The physiology of Clostridium sporogenes NCIB 8053 growing in defined media

The physiology of Clostridium sporogenes was investigated in defined, minimal media. In batch culture, the major end products of glucose dissimilation were acetate, ethanol and formate. When L-proline was present as an electron acceptor, acetate production was strongly enhanced at the expense of ethanol. As judged by assay of the relevant enzymes, glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The growth energetics of Cl. sporogenes were investigated in glucose- or L-valine-limited chemostat cultures. In the former case, the addition of L-proline to the medium caused a significant increase in the molar growth yield (as calculated by extrapolation to infinite dilution rate). This finding adds weight to the view that the reduction of L-proline by Cl. sporogenes is coupled to the conservation of free energy.     

The growth and nutrition of Clostridium sporogenes NCIB 8053 in defined media

Various defined and minimal media are described for the growth of Clostridium sporogenes NCIB 8053. The organism requires 10 amino acids and one vitamin for growth, whilst three other vitamins are growth stimulatory. L-α-hydroxy acid analogues can replace eight, and fatty acid analogues four, of these amino acids. The organism may generate free energy by a variety of Stickland reactions. Most Stickland acceptors, but not glycine, stimulate the growth of this organism on glucose. Nonetheless, cells grown in the presence of glycine will reductively deaminate it. The media described support the growth of several other strains of this species. The simplified growth media which we have developed permit quantitative studies of the physiology of this organism.     

The growth and nutrition of Clostridium sporogenes NCIB 8053 in defined media

Various defined and minimal media are described for the growth of Clostridium sporogenes NCIB 8053. The organism requires 10 amino acids and one vitamin for growth, whilst three other vitamins are growth stimulatory. L-alpha-hydroxy acid analogues can replace eight, and fatty acid analogues four, of these amino acids. The organism may generate free energy by a variety of Stickland reactions. Most Stickland acceptors, but not glycine, stimulate the growth of this organism on glucose. Nonetheless, cells grown in the presence of glycine will reductively deaminate it. The media described support the growth of several other strains of this species. The simplified growth media which we have developed permit quantitative studies of the physiology of this organism.     

Growth energetics of Clostridium sporogenes NCIB 8053: modulation by CO2

The effects of the partial pressure of carbon dioxide on the growth energetics of Clostridium sporogenes NCIB 8053 grown in chemostat culture were investigated in defined minimal media. Both the 'maintenance' requirements and the growth yield coefficients were dependent upon the partial pressure of carbon dioxide in otherwise glucose-limited cultures. Since growth yield coefficients decreased along with the apparent 'maintenance' requirements in essential amino acid/fatty acid medium when the partial pressure of carbon dioxide was increased above 0.5 atm, the occurrence of some type of metabolic uncoupling seemed likely. By contrast, when the organism was grown in amino acid complete medium both the maintenance requirements and the growth yield coefficients were increased when the partial pressure of carbon dioxide was raised above 0.5 atm partial pressure of carbon dioxide, suggesting an increased efficiency of growth. A futile cycle involving carbon dioxide is proposed as a factor contributing to the variable extent of free energy dissipation within this organism.     

Growth of Clostridium tertium and Clostridium septicum in chemically defined media.

Defined media for the growth of Clostridium tertium and Clostridium septicum are described. The requirements for growth of these two species are compared with each other and with those of Clostridium perfringens.     

The 21-acetylation of corticosteroids by Clostridium sporogenes

A strain of Clostridium sporogenes, an anaerobic bacterium, isolated from sewage in New York City synthesizes two constitutive enzymes with action on steroid molecules: (i) an enzyme capable of selectively acetylating the 21-hydroxyl function of certain steroids and (ii) the corresponding esterase. Under our experimental conditions the enzymes have a strict structural requirement for 3-keto-4-ene and C-20-keto or 20 alpha-hydroxyl group and convert their respective substrates to a mixture of free and acetylated products.     

Glucose metabolism in Clostridium sporogenes and Clostridium sticklandii bacteria

Clostridium sporogenes 272 has a high rate of glucose fermentation. Its cell-free extract contains all glycolytic enzymes catalysing glucose degradation to pyruvate and shows the phosphoroclastic activity. C. sticklandii CSG has a low rate of glucose fermentation. Hence, the activity of the following enzymes is lower in this organism comparing to C. sporogenes: phosphohexoisomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1) and glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12). Moreover, it is possible that the system of glucose transport into the cell is damaged in C. sticklandii.     

Sheathed Cells in Cultures of Clostridium sporogenes

Many strains of Clostridium sporogenes were shown to contain two types of cells which exhibited strikingly different growth habits. Over 99% of the population of most strains were motile bacilli which occurred singly or in short chains. Infection by any of several C. sporogenes bacteriophages lysed most of these cells and revealed a minority population component consisting of cells which grew in extremely long chains. Each chain was surrounded by and contained in a long tubular polysaccharide sheath which was ultrastructurally quite separate and distinct from the cell walls of the enclosed cells. The sheathed cells were identical to "normal" cells of C. sporogenes in anaerobiosis, Gram reaction, sporulation, deoxyribonucleic base composition, general morphology, and ultrastructure. They differed from the "normal" cells in having a sheath, in being nonmotile, and in that they were infected by C. sporogenes bacteriophages but not usually lysed by them. The sheathed cells appeared spontaneously in cultures cloned from single colonies and were demonstrably present in cultures before bacteriophage infection. Thus, they were not contaminants but were normal, although inconspicuous, growth forms of C. sporogenes which were selected but not induced by bacteriophage infection.     

Regulation of protease production in Clostridium sporogenes

The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions.     

Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium

A reinvestigation of cellulose degradation by Clostridium cellulolyticum in a bioreactor with pH control of the batch culture and using a defined medium was performed. Depending on cellulose concentration, the carbon flow distribution was affected, showing the high flexibility of the metabolism. With less than 6.7 g of cellulose liter(-1), acetate, ethanol, H(2), and CO(2) were the main end products of the fermentation and cellulose degradation reached more than 85% in 5 days. The electron flow from the glycolysis was balanced by the production of H(2) and ethanol, the latter increasing with increasing initial cellulose concentration. From 6.7 to 29.1 g of cellulose liter(-1), the percentage of cellulose degradation declined; most of the cellulase activity remained on the cellulose fibers, the maximum cell density leveled off, and the carbon flow was reoriented from ethanol to acetate. In addition to that of previously indicated end products, lactate production rose, and, surprisingly enough, pyruvate overflow occurred. Concomitantly the molar growth yield and the energetic yield of the biomass decreased. Growth arrest may be linked to sufficiently high carbon flow, leading to the accumulation of an intracellular inhibitory compound(s), as observed on cellobiose (E. Guedon, M. Desvaux, S. Payot, and H. Petitdemange, Microbiology 145:1831-1838, 1999). These results indicated that bacterial metabolism exhibited on cellobiose was distorted compared to that exhibited on a substrate more closely related to the natural ecosystem of C. cellulolyticum. To overcome growth arrest and to improve degradation at high cellulose concentrations (29.1 g liter(-1)), a reinoculation mode was evaluated. This procedure resulted in an increase in the maximum dry weight of cells (2,175 mg liter(-1)), cellulose solubilization (95%), and end product concentrations compared to a classical batch fermentation with a final dry weight of cells of 580 mg liter(-1) and 45% cellulose degradation within 18 days.     

Thiaminase Activity Amongst Strains of Clostridium sporogenes

All the 28 strains of Clostridium sporogenes type I tested produced thiaminase. Only 2 of the 16 strains of Cl. sporogenes type II tested were positive for the enzyme; these gave a weak positive reaction. The single strain of Cl. sporogenes type III behaved in a manner similar to the strains of type I, giving a strong positive thiaminase reaction. Thiaminase production amongst the strains of Cl. sporogenes does in the main support the cultural, biochemical and immunological properties described earlier.     

Regulation of protease production in Clostridium sporogenes.

The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions.     

Chemically defined media for growing anaerobic bacteria of the genus Veillonella

A chemically defined medium for Veillonella parvula and V. alcalescens is described. Some nutritional aspects of the two strains used were examined: the optimum concentration of reducing agents, the requirement for amino acids, diamines, vitamins and other growth factors, and the conditions needed for well balanced nutrition.No specific requirements for single amino acids were observed. A combination of l-cysteine, dl-aspartic acid, l-glutamic acid, l-serine and l-tyrosine, promoted growth. In V. alcalescens, serine could substitute both arginine and tryptophan (or histidine). No growth was obtained with ammonium salts as the sole N source.Decarboxylation of l-ornithine, l-lysine and l-arginine was not demonstrated in the Veillonella parvula strain, which required putrescine or cadaverine for growth. Spermine, spermidine, l-lysine, l-ornithine and l-arginine, could not substitute putrescine in Veillonella parvula. Veillonella alcalescens, which does not require putrescine in the medium, was able to decarboxylate l-ornithine while forming putrescine.     

Regulation of the arginine dihydrolase pathway in Clostridium sporogenes.

Arginine deiminase activity was induced during the vegetative growth of Clostridium sporogenes. The enzyme was sensitive to catabolite repression. The other enzymes of the arginine dihydrolase pathway, namely, ornithine carbamoyl-transferase and carbamate kinase, did not show such variation.