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Acetoacetic decarboxylase (acetoacetate carboxy lyase, EC 4.1.1.4), as extracted from acetone-powdered Clostridium acetobutylicum, contains some enzyme which is expressed and an equal amount which is latent and which may be measured only after its exposure by heating. Activation of the latent decarboxylase is an effectively irreversible, time- and temperature-dependent process which is kinetically first order. ΔHa for this activation is 25 kcal per mole. The expressed decarboxylase can be irreversibly inhibited by treatment with borohydride plus acetoacetate or 2-oxopropane sulfonate, without affecting the latent decarboxylase. The latent decarboxylase and the expressed decarboxylase sediment identically in a sucrose density gradient and are not separated by the procedures employed in the purification of the enzyme. The initially expressed decarboxylase and that which is exposed by heating, exhibit the same Km for acetoacetate and Ki for 2-oxopropane sulfonate. Monovalent anions protect against the irreversible inhibition of the enzyme by substrate plus BH4- but have no effect on the activation of the latent decarboxylase or on the thermal stability of the enzyme. Acetylacetone is similarly without effect on the process of heat activation but strikingly enhances the heat stability of the decarboxylase.

Glyceryl ether phospholipids were found to exist in various strictly anaerobic bacteria and propionibacteria. The strains included Selenomonas ruminantium, Veillonella gazogenes, Propionibacterium freudenreichii, Propionibacterium shermanii, Clostridium saccharoperbutylacetonicum, Clostridium acetobutylicum, Clostridium perfrigens, and Clostridium kaneboi. Selenomonas ruminantium contained the largest amount of glyceryl ether phospholipids among the tested bacteria. The molar percentage composition of glyceryl ethers in total phospholipids from Selenomonas ruminantium was approximately 3. Glyceryl ether phospholipid was not detected in aerobic and facultative anaerobic bacteria even when they were cultured under strictly anaerobic conditions.

Racemiase, an enzyme which catalyzed racemization of lactic acids, was isolated from culture filtrate of Clostridium acetobutylicum by salting-out, and its purification was attained to about twenty-fold by treating with calcium phosphate gel. It was shown that racemiase requires for its full activity cofactors, pyridoxamine phosphate and ferrous ions. These substances were detected in the racemiasc preparation. Several other properties of racemiase were also investigated.

In studying the nutritive requirements of Clostridium acetobutylicum Rubbo and Gillespie1 found that paraaminobenzoic acid (PAB) was needed by this organism. Small amounts were sufficient to support growth and the compound could apparently be classed as a growth factor. This work was extended to include a more detailed study of the organism and the availability of some related compounds.2 The PAB requirement of Cl. acetobutylicum was confirmed by Lampen and Peterson3 and by Park and Wood.4

L-Arabinose ketol-isomerase (EC 5. 3. 1. 4) was extracted from the L-arabinose-grown cells of Clostridium acetobutylicum and partially purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and the gel filtration on Sephadex G-200. Optimum pH and temperature for the activity of the enzyme were 7.5 and 37°C, respectively. The enzyme was inhibited by EDTA, Na-pyrophosphate and KCN, and its activity was restored by the addition of divalent metal ions such as Mn2+ and Co2+. The Michaelis-Menten's constants for L-arabinose, Mn2+ and Co2+ were 1.08×10-2M, 0.83×10-4M and 0.38×10-4M, respectively. The equilibrium constant (K=ribulose/arabinose) was 3.14 (37◊C, pH 7.5). The thermodynamic quantities, ΔH, ΔG (37°C), ΔS (37°C) and E were +7354cal/mole, -710.7cal/mole, +26.0cal/(deg•mole) and +7471cal/mole, respectively.

An enzyme catalyzing the reversible hydration of crotonylCoA has been obtained in homogeneous form from Clostridium acetobutylicum. The bacterial hydrase has a molecular weight of 158,000 ± 3,000 as determined by sedimentation equilibrium. In contrast, the enzyme has a molecular weight of approximately 43,000 in 6 m guanidine hydrochloride in either the presence or absence of reducing agent. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate yields a molecular weight of 40,000, also about one-fourth that of the native enzyme. Unlike bovine liver crotonase, the bacterial enzyme is specific for short chain fatty acyl-CoA substrates and is sensitive to high concentrations of crotonyl-CoA. It requires a complete coenzyme A thioester substrate for efficient catalysis. It is concluded that crotonase from C. acetobutylicum is composed of 4 subunit polypeptide chains, with each chain containing about 370 residues devoid of any attached carbohydrate, and that the 4 subunits of the native enzyme are combined through noncovalent interactions. There are, however, a number of similarities between the bacterial and mammalian enzymes, which suggest that the two may be structurally related.

Summary: Clostridium acetobutylicum has been studied during batch cultivation at pH 7 and 35° in a glucose + casein hydrolysate + vitamins and salts medium kept (i) anaerobic (E h, – 400 to – 370 mV), (ii) aerated (E h, – 50 to 0 mV; dissolved O2, < 1 μM), and (iii) aerobic (E h, + 100 mV; dissolved O2, 40 to 50 μM). Short-term (4 to 6 h.) exposure to oxygen was not lethal, though at sufficiently high concentrations oxygen decreased the rate of glucose consumption, halted growth and prevented net synthesis of DNA, RNA and protein. Under these aerobic conditions the organism was drained of ‘reducing power’ and starved of energy – as evidenced by cessation of butyrate formation (but not of acetate production), and by a marked fall in intracellular ATP. These consequences of oxygenation were swiftly reversed when anaerobic conditions were re-established; ‘normal’ growth and glucose metabolism then resumed. There was no evidence of H2O2 production, nor could the effects of oxygenation be attributed merely to its elevation of the culture E h. Thus oxygen (40 μM) inhibited growth even in a medium poised with dithiothreitol at – 50 mV, while growth and glucose metabolism continued unchecked when the E h of an anaerobic culture was maintained at +370 mV by addition of potassium ferricyanide.

The acetoacetate decarboxylase obtained from Clostridium acetobutylicum undergoes a biphasic thermal inactivation; the rapid phase of which was temperature sensitive, whereas the slow phase was much less so. ΔHa for the rapid phase of inactivation was 49 kcal per mole. The fraction of the total activity which was lost by the rapid process was increased with increasing temperature. Acetylacetone protected the enzyme against thermal inactivation and was more effective in preventing the rapid phase than in preventing the slow phase of activity loss. When the enzyme was heated until the rapid phase was over and then chilled; subsequent reheating did not result in the reappearance of a rapid phase of inactivation. When the organism was cultured in the presence of benzoylacetone it generated an active decarboxylase which differed from the normal enzyme in several respects. This modified enzyme did not possess any latent activity; was more susceptible to thermal inactivation and exhibited a ΔHa for the rapid phase of thermal inactivation of 104 kcal per mole. A mechanism is proposed to account for the properties of the thermal inactivation of acetoacetic decarboxylase.

Acetoacetate decarboxylase activity of cell suspension of Clostridium acetobutylicum No. 314 was measured under hydrogen gas phase. When acetate buffer was used, the hydrogen uptake was observed at pH 5.0 after almost 90% of acetoacetate had been decarboxylated. This hydrogen uptake was not observed when the phosphate buffer was used instead of acetate buffer. These results suggest that there occurred the synthesis of acetoacetyl derivative (supposedly AcAc-Co A) from acetate and that the hydrogen was used for the reduction of the derivative. When the reaction was carried out at pH 6.8, both hydrogen uptake and decarboxylation took place at the same time and the velocity of the former exceeded the latter. Thus, it was suggested that one of the important factors which regulate the ratio of the both reactions, decarboxylation and reduction, of acetoacetate derivative is hydrogen ion concentration of the medium.

Since the establishment of p-aminobenzoic acid as a member of the B vitamin group, a considerable interest has been shown in methods of determination in natural materials. Since known chemical methods are not sufficiently sensitive, it became evident that microbiological tests should be the most practicable. The organism Clostridium acetobutylicum has been used (1) but no general assay procedure has been presented. Several bacterial strains which respond to p-aminobenzoic acid have been investigated in this laboratory, but satisfactory assay procedures with these organisms have not yet been devised. For the discovery of the test organism used in the procedure described in this paper, we are indebted to Dr. Beadle and Dr. Tatum who kindly furnished us with a culture of their p-aminobenzoic acid requiring a mutant strain of Neurospora crassa, designated by them as Neurospora crassa p-aminobenzoicless No. 1633 (2). This mold will grow optimally on a medium consisting of inorganic salts, ammonium tartrate, sucrose, biotin, and p-aminobenzoic acid. For purposes of assay, however, it has proved advantageous to supplement this basal medium with natural extracts which are either naturally low in p-aminobenzoic acid or have been treated to remove it. With such a complex medium, the possibility of interference by toxic substances or stimulatory substances other than p-aminobenzoic acid which might be present in samples to be assayed is reduced to a minimum. Since the completion of a considerable part of the experimental work described in this paper, microbiological assay methods for p-aminobenzoic acid have been published by Landy and Dicken (3) utilizing the organism Acetobacter suboxydans and by Lewis (4) using Lactobacillus arabinosus 17-5.

THE BACTERIOLOGY, GROWTH FACTOR REQUIREMENTS AND FERMENTATION REACTIONS OF CLOSTRIDIUM ACETOBUTYLICUM (WEIZMANN)

Behaviours of acetone-butanol fermentation bacterium, Clostridium acetobutylicum, towards optically active lactic aci4ls were investigated. As a rule, cells harvested from lactic acid supplemented culture medium surpassed in metabolizing lactic acid as compared with glucosegrown cells. It was pointed out that Cl. acetobutylicum possessed D-lactic dehydrogenase by nature, and that L-lactic acid could be dehydrogenated after its conversion to D-isomer through the action of racemiase. In addition to D-lactic dehydrogenase, it was further indicated that this organism had another type of lactic dehydrogenase or oxidase, which was capable of oxidizing L-isomer with air oxygen in liberating acetic acid and carbon dioxide. Physiological action of racemiase as a mediator between two lactic enzymes, lactic dehydrogenase and. D-lactic acid oxidizing enzyme, was ascertained. In addition, it is mentioned about pyruvic oxidase and partial inhibition by cyanide to lactic dehydrogenase.

1. The enzyme catalyzing the conversion of 8-N-ribityl-6, 7-dimethyllumazine to riboflavin was found in Er. ashbyii, beef liver, L. plantarum, Neurospora crassa, as well as in E. coli and Cl. acetobutylicum, in which its existence has already been reported.2. The enzyme is widely distributed in nature, and among a number of organisms tested, the activity of Er. ashbyii was the highest.3. The activity of the partially purified enzyme of E. coli neapolitanus was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and monoiodoacetate. A remarkable inhibition was also produced by several heavy metal ions at the concentration of 10-2M, i.e., Ag, Hg, Cu, Zn and Fe. KCN, NaF and monofluoroacetate did not exert any inhibition at 0.01M.4. The effect of the concentrations of 6, 7-dimethylribolumazine and of the enzyme as well as that of pH on the rate of riboflavin synthesis were examined.5. For full activation of this enzyme, it requires the addition of such cofactors as ATP, DPNH and CoA. Acetyl phosphate is found to be replaceable for acetate and ATP, when the enzyme preparations of Cl. acetobutylicum and E. coli. were used.

Při studiu fysiologicke cinnosti mikrobaClostridium acetobutylicum v prve a druhe fazi acetonbutanoloveho kvaseni jsme zjistili, ze pouze aceton je produkt charakterisujici přechod kultury do produkcni faze kvaseni. K přechodu do druhe, produkcni faze kvaseni dochazi az po dokonceni mnozeni a růstu buněk v kultuře, nezavisle na mnozstvi nahromaděných volných kyselin v prostředi. Změna v průběhu krivky titracni acidity, vseobecně spojovana s přechodem kvaseni do druhe faze, je podle výsledků nasich pokusů důslědkem změn fysiologicke cinnosti kultury po dokonceni jejiho růstu a mnozeni. It was found, from the physiological activity of Clostridium acetobutylicum in the first and second phase of acetonbutanol fermentation, that only acetone is the product characterising the transition of the culture into the production phase of fermentation. Transition, into the second, production phase of fermentation occurs only after proliferation and growth of the cells in the culture is complete, independently of the amount of free acids which have accumulated in the environment. According to the results of our experiments, the change in the course of the titration curve of acidity, which is generally combined with the transition of fermentation into the second phase, is due to changes in the physiological activity of the culture following completion of growth and proliferation. Исследуя физиологию жизнедеятельности м икроба Clostridium acetobutylicum в первой и во второй фазах ацет онбутанолового брож ения, мы установили, что тольк о ацетон является продуктом, характери зующим вступление ку льтуры в продуктивную фазу бр ожения. Переход во вторую, производит ельную фазу брожения осуществляется толь ко после окончания роста и размножения к леток в культуре, — нез ависимо от количества накопивш ихся в среде свободных кис лот. — Изменения в ходе кривой титруемой кислотнос ти, которые принято сопоставлять со всту плением брожения во в торую фазу, являются, судя по резу льтатам наших опытов, последс твием физиологическ их изменений жизнедеятельности к ультуры после окончания ее роста и р азмножения.

β-Keto acids have been observed to react reversibly in a one to one stoichiometry with boric acid yielding complexes which exhibit intense absorption in the ultraviolet. The effects of pH and temperature on the extent of this reaction have been investigated, and a reaction mechanism has been proposed. The reaction described has been utilized to provide a sensitive and convenient spectrophotometric assay of acetoacetic decarboxylase from Clostridium acetobutylicum . It would appear to be generally applicable to β-keto acid decarboxylases.

The effect of auxins (plant hormones) on microorganisms has been studied by several investigators. The bacteriostatic effects of 2,4-dichlorophenoxyacetic acid (2, 4-D), a growth-regulating substance, was first demonstrated by Stevenson and Mitchell (13) and by Lewis and Hammer (3). Dubos (1) observed that a number of synthetic, unsaturated, ring-containing acids including 2, 4-D, exerted bacteriostatic effects on certain microorganisms.

1. When metronidazole (1-β-hydroxyethyl-2-methyl-5-nitroimidazole) was added at 50 μg/ml to a liquid (35°C) culture of Clostridium acetobutylicum (0.5 mg dry wt of organisms/ml), it inhibited growth for only 2 h; thereafter, growth resumed at a slower and arithmetic rate. Consumption of glucose and excretion of acetate plus butyrate continued, though at diminished rates, during the period of bacteriostasis, and the ATP content of the organisms did not decline. 2. Metronidazole inhibited production of H2 by a culture of Cl. acetobutylicum, yet it only temporarily halted formation of H2 from pyruvate by cell extracts, and had no deleterious effect on the simultaneous production of acetyl phosphate and CO2. After a relatively short time no residual metronidazole was detectable in this cell-free system and H2 production resumed at its former rate. 3. The drug did not inhibit the hydrogenases of either Cl. acetobutylicum or Cl. pasteurianum, but having an E′0 at pH 7 of approx. -0.23 V (on the hydrogen scale), it acted as a preferential acceptor of electrons from reduced ferredoxin (or reduced viologen dyes). Reduction of metronidazole was a 6 electron process compatible with complete reduction of its 5-nitro group. Yet, although the expected decrease in absorbance at 320 nm was observed, the 5-amino derivative did not accumulate (probably due to its intrinsic lability). 4. Since many aryl nitro compounds are similarly able to accept electrons from reduced ferredoxin, it seems that additional reasons might have to be sought to explain the unique effectiveness of metronidazole as an antimicrobial agent.

Crystalline acetoacetate decarboxylase can be prepared from Clostridium acetobutylicum by the following steps: Preparation of an acetone powder from the bacteria, extraction of this powder at pH 5.9, precipitation of the enzyme at pH 3.8, a single fractionation with ammonium sulfate, and chromatography over diethylaminoethylcellulose, followed by crystallization. The yield of crystalline enzyme is about 180 mg from 120 1. of bacterial broth.

SUMMARY: Chloramphenicol did not inhibit growth and protein synthesis when added (100 μg./ml.) to growing cultures of Clostridium acetobutylicum, even though the organism was sensitive to this antibiotic in plate assay. This paradox was explained by the finding that chloramphenicol was rapidly reduced and thus detoxified by actively growing cultures. Cell-free extracts of C. acetobutylicum reduced the aryl nitro group of chloramphenicol via a ferredoxin-dependent enzymic reaction for which pyruvate was the most effective primary electron donor. A number of other aryl nitro-compounds were similarly reduced.