Sinorhizobium meliloti Cells Require Biotin and either Cobalt or Methionine for Growth

Sinorhizobium meliloti is usually cultured in rich media containing yeast extract. It has been suggested that some components of yeast extract are also required for growth in minimal medium. We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation. By fractionation of yeast extract, two required growth factors were identified. Biotin was found to be absolutely required for growth, whereas previously the need for this vitamin was considered to be strain specific. All strains also required supplementation with cobalt or methionine, consistent with the requirement for a vitamin B₁₂-dependent homocysteine methyltransferase for methionine biosynthesis.     

Nutritional studies on a methionine-requiring strain of Paracoccidioides brasiliensis

Strain IVIC-Pb9, unlike other strains ofParacoccidioides brasiliensis, cannot grow on a simple basal medium and requires the addition of casein hydrolyzate or yeast extract. The present study shows that this requirement is limited to very low concentrations of methionine and that methionine concentrations above 0.01% inhibit growth. The levels of glucose and organic nitrogen required for maximum rate of growth of strain IVIC-Pb9 on both basal medium and GGY medium composed of glucose, glycine and yeast extract were also determined. An evaluation of the suitability of the GGY medium revealed that its composition, as commonly used to grow dimorphic fungi, is not adequate to obtain a maximum rate of growth with strain IVIC-Pb9 ofP. brasiliensis.     

Growth requirements of san francisco sour dough yeasts and bakers' yeast

The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.     

Growth Requirements of San Francisco Sour Dough Yeasts and Bakers' Yeast

The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.     

1,4-Naphthoquinone and other nutrient requirements of Succinivibrio dextrinosolvens.

Three strains of Succinivibrio dextrinosolvens isolated from the rumen of cattle or sheep under diverse conditions grew well in a minimal medium containing glucose, minerals, cysteine, methionine, leucine, serine, ammonia, 1,4-naphthoquinone, p-aminobenzoic acid, and bicarbonate-carbonic acid buffer, pH 6.7. When menadione or vitamin K5 was substituted for 1,4-naphthoquinone, the growth rate was somewhat depressed. Growth was poor with vitamin K1 and ammonia, further addition of the amino acids aspartic acid, arginine, histidine, and tryptophan was necessary for good growth of type strain 24, but the other two strains grew well only in media containing ammonia. Strains C18 and 22B produced urease and grew well when ammonia replaced urea. When urea replaced ammonia, strain 24 grew poorly and urease activity could not be detected. Strain 24 required no B-vitamins, but the other two strains were stimulated by p-aminobenzoic acid. The methionine requirement was not placed by vitamin B12, betaine, or homocysteine. Cysteine was replaced by sulfide in strain 24 but less well in the other two strains. Very poor growth was obtained when sulfate replaced cysteine. The half-saturation constant for ammonia during growth of S. dextrinosolvens is more than 500 microM, a much higher value than that of many rumen bacteria.     

Nutrition of Myxococcus xanthus, a fruiting myxobacterium.

The minimal requirements for vegetative growth of Myxococcus xanthus have been sought. Isoleucine, leucine, and valine were required, and vitamin B12 was needed for the synthesis of methionine. Pyruvate was an excellent energy source and an efficient source of cellular carbon. Acetate, aspartate, glutamate, and most tricarboxylic acid cycle intermediates could also be utilized, but were less efficient sources of carbon and energy than was pyruvate. Many mono- and disaccharides were tested, but, in agreement with earlier results, none served as carbon-energy sources. A minimal medium (A1) has been devised that includes the essential amino acids and vitamin B12, with pyruvate and aspartate as carbon-energy sources. In this medium, M. xanthus could propagate indefinitely, and on it vegetative cells formed colonies with greater than 75% efficiency; hence, it is likely that no organic cofactors other than those present in A1 are required in more than trace amounts.     

Cellulolytic cocci isolated from the cecum of guinea pigs (Cavia porcellus).

Five strains of anaerobic, gram-variable cellulolytic cocci, belonging to the genus Ruminococcus, were isolated from the cecum of a guinea pig. They differed from most previously described strains of cellulolytic ruminococci as follows. (i) Lactate was the major fermentation product; lesser amounts of formate and ethanol and a trace of succinate were also produced, along with an uptake of acetate. (ii) No growth occurred at 30 degrees C; however, good growth was observed at 38 and 45 degrees C, (iii) Glucose, cellobiose, cellulose, xylose, arabinose, xylan, sucrose, and lactose were fermented by all strains. Rumen fluid was required for growth in a complete medium containing all nutrients previously found to be required by species in this genus. Limited growth occurred when rumen fluid was replaced by yeast extract, and maximum, but delayed, growth occurred when a water extract of alfalfa was added to the complete medium. No qualitative differences were found in the cell wall amino acids and sugar composition of these strains as compared to Ruminococcus flavefaciens and Ruminococcus albus; however, cell walls of the guinea pig strains appeared to contain a higher proportion of glucose.     

Chemically defined media for the cultivation of Naegleria: pathogenic and high temperature tolerant species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCC 30100, ATCC 30863, and ATCC 30896) and two strains of N. lovaniensis (ATCC 30467 and ATCC 30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCC 30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysin for ATCC 30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCC 30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCC 30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B12, nicotinamide, and Ca pantothenate) was required.     

An investigation of ethanol inhibition and other limitations occurring during the fermentation of concentrated whey permeate byKluyveromyces fragilis

Based on the well-known fact thatKluyveromyces fragilis strains show sub-optimal performance when grown in concentrated whey permeate, previously optimized medium was investigated for possible limitations appearing at high concentrations. Shaken flask cultures showed that no additional vitamin or mineral sources were required when the optimized amount of yeast extract was added to the concentrated permeate. Several aspects of the ethanol inhibition of the growth ofK. fragilis NRRL 665 were investigated in continuous culture. The maximum ethanol concentration tolerated by this yeast, i.e. 45 g/l, was much lower than commonly reported for other strains. Ethanol and biomass production were also influenced by the increased ethanol concentration of the medium. At 31 g/l of alcohol product yield was reduced to 0.23 g/g whereas biomass yield was 0.05 g/g. Some evidence suggested that residence time and residual lactose concentration played a significant role in modulating the toxic effect of ethanol.     

A minimal medium for growth of Pasteurella multocida

Abstract We developed a minimal medium supporting the growth of both toxigenic and nontoxigenic strains of Pasteurella multocida to optical densities of > 0.5 (600 nm ). P. multocida P1059 (ATCC 15742), one of a number of strains which can cause fowl cholera, was used as the model strain in this study. The medium was composed of 17 ingredients including cysteine, glutamic acid, leucine, methionine, inorganic salts, nicotinamide, pantothenate, thiamine, and an energy source. Leucine was not required for growth but was stimulatory, and thiamine could be replaced by adenine. An additional 46 strains of P. multocida were tested, and 40 out of 46 (87%) strains grew as well as strain P1059 through a minimum of 10 serial transfers. P. multocida toxin (PMT) was produced when cells of a known toxigenic strain (P4261) were cultivated in the minimal medium. No growth of Pasteurella haemolytica or Pasteurella trehalosi strains was observed in this minimal medium.     

Mutants of Salmonella typhimurium Lacking Phosphoenolpyruvate Carboxykinase and α-Ketoglutarate Dehydrogenase Activities

Two auxotrophic mutants (SM16 and SM51) of Salmonella typhimurium, which for aerobic growth, with hexoses as carbon source, required lysine and methionine (SM51 required also nicotinic acid), were isolated and characterized. The requirement for the amino acids disappeared in anaerobiosis. Neither lipoate nor 4-hydroxybenzoate was effective in supporting aerobic growth of the mutants. The lysine and methionine requirement for aerobic growth was due to the absence in the mutants of the enzymatic activities of the alpha-ketoglutarate dehydrogenase complex. The mutants could not use succinate as carbon source even after enrichment of the growth medium with acid-hydrolyzed casein and yeast extract. No phosphoenolpyruvate carboxykinase activity was found in the mutants, a phenomenon which explained their inability to use succinate. By interrupted conjugation and by transduction experiments, the positions of the three affected loci, pck, suc, and Nic, were located at approximately 17 to 19 min of the S. typhimurium chromosome; they were found to be closely linked. From different criteria, it appears as if the genetic lesions present in both mutants are due to deletion of a small chromosome fragment.     

Effect of vitamin B12 and folate on biosynthesis of methionine from homocysteine in the nematode Caenorhabditis briggsae.

(i) Omission of L-methionine from the medium resulted in an 80% population reduction. Substitution of D,L-homocysteine corrected methionine deficiency in C. briggsae in the presence of supraoptimal vitamin B12 and folic acid. (ii) An absolute vitamin B12 requirement in C. briggsae developed in the medium containing homocysteine at the second subculture. Concentration of 6 ng/ml of vitamin B12 (at 100 ng/ml of folic acid) was sufficient to support maximum growth of C. briggsae in the medium containing homocysteine. (iii) It was found that either supraoptimal folic acid (2000 ng/ml) or supraoptimal vitamin B12 (3750 ng/ml), with homocysteine, supported very little population growth of C. briggsae. However, supraoptimal folic acid and supraoptimal vitamin B12 together supported a maximum population growth. Therefore, it was concluded that both vitamin B12 and folic acid were required for the biosynthesis of methionine from homocysteine. Studies also showed that the two vitamins spared each other for population growth in the medium containing homocysteine.     

GLUCOSE FERMENTATION AND GROWTH OF SELENOMONAS SPUTIGENA ON A MINIMAL MEDIUM

Very little is known about the growth physiology and metabolic niche of the human oral isolate Selenomonas sputigena. The objective of this study was to devise a minimal medium for comparing growth rates and fermentation of rumen Selenomonas ruminantium strains with S. sputigena. When anaerobically grown on a minimal glucose medium containing yeast extract as the only chemically undefined component, S. sputigena produced acetate, propionate, and succinate while S. ruminantium strains produced primarily lactate. When strains were compared (P < 0.05) for each carbon source that yielded growth, rumen strain HD₄ grew faster than all other strains on glucose, cellobiose and glycerol while strain GA192 grew faster on trehalose. Rumen strains GA192, PC18, and HD₄ grew faster on mannitol than rumen strains D and GA31. S. sputigena grew faster on lactate (0.38 ± 0.04) than any of the S. ruminantium strains. The minimal medium developed in this study should be useful for jurmer physiological studies on fermentation and metabolism in S. sputigena.     

Chemically defined and minimal media forBacteroides gingivalis

A chemically defined medium (BGDM) has been developed specifically forBacteroides gingivalis. The medium contains 4 amino acids, 5 mineral salts, cysteine hydrochloride as a reducing agent, and the growth factors hemin and menadione. Eight strains ofB. gingivalis 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 complex media. The growth rates were about 50% slower than those of cells grown in a complex medium, and the growth rate constants ranged between 0.013 and 0.067 H^–1. When the defined medium was supplemented with protein hydrolysates such as trypticase, proteose peptone, bactocasitone, or yeast extract, at concentrations up to 1.0%, growth increased. No such growth increase was observed in the medium supplemented with casamino acids. Thus a minimal medium can be formulated by adding one of the growth-enhancing protein hydrolysates to the defined medium at varying concentrations depending upon the growth yield required.     

Chemically Defined Media for the Cultivation of Naegleria: Pathogenic and High Temperature Tolerant Species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCCr?30100, ATCCr?30863, and ATCCr?30896) and two strains of N. lovaniensis (ATCCr?30467 and ATCCr?30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCCr?30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysine for ATCCr?30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCCr?30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCCr?30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B₁₂, nicotinamide, and Ca pantothenate) was required.     

Effect of alanine, leucine and fructose on lysyl-transfer ribonucleic acid ligase activity in a mutant of Escherichia coli K-12

A mutant of Escherichia coli K-12 was examined which has growth medium-dependent lysyl-transfer ribonucleic acid (tRNA) ligase activity. In minimal medium or 0.5% yeast extract, the activity of the enzyme in the mutant strain was 5 to 10% of wild type. However, when the mutant was grown in a highly enriched medium, such as AC broth (Difco), the activity of the mutant ligase increased 10- to 20-fold. We found that the supplementation of 0.5% yeast extract by l-alanine plus d-fructose replaces the need for the highly enriched medium. Fructose plus l-leucine and fructose plus l-alpha-amino-n-butyric acid were also stimulatory, but not as effective as fructose and alanine. With minimal medium, a combination of carbohydrate (fructose or glucose) plus alanine and leucine was required to replace the enriched medium. The most effective combination was fructose, glucose, alanine, and leucine. Lysyl-tRNA ligase was stimulated 1.5 to 2-fold in the wild-type strain or Hfr H (Hayes) by fructose plus alanine when these strains were cultured in 0.5% yeast extract. Experiments employing the combined technique of density labeling with D(2)O and isopycnic centrifugation in cesium chloride indicated that the increased activity of lysyl-tRNA ligase observed in AC broth or in the presence of fructose, glucose, alanine, and leucine is due to the synthesis of new enzyme.     

Isolation and characterization of the major nod factor of Bradyrhizobium japonicum strain 532C

Bradyrhizobium japonicum 532C nodulates soybean effectively under cool Canadian spring conditions and is used in Canadian commercial inoculants. The major lipo-chitooligosaccharide (LCO), bacteria-to-plant signal was characterized by HPLC, FAB-mass spectroscopy MALDI-TOF mass spectroscopy and revealed to be LCO Nod Bj-V (C18:1, MeFuc). This LCO is produced by type I strains of B. japonicum and is therefore unlikely to account for this strains superior ability to nodulate soybean under Canadian conditions. We also found that use of yeast extract mannitol medium gave similar results to that of Bergerson minimal medium.     

Novel Escherichia coli K-12 mutants impaired in S-adenosylmethionine synthesis.

S-Adenosylmethionine (AdoMet) plays a myriad of roles in cellular metabolism. One of the many roles of AdoMet in Escherichia coli and Salmonella typhimurium is as a corepressor of genes encoding enzymes of methionine biosynthesis. To investigate the metabolic effects of large reductions in intracellular AdoMet concentrations in growing cells, we constructed and examined mutants of E. coli which are conditionally defective in AdoMet synthesis. Temperature-sensitive mutants in metK, the structural gene for the S-adenosylmethionine synthetase (AdoMet synthetase) expressed in minimal medium, were constructed by in vitro mutagenesis of a plasmid-borne copy of metK. By homologous recombination, the chromosomal copy was replaced with the mutated metK gene. Both heat- and cold-sensitive mutants were examined. At the nonpermissive temperature, two such mutants had 200-fold-reduced intracellular AdoMet levels and required either methionine or vitamin B12 for growth. In the presence of methionine or vitamin B12, the mutants grew at normal rates even though the AdoMet levels remained 0.5% of wild type. A third mutant when placed at nonpermissive temperature had less than 0.2% of the normal AdoMet level and did not grow on minimal medium even in the presence of methionine or vitamin B12. All of these mutants grew normally on yeast-extract-based medium in which an alternate form of S-adenosylmethionine synthetase was expressed.     

Temperature and pH optima for 21 species of thermophilic and thermotolerant fungi.

A glucose-containing mineral medium supplemented with 0.01% yeast extract is described upon which all the species of thermophilic and thermotolerant fungi tested will grow. Thirteen of the 21 species do not require the yeast extract supplement for growth. Using this solid, supplemented mineral medium, the pH and temperature optima for growth of all strains were measured. No correlation was found between temperature optimum and pH optimum among members of the group tested.     

Characterization of unexpected growth of Escherichia coli O157:H7 by modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.