3-Methylindole production is regulated in Clostridium scatologenes ATCC 25775

Aims:  3-Methylindole (3-MI) is a degradation product of l -tryptophan and is both an animal waste malodorant and threat to ruminant health. Culture conditions influencing 3-MI production in Clostridium scatologenes ATCC 25775 were investigated.
Methods and Results:  Extracellular 3-MI levels in cells cultured in brain heart infusion (BHI) medium (pH 7·0) at 33°C and 37°C for 72 h were 907 ± 38 and 834 ± 121  μ mol l⁻¹, respectively. Cells cultured in tryptone-yeast (TY) extract medium at 37°C for 48 h produced 104 ± 86  μ mol l⁻¹ 3-MI; however, addition of 1 mmol l⁻¹  l -tryptophan failed to increase extracellular levels (113 ± 50  μ mol l⁻¹ 3-MI). Specific activity of indole acetic acid decarboxylase measured in BHI, TY and TY plus 1 mmol l⁻¹ tryptophan-grown cells displayed 35-, 33- and 76-fold higher levels than in semi-defined medium-grown cells.
Conclusions:  When cultured in rich medium, at 33°C or 37°C and pH 7·0, Cl. scatologenes ATCC 25775 optimally produced 3-MI. Addition of l- tryptophan to medium did not lead to significant increases in extracellular 3-MI levels. Whole cell assays indicate growth in rich medium significantly up-regulated 3-MI production.
Significance and Impact of the Study:  Information presented here may prove useful in understanding what factors influence 3-MI production in malodorous animal wastes.     

Development of a minimally defined medium for the acetogen Clostridium thermoaceticum

A minimally defined medium was developed for the cultivation of the acetogen Clostridium thermoaceticum. The medium contained glucose as the carbon and energy source, ammonium sulfate as the nitrogen source, nicotinic acid as the sole essential vitamin, reductant, a phosphate-bicarbonate buffer, mineral salts and chelator, and a CO2 gas phase. Adaptation of C. thermoaceticum from undefined medium containing yeast extract and tryptone to the minimally defined medium required sequential passage on defined medium supplemented with amino acids and vitamins. Growth and cell yields were reduced on the minimal medium, but the activities of carbon monoxide dehydrogenase, hydrogenase, and formate dehydrogenase were comparable between undefined and minimal media.     

Reactivity of Indole Derivatives Towards Oxygenated Radicals

The reactivity of a series of indole derivatives was assessed in the following systems: (i) oxidation of the indole derivatives induced by the thermolysis of 2,2′-azobis-(2-amidinopropane) (ABAP); (ii) oxidation of cumene induced by the thermolysis of 2,2′-azobis-(2-methyl propionitrile) (AIBN); (iii) lysozyme inacti vation induced by the thermolysis of ABAP and (iv) brain homogenate autoxidation.

In systems (ii) to (iv), addition of the indole derivatives decreases the rate of the process. The data obtained indicate that common factors (i.e., oxidation potential and presence of N-H bonds) control the reactivity of the indole derivatives in the four systems considered. However, in the brain homogenate autoxidation, hydrophobicity is an additional factor that affects the efficiency of antioxidants, as illustrated by Q_1/2 values (the concentration of additive required to decrease the autoxidation rate to one half that observed in the absence of additive) of 0.1 mM and ? 8 mM for 3-methylindole and tryptophan, respectively.     

A chemically defined and minimal medium for Clostridium difficile

A chemically defined medium (CDCDM) has been developed for Clostridium difficile. The medium contains nine amino acids, five mineral salts, N -acetylglucosamine and the growth factors riboflavin and nicotinic acid. Ten strains of C. difficile have been subcultured repeatedly in this medium with no apparent changes in colonial or cellular morphology. The metabolic end-products of strains grown in this medium were reproducible and yielded patterns similar to those produced by cells cultured in Brain Heart Infusion broth (BHI). The growth rates were approximately 40% slower than those in a complex medium and the growth rate constants ranged between 0·011 and 0·087 h^-1. When the defined medium was supplemented with proteose peptone, yeast extract or caesin hydrolysate at concentrations of 1%, growth increased. No such growth increase was observed when the medium was supplemented with casamino acids or glucose.     

Clear, defined medium for the sporulation of Clostridium perfringens.

A new, defined medium for the sporulation of Clostridium perfringens is presented. Sporulation levels exceeding 10(6) to 10(7) heat-resistant spores per ml were obtained for seven strains: PS49, PS52, FD-1, T-65, NCTC strains 8798, 8238, and 10240. In the presence of theophylline, a methylxanthine, higher levels of heat-resistant spores were attained for strains PS49, PS52, FD-1, ant T-65; photomicrographs demonstrated a higher fraction of sporulating cells when these strains were grown in the presence of methylxanthines. Use of washed, highly diluted (less than 100 cells) inocula resulted in no reduction in spore yield. Strain KA3 grew well but sporulated poorly on this medium. The medium was clear and free of precipitate when small amounts (100 microgram/ml) of methylxanthine were incorporated.     

Clear, defined medium for the sporulation of Clostridium perfringens.

A new, defined medium for the sporulation of Clostridium perfringens is presented. Sporulation levels exceeding 10(6) to 10(7) heat-resistant spores per ml were obtained for seven strains: PS49, PS52, FD-1, T-65, NCTC strains 8798, 8238, and 10240. In the presence of theophylline, a methylxanthine, higher levels of heat-resistant spores were attained for strains PS49, PS52, FD-1, ant T-65; photomicrographs demonstrated a higher fraction of sporulating cells when these strains were grown in the presence of methylxanthines. Use of washed, highly diluted (less than 100 cells) inocula resulted in no reduction in spore yield. Strain KA3 grew well but sporulated poorly on this medium. The medium was clear and free of precipitate when small amounts (100 microgram/ml) of methylxanthine were incorporated.     

Enterotoxin formation by Clostridium perfringens type A in a defined medium

Enterotoxin was produced by 9 of 10 strains of Clostridium perfringens type A when grown in a defined medium. Additional dextrin increased the amount of enterotoxin in extracts of sporulating cells of strain NCTC 10239.     

Development of a chemically defined medium for growth ofNeisseria gonorrhoeae

A chemically defined medium satisfactory for growth of a number of laboratory strains and recent isolates ofNeisseria gonorrhoeae has been devised. It contains inorganic salts, dextrose, guanine, cytosine, B-vitamin supplement, and the following amino acids:l-arginine,l-aspartic acid,l-cystine,l-isoleucine,l-leucine,l-proline,l-threonine, andl-valine.Nine of the eleven strains grew satisfactorily in this medium without being provided supplemental CO₂ during incubation, and a tenth strain grew in the medium supplemented with glutamine. No single B-vitamin or purine or pyrimidine base was essential for growth of any of the strains, but some combinations of them were stimulatory. Riboflavin, however, was inhibitory. The strains showed variations in requirements for amino acids. The amino acids which were either essential or stimulatory for one or more of the strains were included in the medium. Those to which the strains responded differently were used at concentrations intermediate between those optimal for growth of one strain and inhibitory for another. Conventional agar was inhibitory, but a purified agar, having a gel strength twice that of conventional agar, was satisfactory. An aqueous solution of 0.1% cysteine and 0.86% NaCl was satisfactory for preparation of inocula.This investigation was supported by a Public Health Service Predoctoral Fellowship (F-FI-GM-24-755-01A1) from the National Institute of General Medical Sciences of the United States Public Health Service to the senior author.     

Enterotoxin formation by Clostridium perfringens type A in a defined medium.

Enterotoxin was produced by 9 of 10 strains of Clostridium perfringens type A when grown in a defined medium. Additional dextrin increased the amount of enterotoxin in extracts of sporulating cells of strain NCTC 10239.     

Endogenous radiolabeling of enterotoxin from Clostridium perfringens type A on a defined medium

Four enterotoxin-positive strains of Clostridium perfringens were tested for sporulation and enterotoxin production on defined media. The medium described by Sacks and Thompson (Appl. Environ. Microbiol. 35:405-409, 1978) gave the highest enterotoxin production and was selected for the production of endogenously labeled enterotoxin. The specific radioactivity of the enterotoxin was 16,000 dpm/microgram when the tritiated amino acids were added to the growth medium just before the inoculum. Addition of the radioactive amino acids during the growth period gave consistently lower specific radioactivity. When the enterotoxin was produced on the medium described by Duncan and Strong (Appl. Microbiol. 16:82-89, 1968), the highest specific radioactivity of the enterotoxin was found when the radioactive amino acids were added to the growth medium 4 h after inoculation. In this case, the specific activity of the enterotoxin was 10,000 dpm/microgram.     

Development and Characterization of a Gene Expression Reporter System for Clostridium acetobutylicum ATCC 824

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of β-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional β-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the β-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the β-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the β-galactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of β-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.     

Development of a minimal defined medium for recombinant human interleukin-3 production by Streptomyces lividans 66

A systematic approach was developed to identify and optimize the essential amino acids in defined minimal medium for the production of recombinant human interleukin 3 (rHuIL-3) by Streptomyces lividans. Starvation trials were carried out initially to narrow down the number of probable essential amino acids from an initial number of 20 to 8. Then a screening mixture experiment was designed and performed with the eight identified amino acids and distance-based multivariate analysis was employed to rank the probable essential amino acids regarding both growth and product formation. Following this procedure, the search was narrowed to four amino acids (Asp, Leu, Met, and Phe). Finally, a mixture design experiment known as the simplex lattice design was carried out and the composition of the optimum minimal medium was found (Asp 53%, Met 5%, and Phe 42%).     

Endogenous radiolabeling of enterotoxin from Clostridium perfringens type A on a defined medium.

Four enterotoxin-positive strains of Clostridium perfringens were tested for sporulation and enterotoxin production on defined media. The medium described by Sacks and Thompson (Appl. Environ. Microbiol. 35:405-409, 1978) gave the highest enterotoxin production and was selected for the production of endogenously labeled enterotoxin. The specific radioactivity of the enterotoxin was 16,000 dpm/microgram when the tritiated amino acids were added to the growth medium just before the inoculum. Addition of the radioactive amino acids during the growth period gave consistently lower specific radioactivity. When the enterotoxin was produced on the medium described by Duncan and Strong (Appl. Microbiol. 16:82-89, 1968), the highest specific radioactivity of the enterotoxin was found when the radioactive amino acids were added to the growth medium 4 h after inoculation. In this case, the specific activity of the enterotoxin was 10,000 dpm/microgram.     

A defined growth medium with very low background carbon for culturing Clostridium thermocellum

A growth medium was developed for cultivation of Clostridium thermocellum ATCC 27405 in which "background" carbon present in buffers, reducing agents, chelating agents, and growth factors was a small fraction of the carbon present in the primary growth substrate. Background carbon was 1.6% of primary substrate carbon in the low-carbon (LC) medium, whereas it accounts for at least 40% in previously reported media. Fermentation of cellulose in LC medium was quite similar to Medium for Thermophilic Clostridia (MTC), a commonly used growth medium that contains background carbon at 88% of primary substrate carbon. Of particular note, we found that the organism can readily be cultivated by eliminating some components, lowering the concentrations of others, and employing a tenfold lower concentration of reducing agent. As such, we were able to reduce the amount of background carbon 55-fold compared to MTC medium while reaching the same cell biomass concentration. The final mass ratios of the products acetate:ethanol:formate were 5:3.9:1 for MTC and 4.1:1.5:1 for LC medium. LC medium is expected to facilitate metabolic studies involving identification and quantification of extracellular metabolites. In addition, this medium is expected to be useful in studies of cellulose utilization by anaerobic enrichment cultures obtained from environmental inocula, and in particular to diminish complications arising from metabolism of carbon-containing compounds other than cellulose. Finally, LC medium provides a starting point for industrial growth media development.     

Development of a defined minimal medium for the growth of Neisseria gonorrhoeae

This investigation describes the development of a solid and a liquid medium (Gonococcal Genetic Medium; GGM) which support the rapid growth of 41 gonococcal clinical isolates and laboratory strains with a minimum number of nutritional components. The complete medium contains minimal salts, eight amino acids, two nitrogen bases, vitamins, coenzymes, key metabolic intermediates, and some miscellaneous components. Results indicate that GGM can be modified and simplified even further than we described. In liquid GGM, several gonococcal strains grew logarithmically after a 2- to 3-h lag period with generation times ranging from 72 to 115 min, reaching optical densities of 175 to 320 Klett units in the presence of seven amino acids and in the absence of a CO(2) atmosphere. The development of a solid and a liquid defined minimal medium such as GGM should greatly broaden the avenues of experimentation for biochemical genetic studies with N. gonorrhoeae, especially gonococcal genetic transformation. N. gonorrhoeae can be classified into eight major and minor phenotypic groups, depending on its growth responses on GGM to just five amino acids: cysteine and cystine, arginine, proline, isoleucine, and serine. Such results demonstrate the feasibility of using GGM as a simple, sensitive, rapid probe for investigating the epidemiological patterns of gonorrhea.