Effect of Bicarbonate on the Growth of Actinobacillus actinomycetemcomitans in Anaerobic Fructose-limited Chemostat Culture

Abstract

The effect of bicarbonate on the growth and product formation by a periodontopathic bacterium, Actinobacillus actinomycetemcomitans, was examined in an anaerobic chemostat culture with fructose as the limiting nutrient. The chemostat cultures were run at dilution rates between 0.04 and 0.25 h-' and the maximum growth yield ( Y::&,se) was estimated to be 40-3 and 61.7 g dry wt (mol fructose)-' in the absence and presence of bicarbonate, respectively. The major fermentation products in the absence of bicarbonate were formate, acetate, ethanol and succinate, with small amounts of lactate. The addition of bicarbonate to the medium resulted in a marked decrease in ethanol production and in a significant increase in succinate production. Washed cells possessed activity for the cleavage of formate to C 0 2 and H2, which seemed to play a role in supplying CO, for the synthesis of succinate in the absence of bicarbonate. The study of enzyme activities in cell-free extracts suggested that fructose was fermented by the Embden-

Fermentable-sugar-level-dependent regulation of leukotoxin synthesis in a variably toxic strain of Actinobacillus actinomycetemcomitans The GenBank/EMBL/DDBJ accession number for the sequence data in this paper is AB054839.

“…A. actinomycetemcomitans 301-b (serotype a) (Ohta et al, 1987) was used throughout this study. The growth medium for A. actinomycetemcomitans was composed of 50 mM Tris\HCl, various mineral salts and 0n2 or 0n3% (w\v) yeast extract (Difco) (Ohta et al, 1989). After the medium had been adjusted to pH 7n3-7n4 and autoclaved, a filter-sterilized fructose solution (final concentration 1n26 g l − ") was added to the medium aseptically.…”

“…A. actinomycetemcomitans 301-b was grown in an anaerobic chemostat system as described previously (Ohta et al, 1989). The chemostat system was kept under a stream (200-300 ml h − ") of N # which was freed of traces of oxygen by passage over a gas-purifying column (Gas Clean GC-RX ; Nikka Seiko).…”

“…For the measurement of total cellular leukotoxin, cell suspensions (1n9 mg protein ml − ") or cell sonicates (3n5 mg protein ml − ") were mixed 11 : 9 with SDS-containing sample buffer [0n05 M Tris\HCl (pH 6n8), 1 % SDS, 8n8 % glycerol, 2 % 2-mercaptoethanol, 0n0063 % bromphenol blue]. The cell sonicate was prepared as described previously (Ohta et al, 1989). For the determination of extracellular leukotoxin, the culture supernatants resulting from the centrifugation of bacterial cultures were passed through cellulose acetate membrane filters (Advantec Toyo ; pore size, 0n22 µm), then the filtrates were dialysed against deionized water, lyophilized and dissolved in distilled water (1\20 the original volume).…”

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

“…actinomycetemcomitans 301-b (serotype a) (Ohta et al, 1987) was used throughout this study. The growth medium was composed of 50 mM Tris/HCl, various mineral salts, and 0.2 % yeast extract (Difco) (Ohta et al, 1989). Fructose (quantities as indicated in Results and Discussion) was autoclaved separately and added to the medium aseptically.…”

“…Fructose concentrations in the reservoir medium and culture supernatants were determined by using the enzyme system composed of hexokinase, glucose-6-phosphate dehydrogenase and glucose-6-phosphate isomerase (Boehringer Mannheim). Bicarbonate was determined by GLC as described previously (Ohta et al, 1989).…”