The fermentation of glycerol byClostridium butyricum LMG 1212t2 and 1213t1 andC. pasteurianum LMG 3285

Summary In batch culture on reiinforced clostridial medium strain-dependent product profiles from glycerol revealed unusual fermentation products such as propionate and n-propanol with Clostridium butyricum LMG 1213t₁, and 1,3-propanediol with C. butyricum LMG 1212t₂ and C. pasteurianium LMG 3285. Only the latter two strains were able to grow on glycerol in a minimal medium. Nicotinamide adenine dinucleotide (NAD⁺)-dependent dehydrogenase activities were detected with 1,3-propanediol and n-butanol as substrate (the latter only after a lag period) in cell-free extracts of C. butyricum LMG 1212t₂ and with 1,3-propanediol, n-butanol and ethanol in cell-free extracts of C. pasteurianum LMG 3285. The data indicated the existance of a specific 1,3-propanediol dehydrogenase in both organisms. In a chemostat, C. butyricum LMG 1212t₂ converted 65% of the glycerol supplied as sole carbon and energy source to 1,3-propanediol without H₂ production. Increasing concentration of acetate in the inflow medium resulted in less 1,3-propanediol and more butyrate and H₂ production. C. pasteurianum LMG 3285 converted somewhat more than half of the glycerol supplied as sole energy and carbon source to n-butanol with significant concomitant H₂ production. This fermentation pattern was hardly affected by acetate as co-substrate. Offprint requests to: P. De Vos     

Fermentation characteristics of Fusariumoxysporum grown on acetate

In this study, the growth characteristics of Fusariumoxysporum were evaluated in minimal medium using acetate or different mixtures of acetate and glucose as carbon source. The minimum inhibitory concentration (MIC) of acetic acid that F.oxysporum cells could tolerate was 0.8%w/v while glucose was consumed preferentially to acetate. The activity of isocitrate lyase was high when cells were grown on acetate and acetate plus glucose indicating an activation of the glyoxylate cycle. Investigation of the metabolic fingerprinting and footprinting revealed higher levels of intracellular and extracellular TCA cycle intermediates when F.oxysporum cells were grown on mixtures of acetate and glucose compared to growth on only glucose. Our data support the hypothesis that a higher flux through TCA cycle during acetate consumption could significantly increase the pool of NADH, resulting in the activation of succinate-propionate pathway which consumes reducing power (NADH) via conversion of succinate to propionyl-CoA and produce propionate.     

Formate dehydrogenase of Clostridium pasteurianum

Formate dehydrogenase was purified to electrophoretic homogeneity from N2-fixing cells of Clostridium pasteurianum W5. The purified enzyme has a minimal Mr of 117,000 with two nonidentical subunits with molecular weights of 76,000 and 34,000, respectively. It contains 2 mol of molybdenum, 24 mol of nonheme iron, and 28 mol of acid-labile sulfide per mol of enzyme; no other metal ions were detected. Analysis of its iron-sulfur centers by ligand exchange techniques showed that 20 iron atoms of formate dehydrogenase can be extruded as Fe4S4 centers. Fluorescence analysis of its isolated molybdenum centers suggests it is a molybdopterin. The clostridial formate dehydrogenase has a pH optimum between 8.3 and 8.5 and a temperature optimum of 52 degrees C. The Km for formate is 1.72 mM with a Vmax of 551 mumol of methyl viologen reduced per min per mg of protein. Sodium azide competes competitively with formate (K1 = 3.57 microM), whereas the inactivation by cyanide follows pseudo-first-order kinetics with K = 5 X 10(2) M-1 s-1.     

Isolation of mutants of Clostridium pasteurianum

Twenty-nine independent mutants of Clostridium pasteurianum ATCC 6013, including several auxotrophs and UV resistants, have been isolated and characterized. The protoplast formation and regeneration procedure of Minton and Morris (1983) has also been successfully tried with some of these newly obtained mutants. The availability of these mutants together with the possibility of protoplast formation and regeneration will be useful for the development of a genetic exchange system in this species.Abbreviations CFU colony forming unit - DCCP dicyclohexyl carbodiamide - EMS ethylmethane sulfonate - IB isotonic buffer - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PEG polyethylene glycol - UV ultraviolet     

Changes in Membrane Plasmalogens of Clostridium pasteurianum during Butanol Fermentation as Determined by Lipidomic Analysis

Changes in membrane lipid composition of Clostridium pasteurianum NRRL B-598 were studied during butanol fermentation by lipidomic analysis, performed by high resolution electrospray ionization tandem mass spectrometry. The highest content of plasmalogen phospholipids correlated with the highest butanol productivity, which indicated a probable role of these compounds in the complex responses of cells toward butanol stress. A difference in the ratio of saturated to unsaturated fatty acids was found between the effect of butanol produced by the cells and butanol added to the medium. A decrease in the proportion of saturated fatty acids during conventional butanol production was observed while a rise in the content of these acids appeared when butanol was added to the culture. The largest change in total plasmalogen content was observed one hour after butanol addition i.e. at the 7th hour of cultivation. When butanol is produced by bacterial cells, then the cells are not subjected to severe stress and responded to it by relatively slowly changing the content of fatty acids and plasmalogens, while after a pulse addition of external butanol (to a final non-lethal concentration of 0.5 % v/v) the cells reacted relatively quickly (within a time span of tens of minutes) by increasing the total plasmalogen content.     

The biosynthesis of granulose by Clostridium pasteurianum

1. Mutant strains of Clostridium pasteurianum were obtained, which are unable to synthesize granulose (an intracellularly accumulated amylopectin-like alpha-polyglucan). 2. These mutants lacked either (a) ADP-glucose pyrophosphorylase (EC 2.7.7.27), or (b) granulose synthase (i.e. ADP-glucose-alpha-1,4-glucan glucosyltransferase, EC 2.4.1.21). 3. Although both of these enzymes were constitutively synthesized by the wild-type organism, massive deposition of granulose in a sporulating culture coincided with a threefold increase in the specific activity of ADP-glucose pyrophosphorylase. 4. The soluble ADP-glucose pyrophosphorylase was partially purified (33-fold). Its ATP-saturation curve was not sigmoidal and its activity was not enhanced by phosphorylated intermediates of glycolysis, pyruvate, NAD(P)H or pyridoxal 5'-phosphate. ADP at relatively high concentrations acted as a competitive inhibitor (K(i)=19mm). 5. The dependence of granulose synthase on a suitable polyglucan primer was demonstrated by using enzyme obtained from a granulose-free mutant strain (lacking ADP-glucose pyrophosphorylase). 6. Partial purification of granulose synthase from wild-type strains was facilitated by its being bound to the native particles of granulose. No activator was discovered, but ADP, AMP and pyridoxal 5'-phosphate were competitive inhibitors, ADP being most effective (K(i) about 0.2mm). 7. It would appear that the synthesis of granulose in Cl. pasteurianum is not subject to the positive, fine control that is a feature of glycogen biosynthesis in most bacteria.     

Identification of molybdoproteins in Clostridium pasteurianum.

Cells of Clostridium pasteurianum whose N source is switched from NH3 to N2 accumulate large amounts of molybdenum beginning 1.5 h before the detection of nitrogenase activity. Anaerobic multiphasic gel electrophoresis and anion-exchange chromatography were used to identify the molybdoproteins and molybdenum-containing components present in N2-fixing cells. In addition to molybdate, six distinct 99Mo-labeled species were detected, i.e., a membrane fragment, the MoFe protein of nitrogenase, formate dehydrogenase, a Mo "binding-storage" protein, a 30-kilodalton molybdoprotein, and a low-molecular-weight molybdenum species. Of these, the MoFe protein, formate dehydrogenase, and the Mo binding-storage protein were present in more than one zone because of complex formation with other proteins, partial denaturation, and variation in the amount of Mo bound to the protein, respectively. In addition to the six proteins, a soluble "free" Mo cofactor in the cytosol was detected by showing that it reconstituted nitrate reductase activity in crude extracts of the Neurospora crassa mutant nit-1.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Transport of molybdate by Clostridium pasteurianum.

The transport of 99MoO42- into dinitrogen-fixing cells of Clostridium pasteurianum was investigated. Transport of molybdate in this organism is energy dependent; sucrose is required in the minimal media, and the system is inhibited by the glycolysis inhibitors, NaF, iodoacetic acid, and arsenate. The cells accumulate molybdate against a concentration gradient, and the uptake shows a marked dependence on temperature (optimum 37 C) and pH (optimum 6.0). The rate of molybdate uptake with increasing molybdate concentrations shows saturation kinetics with an apparent Km and Vmax of 4.8 X 10(-5) M and 55 nmol/g of dry cells per min, respectively. Inhibition studies with the anions SO42-, S2O32-, WO42-, and VO32- show that SO42- and WO42- competitively inhibit MoO42- uptake (apparent Ki [SO42-] is 3.0 X 10(-5) M; apparent Ki [WO42-] is 2.4 X 10(-5), whereas S2O32- and VO32- have no inhibitory effect. Exchange experiments with MoO42- show that only a small percentage of the 99MoO42- taken up by the cells is exchangeable. Exchange experiments with WO42- and SO42- indicate that once inside the cells WO42- and SO42- cannot substitute for MoO42-.     

Lysine mannitol glycerol agar (LMG) and LMG with sulphamandelate for isolation of Salmonella spp. from clinical specimens

Lysine mannitol glycerol agar (LMG) was compared to xylose lysine deoxycholate agar (XLD), bismuth sulphite agar (BS), and Salmonella-Shigella agar (SS) for the ability to detect Salmonella spp. in clinical specimens, primarily faeces samples. During an 8-month period, 15 salmonellae were isolated from 940 faeces on LMG, while 14 strains were obtained on XLD, 11 on SS and only 3 strains on BS. Salmonella typhi was recovered from two blood cultures in 24 h on LMG, compared to 48 h on BS. LMG was augmented by addition of a sulphacetamide/mandelic acid (sulphamandelate) selective supplement (LMGS). During a 20-month period, 43 salmonellae were isolated from 2622 faeces on LMG and LMGS. The selectivity of LMGS was superior to that of LMG with no decrease in sensitivity of detection; all salmonellae isolated on LMG were isolated on LMGS. Both LMG and LMGS were suitable for routine use in the isolation of Salmonella spp. from clinical specimens.     

Solution structure of reduced Clostridium pasteurianum rubredoxin

The solution structure of reduced Clostridium pasteurianum rubredoxin (MW 6100) is reported here. The protein is highly paramagnetic, with iron(II) being in the S=2 spin state. The Hβ protons of the ligating cysteines are barely observed, and not specifically assigned. Seventy-six percent of the protons have been assigned and 1267 NOESY peaks (of which 1037 are meaningful) have been observed. Nonselective T ₁ measurements have been measured by recording four nonselective 180°-τ-NOESY at different τ values, and fitting the intensity recoveries to an exponential recovery. Thirty-six metal-proton upper and lower distance constraints have been obtained from the above measurements. The use of such constraints is assessed with respect to spin delocalization on the sulfur donor atoms. The solution structure obtained with the program DYANA has been refined through restrained energy minimization. A final family of 20 conformers is obtained with no distance violations larger than 0.24?Å, and RMSD values to the mean structure of 0.58 and 1.03?Å for backbone and all heavy atoms, respectively (measured on residues 3–53). The structure is compared to the X-ray structure of the oxidized and of the zinc substituted protein, and to the available structures of other rubredoxins. In particular, the comparison with the crystal structure and the solution structure of the Zn derivative of the highly thermostable Pyrococcus furiosus rubredoxin suggested that the relatively low thermal stability of the clostridial rubredoxin may be tentatively ascribed to the loosening of its secondary structure elements. This research is a further achievement at the frontier of solution structure determinations of paramagnetic proteins.     

Fermentation of glucose, lactose, galactose, mannitol, and xylose by bifidobacteria

For six strains of Bifidobacterium bifidum (Lactobacillus bifidus), fermentation balances of glucose, lactose, galactose, mannitol, and xylose were determined. Products formed were acetate, l(+)-lactate, ethyl alcohol, and formate. l(+)-Lactate dehydrogenase of all strains studied was found to have an absolute requirement for fructose-1,6-diphosphate. The phosphoroclastic enzyme could not be demonstrated in cell-free extracts. Cell suspensions fermented pyruvate to equimolar amounts of acetate and formate. Alcohol dehydrogenase was shown in cell-free extracts. Possible explanations have been suggested for the differences in fermentation balances found for different strains and carbon sources. By enzyme determinations, it was shown that bifidobacteria convert mannitol to fructose-6-phosphate by an inducible polyol dehydrogenase and fructokinase. For one strain of B. bifidum, molar growth yields of glucose, lactose, galactose, and mannitol were determined. The mean value of Y (ATP), calculated from molar growth yields and fermentation balances, was 11.3.     

The mother-cell-membrane adenosine triphosphatase of sporulating Clostridium pasteurianum

1. Sporulation of Clostridium pasteurianum effects several changes in its proton-translocating cell-membrane H(+)-ATPase. Notable among these are the acquisition of susceptibility to activation by trypsin and a changed protein subunit composition. 2. A protein was isolated from the mother-cell membrane that inhibited the ATP phosphohydrolase activity of purified vegetative-cell-membrane H(+)-ATPase [BF(0)F(1) complex, which consists of soluble ATPase (BF(1)) and the proton-channel component (BF(0))] and rendered it susceptible to trypsin activation. 3. This trypsin-sensitive inhibitor protein had a molecular weight of 10000 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was indistinguishable from the novel protein subunit e of the mother-cell-membrane ATPase 4. In bacteriorhodopsin-containing everted membrane vesicles, the specific ATP synthetase activity of the mother-cell-membrane ATPase was significantly greater than that of the vegetative-cell-membrane ATPase. 5. Treatment with trypsin-sensitive inhibitor protein of artificial proteoliposomes containing bacteriorhodopsin and vegetative-cell-membrane H(+)-ATPase (BF(0)F(1)) significantly increased the specific ATP synthetase activity of this enzyme. 6. The ATP synthetase activity of crude cell-membrane preparations from cultures of Clostridium pasteurianum increased during that period in the course of sporulation when the membrane ATP phosphohydrolase was both most rapidly decreasing in specific activity and acquiring its susceptibility to activation by trypsin.