Nutritional Requirements for Growth and Sporulation of Clostridium perfringens

Two strains of Clostridium perfringens grew in a chemically defined medium consisting of L-tryptophan, L-arginine, L-glutamic acid, L-histidine HCl, L-leucine, DL-threonine, DL-phenylalanine, DL-tryrosine, DL-valine, L-cystine, ascorbic acid, Ca d -pantothenate, pyridoxine, biotin, adenine HCl, glucose, salts and mercaptoacetic acid. Alanine, aspartic acid and methionine were highly stimulatory but not essential for growth. Growth did not occur in the absence of glucose, but other fermentable carbohydrates were not tested. Acetone, isopropyl alcohol, succinic acid, acetic acid, butanol, butyric acid, lactic acid, pyruvic acid, oxaloacetic acid or acetaldehyde did not eliminate the requirement for glucose. Methionine was required for sporulation; one strain also required riboflavin, isoleucine, serine and lysine. Butanol increased the degree of sporulation in a complex thioglycolate medium. Failure of Cl. perfringens to sporulate in inadequately buffered media containing glucose was shown to be caused by the high H-ion concentration developing in the culture medium. In addition, some possible end-products of glucose metabolism such as lactic acid, oxaloacetic acid and acetaldehyde, reduced sporulation in one strain appreciably.     

Enterotoxin synthesis by nonsporulating cultures of Clostridium perfringens

Chemostat-cultured Clostridium perfringens ATCC 3624 and NCTC 10240, and a nonsporulating mutant strain, 8-5, produced enterotoxin in the absence of sporulation when cultured in a chemically defined medium at a 0.084-h-1 dilution rate at 37 degrees C. The enterotoxin was detected by serological and biological assays. Examination of the chemostat cultures by electron microscopy did not reveal sporulation at any stage. The culture maintained enterotoxigenicity throughout cultivation in a continuous system. The enterotoxin was detected in batch cultures of each strain cultivated in fluid thioglycolate medium and a chemically defined medium. No heat-resistant or light-refractile spores were detected in batch cultures during the exponential growth.     

Enterotoxin synthesis by nonsporulating cultures of Clostridium perfringens.

Chemostat-cultured Clostridium perfringens ATCC 3624 and NCTC 10240, and a nonsporulating mutant strain, 8-5, produced enterotoxin in the absence of sporulation when cultured in a chemically defined medium at a 0.084-h-1 dilution rate at 37 degrees C. The enterotoxin was detected by serological and biological assays. Examination of the chemostat cultures by electron microscopy did not reveal sporulation at any stage. The culture maintained enterotoxigenicity throughout cultivation in a continuous system. The enterotoxin was detected in batch cultures of each strain cultivated in fluid thioglycolate medium and a chemically defined medium. No heat-resistant or light-refractile spores were detected in batch cultures during the exponential growth.     

Sporulation of Clostridium perfringens in a Modified Medium and Selected Foods

A modified sporulation medium for Clostridium perfringens was formulated in which a larger number of spores were produced than in SEC broth and in which spores of greater heat resistance were produced than in Ellner's medium when it was also used as the suspending medium. This modified medium consisted of 1.5% peptone; 3.0% Trypticase; 0.4% starch; 0.5% NaCl; and 0.02% MgSO(4). The addition of 0.1% sodium thioglycolate and 0.0001% thiamine hydrochloride was optional. The optimal temperature for sporulation of five strains was 37 C in comparison with 5, 22, and 46 C. Sporulation had occurred by 6 hr and was essentially complete after 20 hr at 37 C. Noyes veal broth without glucose also supported the formation of heat-resistant spores but in smaller numbers than did the modified medium. Very low numbers of spores, or none, were produced under the same conditions in pea or tuna slurries.     

Influence of carbohydrates on growth and sporulation of Clostridium perfringens in a defined medium with or without guanosine.

Clostridium perfringens strains NCTC 8238, NCTC 8798, NCTC 8679, 8-6, FD-1, and PS52 formed high levels of heat-resistant spores in a defined medium (D) with various sugars as energy sources. Strain PS49 formed high levels of heat-resistant spores when grown with dextrin and methylxanthines. The experiments showed the possibility of carrying out experiments on the sporulation of certain C. perfringens strains in a completely defined medium, without using the ill-defined polysaccharide dextrin. The addition of guanosine and sucrose to D medium generally suppressed sporulation in most strains and made it possible to prepare overnight cultures consisting mainly of vegetative cells. These cultures could be used to inoculate D medium directly, eliminating both the need to wash cells and the lag which normally occurs when cells have been grown in a different medium. Except for strains PS52 and NCTC 8238, guanosine generally increased growth rates and reduced sporulation for all strains when grown on simple sugars. Methylxanthines decreased growth rates and increased sporulation of NCTC 8679 and PS49 when present in D medium with dextrin. In the absence of guanosine, strains NCTC 8798 and 8-6 grew much slower on glucose than on disaccharides. Strain PS52 grew on lactose only after a prolonged lag. For strains requiring dextrin for good sporulation, a commercial dextrin (Difco Laboratories) was found to be readily filter sterilized, making it possible to prepare large amounts of media for use in the production of spores (or enterotoxin).     

Influence of carbohydrates on growth and sporulation of Clostridium perfringens in a defined medium with or without guanosine

Clostridium perfringens strains NCTC 8238, NCTC 8798, NCTC 8679, 8-6, FD-1, and PS52 formed high levels of heat-resistant spores in a defined medium (D) with various sugars as energy sources. Strain PS49 formed high levels of heat-resistant spores when grown with dextrin and methylxanthines. The experiments showed the possibility of carrying out experiments on the sporulation of certain C. perfringens strains in a completely defined medium, without using the ill-defined polysaccharide dextrin. The addition of guanosine and sucrose to D medium generally suppressed sporulation in most strains and made it possible to prepare overnight cultures consisting mainly of vegetative cells. These cultures could be used to inoculate D medium directly, eliminating both the need to wash cells and the lag which normally occurs when cells have been grown in a different medium. Except for strains PS52 and NCTC 8238, guanosine generally increased growth rates and reduced sporulation for all strains when grown on simple sugars. Methylxanthines decreased growth rates and increased sporulation of NCTC 8679 and PS49 when present in D medium with dextrin. In the absence of guanosine, strains NCTC 8798 and 8-6 grew much slower on glucose than on disaccharides. Strain PS52 grew on lactose only after a prolonged lag. For strains requiring dextrin for good sporulation, a commercial dextrin (Difco Laboratories) was found to be readily filter sterilized, making it possible to prepare large amounts of media for use in the production of spores (or enterotoxin).     

A new growth and in vitro sporulation medium for Clostridium perfringens

Growth and in vitro sporulation capabilities of three related Clostridium perfringens strains (NCTC 8798, 8-6 and R3) were followed in a new sporulation medium (NSM), with notable changes from a maintenance medium originally designed for strictly anaerobic bacteria. Compared with thioglycollate (FTG) medium, the new sporulation medium promoted growth of Cl. perfringens with a shorter lag phase and a 20% higher biomass production. The age of inoculum did not change Cl. perfringens growth kinetics. When compared with reference conditions, in vitro spore production kinetics were different in the new sporulation medium, but both conditions led routinely to 100% sporulation and spore counts of approximately 10(8) ml-1. The ease of preparation of the NSM, and the use of the same culture medium for good growth, high sporulation yields and spore production, represent an attractive alternative to the complex media routinely used for in vitro studies of Cl. perfringens physiology.     

Acid exposure enhances sporulation of certain strains of Clostridium perfringens

A gastroenteritis results when Clostridium perfringens is ingested in high numbers and sporulates releasing enterotoxin in the intestines. Since the organism must pass through the stomach, its ability to form spores may be affected by the acidic environment. Five strains of C. perfringens were exposed to acidic conditions and then assessed for survival and their ability to form spores. An acidic pH environment kills the bacteria over time but surviving cells are able to recover and form spores. Two of the five strains demonstrated enhanced sporulation following a 30-min exposure to a pH 2 environment. For four of the strains tested, enterotoxin concentrations were higher from acid-exposed cells than from untreated cells. Exposure to a pH 3.5 environment did not affect sporulation when compared to an untreated control. Bacteria in the stationary phase of growth were the most able to resist the acid and sporulate. The results indicate that some strains will produce more spores and enterotoxin following exposure to an acidic environment.     

Repression of sporulation in Bacillus subtilis by L-malate.

L-Malate repressed sporulation in the wild-type strain of Bacillus subtilis. When 75 mM L-malate was added to the growth medium at the time of inoculation, the appearance of heat-resistant spores was delayed 6 to 8 h. The synthesis of extracellular serine protease, alkaline phosphatase, glucose dehydrogenase, and dipicolinic acid was similarly delayed. Sporulation was not repressed when malate was added to the culture at t4 or later. A mutant was selected for ability to sporulate in the presence of malate. This strain could also sporulate in the presence of glucose. The malate-resistant mutant grew poorly with malate as sole carbon source, although it possessed an intact citric acid cycle, and it showed increased levels of malic enzyme. This indicates a defect in the metabolism of malate in the mutant. A mutant lacking malate dehydrogenase activity was also able to sporulate in the presence of malate. A model for the regulation of sporulation by malate is presented and discussed. Citric acid cycle intermediates other than malate did not affect sporulation. In contrast to previous results, sporulation of certain citric acid cycle mutants could be greatly increased or completely restored by the addition of intermediates after the enzymatic block. The results indicate that the failure of citric acid cycle mutants to sporulate can be adequately explained by lack of energy and lack of glutamate.     

Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A.

Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.     

Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A.

Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.     

Effects of media composition of delta-endotoxin production and morphology of Bacillus thuringiensis in wild types and spontaneously mutated strains

Bacillus thuringiensis strains HD-73 and 4412, and two spontaneous mutants termed 4412aa-ind and 4412sph-cry were studied for the ability to produce crystals of different size and shape when grown in a rich medium and in an appropriate minimal medium defined during this study. Strain 4412aa-ind showed medium-dependent variation in the crystal phenotype. Scanning electron microscopy was utilized in order to show crystal variations in size and shape. B. thuringiensis strains 4412aa-ind and 4412sph-cry grown in rich and in minimal media produced differences in crystal morphology, and SDS-PAGE gel indicated that crystal variation was only at the morphological level and not in composition of the amino acids. A further nineteen B. thuringiensis strains were tested for the ability to sporulate in two simple defined media. Of these strains thirteen were able to complete sporulation with crystal production in one or both media. All strains grew and sporulated in a medium containing the usual twenty amino acids, and no vitamins or other growth factors were required.     

Properties of a Bacillus subtilis mutant able to sporulate continually during growth in synthetic medium

Several mutants of Bacillus subtilis were isolated which sporulate continually during exponential growth in glucose medium. The spdA1 mutation, responsible for the continual sporulation of one of the mutants, mapped near thr. When an exponentially growing culture of a strain containing spdA1 was maintained at essentially constant turbidity, 5% of the viable cells contained heat-resistant spores. The continual sporulation depended on the stringent response since it was absent in spdA relA double mutants. Genetic and biochemical analysis indicated that the continual sporulation of spdA1 strains was associated with a lower specific activity of pyruvate carboxylase, which limited the rate of oxaloacetate synthesis from glucose via pyruvate and thereby the supply of compounds depending on the citrate cycle, especially aspartate. Therefore, the mild stringent response caused by the spdA1 mutation seems to result from a partial deficiency of aspartyl-tRNA which may exert its sporulation-initiating effect during a limited time interval in each growth cycle. A mutant blocked in fumarase activity (citG) behaved similarly. It grew only slowly in glucose medium because much of the limiting oxaloacetate was wasted for the excretion of fumarate. The mutant produced little aspartate and sporulated at a high frequency in glucose medium, even in the presence of glutamate; the sporulation was again prevented by aspartate or malate or by introduction of the relA marker into the strain.     

Sporulation and Enterotoxin Production by Mutants of Clostridium perfringens

The ability of Clostridium perfringens type A to produce an enterotoxin active in human food poisoning has been shown to be directly related to the ability of the organism to sporulate. Enterotoxin was produced only in a sporulation medium and not in a growth medium in which sporulation was repressed. Mutants with an altered ability to sporulate were isolated from an sp(+) ent(+) strain either as spontaneous mutants or after mutagenesis with acridine orange or nitrosoguanidine. All sp(0) (-) mutants were ent(-). Except for one isolate, these mutants were not disturbed in other toxic functions characteristic of the wild type and unrelated to sporulation. A total of four of seven osp(0) mutants retained the ability to produce detectable levels of enterotoxin. None of the ent(-) mutants produced gene products serologically homologous to enterotoxin. A total of three sp(-) mutants, blocked at intermediate stages of sporulation, produced enterotoxin. Of these mutants, one was blocked at stage III, one probably at late stage IV, and one probably at stage V. A total of three sp(+) revertants isolated from an sp(-) ent(-) mutant regained not only the ability to sporulate but also the ability to produce enterotoxin. The enterotoxin appears to be a sporulation-specific gene product; however, the function of the enterotoxin in sporulation is unknown.     

Sporulation Synchrony of Saccharomyces cerevisiae Grown in Various Carbon Sources

Sporulation of several strains of Saccharomyces cerevisiae grown in a variety of carbon sources that do not repress the tricarboxylic acid cycle enzymes was more synchronous than the sporulation of cells grown in medium containing dextrose which does repress those enzymes. Dextrose-grown cells showed optimal sporulation synchrony when inoculated into sporulation medium from early stationary phase when the dextrose in the medium is exhausted. Logarithmic-phase cells grown in either non-fermentable carbon sources (acetate and glycerol) or a fermentable carbon source that does not repress tricarboxylic acid cycle enzymes (galactose) sporulated more synchronously than the early stationary-phase dextrose cells. Attempts were made to sporulate cells taken from both complex and semidefined media. The semidefined acetate medium failed to support the growth of a number of strains. However, cells grown in the complex acetate medium, as well as both complex and semidefined glycerol and galactose media, sporulated with better synchrony than did the dextrose-grown cells.     

Improved Medium for Sporulation of Clostridium perfringens

An improved sporulation medium has been developed in which all five strains of Clostridium perfringens tested exhibited a 100- to 10,000-fold increase in numbers of spores when compared with spore yields in SEC medium under comparable conditions. In addition, three of five strains produced a 100- to 1,000-fold increase, with the remaining two strains yielding approximately the same numbers of spores, when compared with strains cultured in Ellner medium. At the 40-hr sampling time, 18 of 27 strains produced a 10- to 100-fold increase in numbers of spores in our medium, when compared to spore production obtained in a medium recently reported by Kim et al. The new medium contained yeast extract, 0.4%; proteose peptone, 1.5%; soluble starch, 0.4%; sodium thioglycolate, 0.1%; and Na(2)HPO(4). 7H(2)O, 1.0%. In some cases, the spore yield could be increased by the addition of activated carbon to the new medium. The inclusion of activated carbon in the medium resulted in spores with slightly greater heat resistance than spores produced in the new medium without added carbon or in SEC or in Ellner medium. The major differences in heat resistance of the various strains appeared to be genetically determined rather than reflections of a particular sporulation medium. A definite heat-shock requirement was shown for four of four strains, with the optimal temperature ranging from 60 C for a heat-sensitive strain to 80 C for a heat-resistant strain. Heating for 20 min at the optimal temperature resulted in a 100-fold increase over the viable count obtained after heating for 20 min at 50 C.     

Remarks on the modifications of cell wall during the sporulation of Saccharomyces cerevisiae Hansen (author's transl)]

Evolution of cell wall during sporulation was studied by means of scanning electron microscopy and by immunological techniques. Experiments were done simultaneously with a strain a/alpha able to sporulate and a strain alpha/alpha unable to sporulate. Under such conditions it was possible to clarify whether the changes observed were related to the sporulation or to the culture conditions. Cell wall structure modifications during sporulation were not obvious morphologically but have been revealed by immunological methods. During vegetative growth, antigenic sites of strains a/alpha and alpha/alpha were different. During incubation in the sporulation medium, antigenic structure of the cell wall was modified. Some antigenic sites seem to be specific of sporulation.     

Influence of glutamate on growth, sporulation, and spore properties of Bacillus cereus ATCC 14579 in defined medium

A chemically defined medium in combination with an airlift fermentor system was used to study the growth and sporulation of Bacillus cereus ATCC 14579. The medium contained six amino acids and lactate as the main carbon sources. The amino acids were depleted during exponential growth, while lactate was metabolized mainly during stationary phase. Two concentrations of glutamate were used: high (20 mM; YLHG) and low (2.5 mM; YLLG). Under both conditions, sporulation was complete and synchronous. Sporulation started and was completed while significant amounts of carbon and nitrogen sources were still present in the medium, indicating that starvation was not the trigger for sporulation. Analysis of amino acids and NH4+ in the culture supernatant showed that most of the nitrogen assimilated by the bacteria was taken up during sporulation. The consumption of glutamate depended on the initial concentration; in YLLG, all of the glutamate was used early during exponential growth, while in YLHG, almost all of the glutamate was used during sporulation. In YLLG, but not in YLHG, NH4+ was taken up by the cells during sporulation. The total amount of nitrogen used by the bacteria in YLLG was less than that used by the bacteria in YLHG, although a significant amount of NH4+ was present in the medium throughout sporulation. Despite these differences, growth and temporal expression of key sigma factors involved in sporulation were parallel, indicating that the genetic time frames of sporulation were similar under both conditions. Nevertheless, in YLHG, dipicolinic acid production started later and the spores were released from the mother cells much later than in YLLG. Notably, spores had a higher heat resistance when obtained after growth in YLHG than when obtained after growth in YLLG, and the spores germinated more rapidly and completely in response to inosine, l-alanine, and a combination of these two germinants.     

Characterization of a rifampin-resistant, conditional asporogenous mutant of Bacillus subtilis.

A rifampin-resistant, conditionally asporgoenous mutant of Bacillus subtilis was isolated that sporulates poorly in Sterlini-Mandelstam sporulation medium, but that sporulates normally in modified Difco sporulation medium. Rifampin-resistant (Rif-r) and conditional asporogenous (Spo-c) phenotypes co-transformed at 100% frequency. Preliminary genetic studies indicated the Rif-r trait to lie between cysA14 and ery, a locus (rnp) common to Rif-r mutants. Ribonucleic acid polymerase from strains bearing this mutation was found to be rifampin resistant in vitro. The loss of ability to sporulate in Sterlini-Mandelstam medium was found to be corrected, to a large extent, by addition to the medium of arginine, methionine, valine, and isoleucine. Several other amino acids had small effects, whereas others had no effect at all. The restorative effect is approximately additive. Growth studies indicated that Rif-r strains grew more rapidly than the corresponding parent in minimal medium at temperatures higher than 37 C. Addition of certain amino acids to the medium resulted in identical growth rates at these temperatures. Extracellular protease and esterase activities of the Rif-r Spo-c mutant were normal. A slight difference was found in the heat sensitivity of partially purified ribonucleic acid polymerase preparations of this mutant compared to the wild type.     

Phospholipid fatty acid and lipopolysaccharide fatty acid signature lipids in methane-utilizing bacteria

The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined. Each bile salt inhibited growth to a different degree. A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism. Human bile juice completely inhibited the growth of all the strains at a dilution of 1:320. A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C. perfringens strains NCTC 8239 and NCTC 8679. The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested. No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.