The nitrogen-fixing symbiotic bacterium Mesorhizobium loti has and expresses the gene encoding pyridoxine 4-oxidase involved in the degradation of vitamin B6

The gene product of mll6785 of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 was identified as pyridoxine 4-oxidase, the first enzyme in the vitamin B6-degradation pathway. The gene was cloned and ligated into pET-21a+. Escherichia coli BL21(DE3) was co-transformed with the constructed plasmid plus pKY206 containing groESL genes encoding chaperonins. The overexpressed protein was purified to homogeneity by the ammonium sulfate fractionation and three chromatography steps. The enzymatic properties of the purified protein, such as K(m) values for pyridoxine (213+/-19 microM) and oxygen (78+/-10 microM), were compared to those of pyridoxine 4-oxidase from two bacteria with known vitamin B6-degradation pathway. M. loti grown in a Rhizobium medium showed the enzyme activity. The results suggest that M. loti also contains the degradation pathway of vitamin B6.     

Production of Indole-3-Acetic Acid by Bradyrhizobium japonicum: A Correlation with Genotype Grouping and Rhizobitoxine Production

Bioassays show that rhizobitoxine-producing strains of Bradyrhizobiumjaponicum excreted another phytotoxic compound into their culturefluid. This compound was purified and identified by HPLC andmass spectrometry as indole-3-acetic acid (IAA). The levelsof IAA produced by the different strains of B. japonicum, forwhich the genotype groups have been determined with respectto the degree of base substitution in and around nifDKE, werequantified using gas chromatography/mass spectrometry and adeuterated internal standard. Genotype II strains, which producerhizobitoxine, excreted more than 20µof IAA into theirculture fluid. However, no IAA was detected in the culture supernatantsof genotype I strains, which do not produce rhizobitoxine. Thiswas true even when tryptophan was added to the medium. Moreover,cells of genotypes I and II strains, which were grown underour culture conditions, did not show IAA degradation activity.These results suggest that, in wild-type B. japonicum strains,complete IAA biosynthesis is confined exclusively to genotypeII strains that produce rhizobitoxine. (Received April 9, 1990; Accepted October 6, 1990)     

Characterization of an intracellular serine protease from sporulating cells of Bacillus brevis.

Sporulating cells of Bacillus brevis ATCC 9999 produced a high level of an intracellular serine protease when grown in nutrient medium. The protease activity in the crude extracts of this strain appeared at hour 5 (t5) after the end of exponential growth and increased gradually during sporulation, reaching a maximum at t12 to t13. The enzyme isolated in a partially purified state showed a pH optimum between 7.3 and 9.0 and had an apparent molecular weight of about 60,000. The activity was completely inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, EDTA, and ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. The protease possessed a high activity for azocoll and low activities for azocasein and 14C-labeled hemoglobin. It cleaved the cyclic decapeptide gramicidin S specifically at the peptide linkage between valine and ornithine and hydrolyzed the oxidized insulin B-chain mainly at peptide bonds 4-5 (Glu-His), 6-7 (Leu-CysSO3H), and 15-16 (Leu-Tyr). No catalysis of bond cleavage by the enzyme on a variety of small peptides or esters was detected. Unlike other Bacillus species, B. brevis ATCC 9999 grown in nutrient medium excreted no extracellular proteases.     

Calvarial cells synthesize 1 alpha,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3

A metabolite of vitamin D has been isolated in pure form from incubation of 25-hydroxyvitamin D3 with embryonic chick calvarial cells that had been grown on Cytodex 1 microcarrier beads. The isolation involved dichloromethane extraction of the cells and incubation medium, followed by Sephadex LH-20 column chromatography and high-performance liquid chromatography of the extract. The metabolite was identified as 1 alpha,25-dihydroxyvitamin D3 by means of ultraviolet absorption spectroscopy, mass spectrometry, and sensitivity to oxidation by periodate. This metabolite was not produced by cell-free medium or by cells from embryonic chick liver, skin, or heart. In conclusion, (1) kidney cells are not unique in having 25-hydroxyvitamin D3:1 alpha-hydroxylase activity as previously believed and (2) vitamin D target tissues such as the skeleton may play a direct role in mediating the metabolism of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3, a vitamin D metabolite active at those sites.     

Studies on the biosynthesis of biotin. Production of biotin and biotin-like compounds by a pseudomonad

1. Filtrates from cultures of a strain of Pseudomonas aeruginosa, grown in a basal glucose-ammonium chloride-vitamins-salts medium, possessed biotin activity as detected by microbiological assays. Exponential-phase culture filtrates contained biotin and desthiobiotin in the approximate ratio 1:3, with smaller amounts of biotin sulphoxide and three unidentified compounds with biotin activity. 2. The addition of malonate, adipate or pimelate to the basal medium stimulated the production of compounds with biotin activity; this effect was enhanced when these compounds were included in the medium as the major carbon source. Succinate, glutarate, suberate, fumarate or oxaloacetate did not stimulate the production of compounds with biotin activity. The ratio of biotin to desthiobiotin in filtrates from cultures grown in medium containing malonate as the carbon source was about 1:1. Experiments in which mixtures of malonate and pimelate were included in the medium as the carbon sources showed that these acids probably make a similar contribution in biotin biosynthesis. 3. A number of heterocyclic compounds, including several containing the ureido group (-NH-CO-NH-), were included in the basal medium but none of them stimulated the production of compounds with biotin activity to any marked degree. 4. Several amino acids, particularly cysteine (or cystine) and lysine, when added individually as supplements to the basal medium, stimulated the production of compounds with biotin activity. Filtrates from cultures grown in medium supplemented with cysteine contained approximately equal proportions of biotin and desthiobiotin. A much greater stimulation in the production of compounds with biotin activity was obtained when certain amino acids were included in the medium as the major source of nitrogen or carbon and nitrogen; ornithine, citrulline and argininosuccinate had the most marked effect. The ratio of biotin to desthiobiotin in filtrates from these cultures was usually greater than in filtrates from cultures grown in basal medium. 5-Aminovalerate also caused some stimulation when used as the nitrogen source, but urea was inactive. The effect of binary mixtures of certain amino acids was also examined. 5. The results are discussed in relation to the possible role of the stimulatory compounds during biotin biosynthesis.     

EFFECTS OF CERTAIN LIMITING CONDITIONS ON THE SYNTHESIS OF B VITAMINS BY YEAST

In yeast crops which were grown in the presence of various inhibitors, there was considerable variation in content of the various B vitamins. A higher degree of parallelism in variation in content was found to exist between thiamine and niacin than between any other pair of vitamins; this has been interpreted as indicating that the predominant functions of these two vitamins are their established rôles in fermentation. Values for inositol indicate that it may be involved in fermentation processes, but this is not the case for other members of the B complex. Biotin appears to be unique since in no case did the biotin content of yeast grown in the presence of an inhibitor fall below that of the control yeast. There was some evidence of synthesis of biotin, or a material with biotin activity, in the presence of certain inhibitors, the most striking instance being with sulfaguanidine. An exogenous supply of biotin was essential for extensive proliferation of F. B. yeast, and yeast grown in a medium to which biotin was the only added vitamin contained the B vitamins in amounts very similar to those found in the control yeast, the most marked differences being in increased vitamin B₆ and p-aminobenzoic acid contents. In the absence of biotin, significant amounts of all of the B vitamins except biotin were synthesized, both in the presence and absence of certain other members of the B complex. The addition of thiamine, pyridoxine, inositol, and β-alanine to the culture medium caused a reduction in the amounts of vitamin B₆ and p-aminobenzoic acid synthesized. F. B. yeast was able to grow in a xylose medium only when certain of the B vitamins were present, and even then growth was limited. Evidence was obtained for some synthesis of all of the vitamins investigated except biotin and vitamin B₆. The most significant differences in vitamin content between galac yeast and the parent F. B. strain were in folic acid and vitamin B₆, the former being considerably reduced in amount, the latter being increased.     

Effect of vitamin B6 availability on serine hydroxymethyltransferase in MCF-7 cells

Folate-activated one-carbon units are derived from serine through the activity of the pyridoxal-phosphate (PLP)-dependent isozymes of serine hydroxymethyltransferase (SHMT). The effect of vitamin B(6) availability on the activity and expression of the human mitochondrial and cytoplasmic SHMT isozymes was investigated in human MCF-7 cells. Cells were cultured for 6 months in vitamin B(6) replete (4.9 microM pyridoxine) minimal essential medium (alphaMEM) or vitamin B(6)-deficient medium containing 49, 4.9 or 0.49 nM pyridoxine. Total cellular PLP levels and SHMT activity were reduced 72% and 7%, respectively, when medium pyridoxine was decreased from 4.9 microM to 49 nM. Cells cultured in medium containing 4.9 nM pyridoxine exhibited 75%, 27% and 60% reduced levels of PLP, SHMT activity and S-adenosylmethionine, respectively, compared to cells cultured in alphaMEM. Cytoplasmic SHMT activity and protein levels, but not mRNA levels, were decreased in cells cultured in vitamin B(6) deficient medium, whereas mitochondrial SHMT activity and protein levels were less sensitive to vitamin B(6) availability. PLP bound to cytoplasmic SHMT with a K(d)=850 nM, a value two orders of magnitude lower than previously reported for the bovine cytoplasmic SHMT isozyme. Collectively, these data indicate that vitamin B(6) restriction decreases the activity and stability of SHMT, and that the cytoplasmic isozyme is more sensitive to vitamin B(6) deficiency than the mitochondrial isozyme in MCF-7 cells.     

Indole compounds released by the ectendomycorrhizal fungal strain MrgX isolated from a pine nursery

The production and metabolism of indole compounds in pure cultures of the ectendomycorrhizal strain MrgX, a common symbiont of Scots pine in forest nurseries, were investigated. Different indole compounds produced by this fungus were purified and identified by thin-layer chromatography, high-performance liquid chromatography and mass spectrometry. Indole-3-acetic acid (IAA) and indole-3-carboxylic acid were the most abundant. Although MrgX is able to synthesize IAA when cultivated on a medium without tryptophan, much higher IAA production was obtained when 1 mM tryptophan was added. Buffering of the medium at pH 5.8 was shown to be essential for IAA accumulation in the culture filtrate. In vitro IAA-synthesizing activity of the enzymes extracted from the mycelia of MrgX was also maximal when mycelia were grown in a buffered, tryptophan-supplemented medium. The hydrogen ion concentration strongly affected in vivo activity of IAA-synthesizing enzymes. This activity was rather weak at acid pH and was stimulated by increase in pH up to 8.5. These results and their possible significance for ectendo-mycorrhizal symbiosis are discussed with reference to the hormonal metabolism of ectomycorrhizal fungi and ectomycorrhizae.     

Identification of pyridoxine 3-sulfate, pyridoxal 3-sulfate, and N-methylpyridoxine as major urinary metabolites of vitamin B6 in domestic cats

Preliminary studies of vitamin B6 metabolism in three adult domestic cats detected very little pyridoxic acid in the urine. At oral doses of 49 to 490 mumol of [14C]pyridoxine hydrochloride, 50% of the excreted dose occurred as pyridoxine 3-sulfate and 25% as N-methylpyridoxine. The identity of these two metabolites was confirmed by isolation from urine and comparison with known compounds. A third compound was identified as pyridoxal 3-sulfate on the basis of chromatographic behavior and fluorescent properties before and after hydrolysis. At pyridoxine intakes of 0.97 mumol/day, the concentration of pyridoxal 3-sulfate in the urine sometimes exceeded the concentration of pyridoxine 3-sulfate. Pyridoxic acid remained a minor urinary metabolite at pyridoxine intakes ranging from 0.97 to 490 mumol/day. Although sulfation of phenol groups and methylation of the ring nitrogen are well-known detoxication reactions, this appears to be the first time such reactions have been observed in normal metabolism of vitamin B6. These observations provide further evidence of the diversity of vitamin B6 metabolism between species. While such diversity complicates the extrapolation of data from animal studies to humans, it does provide a variety of models for examining the influences of various factors on vitamin B6 metabolism.     

Metabolism of phenanthrene by Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium metabolized phenanthrene when it was grown for 7 days at 37 degrees C in a medium containing malt extract, D-glucose, D-maltose, yeast extract, and Tween 80. After cultures were grown with [9-14C]phenanthrene, radioactive metabolites were extracted from the medium with ethyl acetate, separated by high-performance liquid chromatography, and detected by liquid scintillation counting. Metabolites from cultures grown with unlabeled phenanthrene were identified as phenanthrene trans-9,10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol, 9-phenanthrol, 3-phenanthrol, 4-phenanthrol, and the novel conjugate 9-phenanthryl beta-D-glucopyranoside. Identification of the compounds was based on their UV absorption, mass, and nuclear magnetic resonance spectra. Since lignin peroxidase was not detected in the culture medium, these results suggest the involvement of monooxygenase and epoxide hydrolase activity in the initial oxidation and hydration of phenanthrene by P. chrysosporium.     

Metabolism of phenanthrene by Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium metabolized phenanthrene when it was grown for 7 days at 37 degrees C in a medium containing malt extract, D-glucose, D-maltose, yeast extract, and Tween 80. After cultures were grown with [9-14C]phenanthrene, radioactive metabolites were extracted from the medium with ethyl acetate, separated by high-performance liquid chromatography, and detected by liquid scintillation counting. Metabolites from cultures grown with unlabeled phenanthrene were identified as phenanthrene trans-9,10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol, 9-phenanthrol, 3-phenanthrol, 4-phenanthrol, and the novel conjugate 9-phenanthryl beta-D-glucopyranoside. Identification of the compounds was based on their UV absorption, mass, and nuclear magnetic resonance spectra. Since lignin peroxidase was not detected in the culture medium, these results suggest the involvement of monooxygenase and epoxide hydrolase activity in the initial oxidation and hydration of phenanthrene by P. chrysosporium.     

Aerobic biodegradation of 2,2'-dithiodibenzoic acid produced from dibenzothiophene metabolites

Dibenzothiophene is a sulfur heterocycle found in crude oils and coal. The biodegradation of dibenzothiophene through the Kodama pathway by Pseudomonas sp. strain BT1d leads to the formation of three disulfides: 2-oxo-2-(2-thiophenyl)ethanoic acid disulfide, 2-oxo-2-(2-thiophenyl)ethanoic acid-2-benzoic acid disulfide, and 2,2'-dithiodibenzoic acid. When provided as the carbon and sulfur source in liquid medium, 2,2'-dithiodibenzoic acid was degraded by soil enrichment cultures. Two bacterial isolates, designated strains RM1 and RM6, degraded 2,2'-dithiodibenzoic acid when combined in the medium. Isolate RM6 was found to have an absolute requirement for vitamin B12, and it degraded 2,2'-dithiodibenzoic acid in pure culture when the medium was supplemented with this vitamin. Isolate RM6 also degraded 2,2'-dithiodibenzoic acid in medium containing sterilized supernatants from cultures of isolate RM1 grown on glucose or benzoate. Isolate RM6 was identified as a member of the genus Variovorax using the Biolog system and 16S rRNA gene analysis. Although the mechanism of disulfide metabolism could not be determined, benzoic acid was detected as a transient metabolite of 2,2'-dithiodibenzoic acid biodegradation by Variovorax sp. strain RM6. In pure culture, this isolate mineralized 2,2'-dithiodibenzoic acid, releasing 59% of the carbon as carbon dioxide and 88% of the sulfur as sulfate.     

Effect of Intracellular Concentration of Vitamin B6 on Tryptophanase Activity in Escherichia coli

The intracellular concentration of vitamin B6 (B6) of a wild type strain (WG1) of Escherichia coli B remained almost constant (0.25 to 0.35 nmol per mg cells) when different amounts of B6 were extracellularly added. However, B6-requiring mutants were strongly affected by extracellular B6.

Activities of tryptophanase in a B6-requiring mutant, strain WG3, grown under various conditions were measured. A clear correlation between intracellular B6 concentration and the ratio of holo-tryptophanase activity (i.e., holo-activity/total activity) was observed.

Tryptophanase of strain WG3 grown under B6-deficient conditions and the enzyme of strain WG1 were purified and their properties compared. The purified enzymes of both strains WG3 and WG1 showed similar characteristics, but a difference was observed in the antigen activities.     

Identification of cyclic intermediates in Azorhizobium caulinodans nicotinate catabolism.

In wild-type Azorhizobium caulinodans ORS571, nicotinate served both as anabolic substrate for NAD+ production and as catabolic substrate for use as the N source. Catabolic enzyme activities were greatest from cultures grown with nicotinate as the N source and least when cultures were grown with ammonium as the N source. Vector insertion mutants unable to catabolize nicotinate (nic::Vi mutants) still required micromolar quantities of this compound for growth. Therefore, A. caulinodans wild type is NAD+ auxotrophic. As the first two intermediates in A. caulinodans nicotinate catabolism, two cyclic compounds, 6-hydroxynicotinate and 1,4,5,6-tetrahydro-6-oxonicotinate, were identified. These compounds were purified from the growth medium of strain 61009 (a nic::Vi mutant) by high-performance liquid chromatography; their identities were subsequently confirmed by UV absorbance, nuclear magnetic resonance, and mass spectra. The conversion of 1 mol of nicotinate to 6-hydroxynicotinate consumed 0.5 mol of O2. From 18O isotopic incorporation experiments, water was the hydroxyl-equivalent source. A nicotinate hydroxylase activity proved to be cell wall-membrane associated; this activity served as direct electron donor (not indirect via NADP+) to O2 via membrane electron transport. These catabolic reactions have not previously been witnessed together in the same organism. A. caulinodans nicotinate catabolism seems coupled to N2 fixation, although the explicit mechanism of this coupling remains to be determined.     

Nutritionally variant streptococci from patients with endocarditis: growth parameters in a semisynthetic medium and demonstration of a chromophore.

Nutritionally variant streptococci have been characterized in the past by their growth as satellite colonies and by their nutrient requirements of cysteine or vitamin B6 for growth in complex media. To further understand the growth characteristics of these strains, we studied fresh isolates from patients with endocarditis by using chemically defined medium enriched with 2% Todd-Hewitt dialysate. Under anaerobic conditions, growth yields of the strains in this medium were comparable to those obtained from a complex medium supplemented with vitamin B6, whereas under aerobic conditions, most of the strains had higher growth yields in the semisynthetic medium. Furthermore, the requirement for cysteine and vitamin B6 in the semisynthetic medium was no greater than that of other Streptococcus species. Electron microscopic studies demonstrated normal cell wall structures in organisms grown in the semisynthetic medium as compared with abnormal and irregular cell wall thickening in organisms grown in supplemented complex medium. Finally, these strains appeared to contain a common component when grown in the semisynthetic medium as demonstrated by the appearance of a chromophore after boiling the bacteria at pH 2. Therefore, the demonstration of a medium which permits adequate growth with a normal ultrastructure of nutritionally variant streptococci will permit the further study of this group of important streptococci.     

Direct measurement of vitamin K-dependent enzymes in various isolated and cultured tumor and non-tumor cells

A modification of the assay for vitamin K-dependent carboxylase is described with which the enzyme could be detected in relatively low amounts of cells (n = 10⁶). Using this assay, we could demonstrate vitamin K-dependent carboxylase activity in hepatocytes, renal tubular cells, osteoblasts, endothelial cells and macrophages, but not in lymphocytes or platelets. The cultured tumor cells UMR-106, B16 and 5583 also contained vitamin K-dependent carboxylase activity. Vitamin K epoxide reductase activity was demonstrated only in cells where vitamin K-dependent carboxylase activity was present. The tumor cells possessed remarkably less K epoxide reductase activity than the normal cells. When cells were cultured in medium containing warfarin, the K epoxide reductase activity was found to be decreased and the amount of non-carboxylated precursor protein and increased, suggesting an analogous vitamin K mechanism as in liver.     

Glucose-stimulated acidogenesis by Streptomyces peucetius

Cultures of Streptomyces peucetius often were observed to divert spontaneously from the production of anthracycline antibiotics to the excretion of acidic metabolites, whether grown in a defined medium or a nutrient-rich medium. The predominant excreted acidic metabolites were determined by high-performance liquid chromatography to be pyruvate and 2-oxoglutarate. The acidification occurred at high frequencies when glucose was the primary carbon source in a defined medium, at intermediate frequencies when fructose was the primary carbon source, and did not occur when either maltose, starch, or dextrin were used as primary carbon sources.     

Liquid Nitrogen Preservation of Saccharomyces carlsbergensis and its Use in a Rapid Biological Assay of Vitamin B6 (Pyridoxine)

Growth medium as well as freezing menstruum greatly influenced the recovery of Saccharomyces carlsbergensis when it was quickly frozen in liquid nitrogen at - 196 C and quickly thawed at 40 C. Nearly 90% recovery in viability was obtained when S. carlsbergensis was grown in Trypticase Soy Broth and frozen in vitamin B(6) basal assay medium. The growth phase of S. carlsbergensis also influenced recovery after freezing. When S. carlsbergensis was grown in Trypticase Soy Broth and frozen in the broth at the logarithmic-growth phase, only 7% viability was retained; the recovery rate increased to 81% when the culture was frozen in the maximal stationary phase. To have the least possible lag period of growth after thawing, a technique called growth-phase conditioning was introduced. After 1 hr of growth-phase conditioning, S. carlsbergensis was clearly out of lag phase, and budding was observed. A vitamin B(6) microbiological assay with a 6-hr incubation period and with the use of liquid nitrogen-frozen S. carlsbergensis is described.     

Mechanism of thiamine-induced respiratory deficiency in Saccharomyces carlsbergensis.

Cells of Saccharomyces carlsbergensis 4228 grown aerobically with added thiamine (1 microgram . ml-1) in a vitamin B6-free medium contained no detectable heme precursors, such as delta-aminolevulinate, coproporphyrin III, or protoporphyrin IX. The deficiency in heme precursors in the thiamine-grown cells was accompanied by previously reported phenomena, i.e., growth depression, vitamin B6 deficiency, and respiratory deficiency due to a marked decrease in the activities of heme-containing enzymes and cytochrome level (I. Nakamura et al., FEBS Lett. 62: 354-358, 1976). It has been reported that all of the effects of thiamine are abolished by adding pyridoxine to the medium. delta-Aminolevulinate was found to have quite similar effects to those of pyridoxine, except that growth was partially improved by delta-aminolevulinate, whereas it was fully restored by pyridoxine. Incubation of the thiamine-grown cells with delta-aminolevulinate resulted in the appearance of the heme precursors and the heme-containing enzymes. Consistent with the lowered amount of vitamin B6, the thiamine-grown cells had a lowered activity of delta-aminolevulinate synthase, a pyridoxal phosphate-dependent enzyme. Not only the holoenzyme activity but also the apoenzyme activity was very low in these cells. These results indicate that the thiamine-induced vitamin B6 deficiency brings about the decrease in delta-aminolevulinate synthase activity, which leads to heme deficiency and therefore to respiratory deficiency.     

Biofilm-specific cross-species induction of antimicrobial compounds in bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10(T) and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10(T) and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.