Anaerobic growth of Aerobacter aerogenes on citrate as a carbon source required the presence of Na+. The growth rate increased with increasing Na+ concentration and was optimal at 0.10 M Na+. The requirement was specific for Na+, which could not be replaced by K+, NH4+, Li+, Rb+, or Cs+. K+ was required for growth in the presence of Na+, the optimal K+ concentration being 0.15 mm. Enzyme profiles were determined on cells grown in three different media: (i) intermediate Na+, high K+ concentration, (ii) high Na+, high K+ concentration, and (c) high Na+, low K+ concentration. All cells contained the enzymes of the citrate fermentation pathway, namely, citritase and the Na+-requiring oxalacetate (OAA) decarboxylase. All of the enzymes of the citric acid cycle were present, except a-ketoglutarate dehydrogenase which could not be detected. The incomplete citric acid cycle was, in effect, converted into two biosynthetic pathways leading to glutamate and succinate, respectively. The specific activities of citritase and OAA decarboxylase were lowest in medium (i), and under these conditions the activity of OAA decarboxylase appeared to be limited in vivo by the availability of Na+. Failure of A. aerogenes to grow anaerobically on citrate in the absence of Na+ can be explained at the enzymatic level by the Na+ requirement of the OAA decarboxylase step of the citrate fermentation pathway and by the absence of an alternate pathway of citrate catabolism.
The oxalacetate (OAA) decarboxylase present in extracts of Aerobacter aerogenes did not require added divalent cation for its activity and was not inhibited by 0.1 µ ethylenediaminetetracetate (EDTA). The decarboxylase was inhibited 95% by avidin (0.5 unit), but not by avidin pretreated with biotin, indicating that it is a biotinoprotein. After dialysis or salt fractionation, the enzyme was largely (90%) inactivated and could be reactivated by the T A he anaerobic dissimilation of citric acid by cell suspensions of Aerobacter indologenes (Brewer and Werkman, 1939) and by cell-free extracts of Aerobacter aerogenes (Dagley and Dawes, 1935a) involves the cleavage of citrate to oxalacetate (OAA)* 1 *and acetate followed by the decarboxylation of OAA to pyruvate. This paper demonstrates that the OAA decarboxylase (OAA 4-carboxy-lyase) of A. aerogenes is a novel inducible enzyme of this type which is sensitive to avidin but not to EDTA. Moreover, it specifically requires sodium ion for activity.
Experimental SectionCell Growth and Extraction. A. aerogenes NCTC 418 was grown without aeration at 37°in 20-1. bottles filled to the neck with medium above which a 1-in. layer of sterile mineral oil was placed. The medium used contained per liter of distilled water: monobasic potassium phosphate, 2 g; ammonium sulfate, 1 g; magnesium sulfate-7H20, 0.4 g; and 21 g of trisodium citrate-2H20 (Merck, reagent grade). It was adjusted to pH 7.0 with 7 n sodium hydroxide. A 1 % inoculum of similarly grown cells was added and after 24 hr the newly grown cells were cooled to stop gas production and harvested with a refrigerated Sharpies centrifuge. The harvested unwashed cells were stored at -20°for up to 8 months before extraction without
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