was found that addition of external cAMP to the culture with excess fructose resulted in an apparent recovery of leukotoxin production. Taken together, these findings indicate that a cAMP-dependent mechanism, possibly a catabolite-repression-like system, may be involved in the regulation of leukotoxin production in this bacterium.

This study quantitatively compares the inhibition of calcium phosphate (CaP) precipitation by the salivary acidic proline-rich proteins (PRPs) statherin and histatin-1. Saliva and CaCl2 in 125 mM imidazole buffer (pH 7.0) were incubated with potassium phosphate and a hydroxyapatite (HAP) suspension, for 30 min at 25 degrees C, then filtered through nitrocellulose. The calcium (Ca) concentration in the filtrate was measured by atomic absorption spectrophotometry, then deducted from that in the initial solution to determine the amount of CaP precipitation after 30 min. The values of the inhibitory activities on CaP precipitation relative to crude parotid saliva were 4.7, 4.9, 6.9, and 65.8 for histatin-1, large PRPs, small PRPs, and statherin, respectively.