- Volume 147, Issue 9, 2001

Actinobacillus actinomycetemcomitans, a member of the gamma subclass of the Proteobacteria, has been implicated as the agent responsible for human periodontitis. In this study, A. actinomycetemcomitans 301-b was grown in fructose-limited chemostat cultures under anaerobic [redox potential (E h)<−400 mV] and microaerobic (E h=−200 mV) conditions to characterize its energy metabolism. Effects of K+ and Na+ on growth and metabolism were also examined. In a control medium containing 5·2 mM K+ and 24 mM Na+, the molar growth yield on fructose (Y fructose) of microaerobic cultures was 1·3 times higher than the yield of anaerobic cultures at D≤0·10 h−1, but the difference in the Y fructose between microaerobic and anaerobic cultures decreased at D≤0·10 h−1. When the ATP yield from fermentation was estimated from the amounts of fructose consumed and acetate formed, the value of the microaerobic culture (2·49 mol ATP produced per mol fructose consumed) was lower than the anaerobic value [3·13 mol ATP (mol fructose)−1]. Therefore, ATP production from fermentation could not account for the increase in the Y fructose at D>0·10 h−1 and thus additional ATP was expected to be generated via respiration. Assuming that the Y ATP (g cells formed per mol ATP synthesized) was similar between anaerobic and microaerobic cultures, the estimated ATP yield from respiration was between 1·2 and 2·0 mol ATP (mol fructose)−1 below D=0·10 h−1 and decreased to 0·3 mol ATP (mol fructose)−1 when D was increased to 0·19 h−1. Such growth-rate-dependent decreases in the Y fructose and the estimated ATP production from respiration were also observed in a high-Na+ (5·2 mM K+ and 106 mM Na+) culture but not in a high-K+ (81 mM K+ and 24 mM Na+) culture. In the high-K+ culture, the microaerobic Y fructose was 1·4–2·0 times higher than the anaerobic value and the respiration-derived ATP yield was estimated to be between 1·2 and 1·9 mol ATP (mol fructose)−1 over a wide range of dilution rate. These results suggest that higher concentrations of extracellular K+ are required for the respiration to occur in rapidly growing cells of A. actinomycetemcomitans.

The effect of aflastatin A (AsA), a novel inhibitor of aflatoxin production, on melanin biosynthesis of Colletotrichum lagenarium was examined. Addition of a low concentration of AsA (0·5 μg ml−1) to the culture medium almost completely inhibited the melanin production of this organism. AsA also inhibited the production of scytalone, an early intermediate of melanin biosynthesis. Melanin production was restored by addition of exogenous scytalone in the presence of AsA, suggesting that the late steps after the synthesis of scytalone were not significantly affected by AsA. This was confirmed by the results from RT-PCR analysis of the expression of genes encoding melanin biosynthetic enzymes (SCD1, THR1) and a regulatory gene (CMR1). By contrast, expression of PKS1 was severely impaired by AsA, although catalytic activity of a polyketide synthase (PKS1) was not inhibited by AsA. These results indicate that AsA inhibits an early step in melanin production, which suppresses the expression of PKS1.

A 453 bp fragment of infB, the gene encoding translation initiation factor 2, was sequenced and compared from 66 clinical isolates and type strains of Haemophilus species and related bacteria. Analysis of the partial infB sequences obtained suggested that the human isolates dependent on X and V factor, H. influenzae, H. haemolyticus, H. aegyptius and some cryptic genospecies of H. influenzae, were closely related to each other. H. parainfluenzae constituted a heterogeneous group within the boundaries of the genus, whereas H. aphrophilus/paraphrophilus and Actinobacillus actinomycetemcomitans were only remotely related to the type species of the genus Haemophilus. H. parahaemolyticus and H. paraphrohaemolyticus took up an intermediary position and may not belong in the genus Haemophilus sensu stricto. Ambiguous results were obtained with seven isolates tentatively identified as H. segnis, which fell into two discrete clusters. The delineation of ‘Haemophilus sensu stricto’ as suggested by infB analysis supports previous results obtained by DNA hybridization, in contrast to the delineation inferred from 16S rRNA sequence comparison.