The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum-sensing receptor

Many bacteria use quorum sensing (QS) as an intercellular signaling mechanism to regulate gene expression in local populations. Plant and algal hosts, in turn, secrete compounds that mimic bacterial QS signals, allowing these hosts to manipulate QS-regulated gene expression in bacteria. Lumichrome, a derivative of the vitamin riboflavin, was purified and chemically identified from culture filtrates of the alga Chlamydomonas as a QS signal-mimic compound capable of stimulating the Pseudomonas aeruginosa LasR QS receptor. LasR normally recognizes the N-acyl homoserine lactone (AHL) signal, N-3-oxo-dodecanoyl homoserine lactone. Authentic lumichrome and riboflavin stimulated the LasR receptor in bioassays and lumichrome activated LasR in gel shift experiments. Amino acid substitutions in LasR residues required for AHL binding altered responses to both AHLs and lumichrome or riboflavin. These results and docking studies indicate that the AHL binding pocket of LasR recognizes both AHLs and the structurally dissimilar lumichrome or riboflavin. Bacteria, plants, and algae commonly secrete riboflavin or lumichrome, raising the possibility that these compounds could serve as either QS signals or as interkingdom signal mimics capable of manipulating QS in bacteria with a LasR-like receptor.     

Dodecins: a family of lumichrome binding proteins

Dodecin is a small dodecameric flavoprotein from Halobacterium salinarum that contains two flavins stacked between two tryptophan residues to form an aromatic tetrade. The functional properties of heterologously expressed dodecin were investigated by fluorescence spectroscopy, which allowed the determination of dissociation constants for a number of protein-ligand complexes. The values obtained were in the nanomolar to micromolar range and correlate positively with the ligand size. These data were supplemented by X-ray crystal structures of the apododecin and holocomplexes with lumichrome, lumiflavin, riboflavin and FMN at resolutions between 1.55 to 1.95 A to unravel a gating mechanism as the structural basis for the preferential binding of the small ligands lumichrome and lumiflavin. The detailed analysis of the dodecin manifold for preferential binding of lumichrome and lumiflavin provides insight on a subatom level into a protein's strategy to gain selectivity for low molecular mass compounds by steric restrictions rather than specific interactions. Investigations on the ligand composition of a wild-type dodecin crystal (1.32 A resolution) support conclusions of functional and structural investigations on heterologously expressed dodecin, and strongly suggest that lumichrome, a molecule associated with the flavin metabolism, is a ligand of dodecin in vivo. Studies on mutant protein and a Halorhodospira halophila homologue spread the idea of a lumichrome binding system as a possible "waste"-trapping device, widely distributed in prokaryotes.     

In vivo metabolism of leukotriene C4 in man: urinary excretion of leukotriene E4

Five - 20 nmoles of [5,6,8,9,11,12,14,15-3H8]leukotriene C4 was injected into three male volunteers. Forty-eight percent of the administered 3H was recovered from urine and 8% from feces, within a 72 hr period. Of the total urinary radioactivity 44% was excreted during the first hour after injection. This activity was mainly found in one compound, designated "I". The radioactivity excreted into urine later than one hour after injection, consisted partly of Compound I and two additional components, and partly of polar, non-volatile material. Compound I was identified as leukotriene E4 by UV-spectroscopy and cochromatographies in three high performance liquid chromatography systems with synthetic reference compounds. A total of 13% of administered radioactivity was excreted in urine as leukotriene E4.     

MPTP in mice: treatment, distribution and possible source of contamination

Mice were treated daily with [3H]MPTP (30 mg/kg, 1 uCi, s.c.) for 1, 3, and 10 days to determine the fate and localization of tritiated compounds. An untreated mouse was housed either in the same cage ("cage-mate control") or in an adjacent cage separated by mesh-wire ("near-neighbor control"). The radioactivity measured in blood, brain, liver, and remaining body of [3H]MPTP-treated mice was dependent on the total dose of the drug the animals received and did not vary with the type of tissue analyzed. Significant amounts of radioactivity were found in the tissues of the "cage-mate control" mice, but not of the "near-neighbor control" mice. The route of transmission appears to be through the urine, as the urine of [3H]MPTP-treated mice was highly radioactive after the drug injection. Only traces of radioactivity were found in their feces and there was no increase in the background radiation in the environment of the cages, indicating that the tritiated compounds were not exhaled. Proper disposal of urinary products of MPTP-treated animals is therefore necessary to reduce the risk of possible drug contamination in humans.     

Metabolic fate of radiolabeled prostaglandin D2 in a normal human male volunteer

50 microCi of [3H]prostaglandin D2 tracer (100 Ci/mmol) was infused intravenously into a normal human male volunteer. 75% of the infused radioactivity was excreted into the urine within 5 h. This urine was added to urine obtained from two mastocytosis patients with marked overproduction of prostaglandin D2. Radiolabeled prostaglandin D2 urinary metabolites were chromatographically isolated and purified and subsequently identified by gas chromatography-mass spectrometry. 25 metabolites were identified. 23 of these compounds comprising 37% of the recovered radioactivity had prostaglandin F-ring structures, and only two metabolites comprising 2.7% of the recovered radioactivity retained the prostaglandin D-ring structure. The single most abundant metabolite identified was 9,11-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid which was isolated in a tricyclic form as a result of formation of a lower side chain hemiketal followed by lactonization of the terminal carboxyl and the hemiketal hydroxyl. Different isomeric forms of several prostaglandin F-ring metabolites were identified. An isomer of prostaglandin F2 alpha was also excreted intact into the urine as a metabolite of prostaglandin D2. 15 PGF-ring compounds were treated with n-butylboronic acid and 13 failed to form a boronate derivative, suggesting that the orientation of the hydroxyl group at C-11 in these 13 metabolites is beta. This study documents that prostaglandin D2 is metabolized to prostaglandin F-ring metabolites in vivo in humans. These results also bring into question the accuracy of quantifying prostaglandin F2 alpha metabolites as a specific index of endogenous prostaglandin F2 alpha biosynthesis, as well as quantifying urinary prostaglandin F2 alpha as an accurate index of renal production of prostaglandin F2 alpha.     

Xylem transport and shoot accumulation of lumichrome, a newly recognized rhizobial signal, alters root respiration, stomatal conductance, leaf transpiration and photosynthetic rates in legumes and cereals

* Root respiration, stomatal conductance, leaf transpiration and photosynthetic rates were measured in phytotron and field-grown plants following the application of 5 or 10 nM lumichrome, 10 nM ABA (abscisic acid) and 10 ml of 0.2 OD600 infective rhizobial cells. * Providing soybean and cowpea roots with their respective homologous rhizobia and/or purified lumichrome increased the concentration of this molecule in xylem sap and leaf extracts. Relative to control, rhizobial inoculation and lumichrome application significantly increased root respiration in maize, decreased it in lupin, but had no effect on the other test species. * Applying either lumichrome (10 nM), infective rhizobial cells or ABA to roots of plants for 44 h in growth chambers altered leaf stomatal conductance and transpiration in cowpea, lupin, soybean, Bambara groundnut and maize, but not in pea or sorghum. Where stomatal conductance was increased by lumichrome application or rhizobial inoculation, it resulted in increased leaf transpiration relative to control plants. Treating roots of field plants of cowpea with this metabolite up to 63 d after planting showed decreased stomatal conductance, which affected CO2 intake and reduction by Rubisco. * The effect of rhizobial inoculation closely mirrored that of lumichrome application to roots, indicating that rhizobial effects on these physiological activities were most likely due to lumichrome released into the rhizosphere.     

The metabolism of labelled hexadecyl sulphate salts in the rat, dog and human.

The metabolic fate of [1-14-C]hexadecylsulphate and hexadecyl[35-S]sulphate, administered intravenously as the sodium and trimethylammonium salt to dogs and orally as the erythromycin salt to dogs, rats and humans, was studied. Studies with rats indicated that the compounds were well absorbed and rapidly excreted in the urine. However, after oral administration of the 14-C-and 35-S-labelled hexadecyl sulphate erythromycin salt to dogs, considerable amounts of radioactivity were excreted in the faeces as unmetabolized hexadecyl sulphate. Studies with two humans showed that orally administered erythromycin salt of [1-14C]hexadecyl sulphate was well absorbed in one person but poorly absorbed in the other. Radioactive metabolites in urine were separated by t.l.c. in two solvent systems. The main metabolite of hexadecyl sulphate in the dog, rat and human was identified as the sulphate ester of 4-hydroxybutyric acid. In addition, psi-[14-C]butyrolactone as a minor metabolic product of [1-14-C]hexadecyl sulphate was also isolated from the urine of rat, dog and man. However, there was still another metabolite in dog urine, which comprised about 20% of the total urinary radioactivity and carried both 14-C and 35-S labels. This metabolite was absent from rat urine. The metabolite in dog urine was isolated and subsequently identified by t.l.c. and g.l.c. and by isotope-dilution experiments as the sulphate ester of glycollic acid. Small amounts (about 5% of the total recovered radioactivity in excreta) of labelled glycollic acid sulphate were also found in human urine after ingestion of erythromycin [1-14-C]hexadecyl sulphate.     

Efficient Production of Lumichrome by Microbacterium sp. Strain TPU 3598

Lumichrome is a photodegradation product of riboflavin and is available as a photosensitizer and fluorescent dye. To develop new efficient methods of lumichrome production, we isolated bacterial strains with high lumichrome productivity from soil. The strain with highest productivity was identified as Microbacterium sp. strain TPU 3598. Since this strain inductively produced lumichrome when cultivated with riboflavin, we developed two different methods, a cultivation method and a resting cell method, for the production of large amounts of lumichrome using the strain. In the cultivation method, 2.4 g (9.9 mmol) of lumichrome was produced from 3.8 g (10.1 mmol) of riboflavin at the 500-ml scale (98% yield). The strain also produced 4.7 g (19.4 mmol) of lumichrome from 7.6 g (20.2 mmol) of riboflavin (96% yield) by addition of riboflavin during cultivation at the 500-ml scale. In the resting cell method, 20 g of cells (wet weight) in 100 ml of potassium phosphate buffer, pH 7.0, produced 2.4 g of lumichrome from 3.8 g of riboflavin (98% yield). Since the lumichrome production by these methods was carried out in suspension, the resulting lumichrome was easily purified from the cultivation medium or reaction mixture by centrifugation and crystallization. Thus, the biochemical methods we describe here are a significant improvement in terms of simplicity and yield over the existing chemical, photolytic, and other biochemical methods of lumichrome production.     

The metabolism of [14C]nicotine in the cat

The metabolism of [2'-(14)C]nicotine given as an intravenous injection in small doses to anaesthetized and unanaesthetized cats has been studied. A method is described for the quantitative determination of [(14)C]nicotine and [(14)C]cotinine in tissues and body fluids. Nanogram amounts of these compounds have been detected. After a single dose of 40mug. of [(14)C]nicotine/kg., 55% of the injected radioactivity was excreted in the urine within 24hr., but only 1% of this radioactivity was unchanged nicotine. [(14)C]Nicotine is metabolized extremely rapidly, [(14)C]cotinine appearing in the blood within 2.5min. of intravenous injection. [(14)C]Nicotine accumulates rapidly in the brain and 15min. after injection 90% of the radioactivity still represents [(14)C]nicotine. Metabolites of [(14)C]nicotine have been identified in liver and urine extracts. [(14)C]Nicotine-1'-oxide has been detected in both liver and urine.     

Steroid metabolism in the rabbit. Biliary and urinary excretion of metabolites of [4-14C]progesterone

1. [4-(14)C]Progesterone was administered intravenously to anaesthetized male and female New Zealand White rabbits as a single injection or as a 45-60min. infusion. 2. After a single dose about 60% of the radioactivity was recovered in 6hr., and twice as much radioactivity was present in bile as in urine. After infusion total recovery of radioactivity was only about 40% in 6hr., but the relative proportions of metabolites in bile and urine were about the same as after a single dose. 3. Bile and urine samples were hydrolysed successively by beta-glucuronidase, cold acid and hot acid. 4. In bile the major proportion of metabolites appeared in the glucuronide fraction; in urine beta-glucuronidase hydrolysis yielded the greatest amounts of ether-extractable radioactivity, but the greatest proportion of radioactivity could not be extracted by ether from an alkaline solution of the hydrolysed urine. 5. There was no apparent difference in the quantity or distribution of metabolites excreted by male and female animals.     

The rhizosphere signal molecule lumichrome alters seedling development in both legumes and cereals

The stimulatory role of lumichrome, a rhizosphere metabolite, was assessed on the growth of legume and cereal seedlings. At a very low nanomolar concentration (5 nm), lumichrome elicited growth promotion in cowpea, soybean, sorghum, millet and maize, but not in common bean, Bambara groundnut and Sudan grass. In soybean and cowpea only, 5 nm lumichrome caused early initiation of trifoliate leaf development, expansion in unifoliate and trifoliate leaves, increased stem elongation and, as a result, an increase in shoot and plant total biomass relative to control. Lumichrome (5 nm) also increased leaf area in maize and sorghum, and thus raised shoot and total biomass but there was no effect on the leaf area of the other cereals. Root growth was also stimulated in sorghum and millet by the supply of 5 nm lumichrome. By contrast, the application of a higher dose of lumichrome (50 nm) depressed development of unifoliate leaves in soybean, the second trifoliate leaf in cowpea, and shoot biomass in soybean. The 50 nm concentration also consistently decreased root development in cowpea and millet, but had no effect on the other species. These data show that lumichrome is a rhizosphere signal molecule that affects seedling development in both monocots and dicots.     

Uracil metabolism in golden hamster after irradiation.

The catabolism of uracil and the total balance of excreted radioactivity were studied in golden hamsters after a peroral application of 14C-uracil. Twenty-four hours after administration most of the radioactivity taken up appeared in expired carbon dioxide. The percent proportion of radioactivity in carbon dioxide was independent of the amount of uracil administered. On the other hand, the percentage of radioactivity excreted in urine depended on the amount of uracil taken up, high doses of the compound causing up to eight-fold increase in urine-excreted radioactivity. Most of the exogenously-administered uracil was catabolized within the first 5 hours. Irradiation had no substantial effect on the dynamics of uracil catabolism. Analysis of urine revealed that most urine-excreted radioactivity is in the form of uracil. On peroral application of high doses of uracil to irradiated hamsters, their urine was found to contain barbituric acid which originated from uracil.     

Urinary metabolites of polyamines in rats

Urinary metabolites of polyamines in rats were studied systematically by the intraperitoneal injection of radioactive polyamines. Urinary metabolites were fractionated into 4 fractions containing non-polar and acidic compounds, acidic and neutral ampholytes, basic ampholytes and polyamines. A large amount of radioactivity was detected in the fractions containing non-polar and acidic compounds and polyamines of urine of rats injected with radioactive putrescine, while in the case of the injection of radioactive spermidine or spermine a relatively large amount of radioactivity was found in the basic ampholyte fraction as well as in the polyamine fraction. Analysis of these fractions indicated that gamma-aminobutyric acid, N-monoacetylputrescine, 2(3)-hydroxyputrescine, putreanine, N-(3-aminopropyl)-4-aminobutyric acid (isoputreanine), spermic acid, N-(3-aminopropyl), N'-(2-carboxyethyl)-1,4-diaminobutane, and N-monoacetylspermidine A and B were excreted as urinary metabolites of the polyamines in addition to putrescine, spermidine and spermine.     

Photoirradiation products of flavin derivatives,and the effects of photooxidation on guanine

Photoirradiation in the presence of riboflavin led to guanine oxidation and the formation of imidazolone. Meanwhile, riboflavin itself was degraded by ultraviolet light A (UV-A) and visible light (VIS) radiation, and the end product was lumichrome. VIS radiation in the presence of riboflavin oxidized guanine similarly to UV-A radiation. Although UV-A radiation with lumichrome oxidized guanine, VIS radiation with lumichrome did not. Thus, UV-A radiation with riboflavin can oxidize guanine even if riboflavin is degraded to lumichrome. In contrast, following VIS radiation degradation of riboflavin to lumichrome, VIS radiation with riboflavin is hardly capable of oxidizing guanine. The consequences of riboflavin degradation and guanine photooxidation can be extended to flavin mononucleotide and flavin adenine dinucleotide. In addition, we report advanced synthesis; carboxymethylflavin was obtained by oxidation of formylmethylflavin with chlorite and hydrogen peroxide; lumichrome was obtained by heating of formylmethylflavin in 50% AcOH; lumiflavin was obtained by incubation of formylmethylflavin in 2 M NaOH, followed by isolation by step-by-step concentration.     

Reduction of lumichrome by the radical anions of CO2 and lipoamide

The uptake of reducing equivalents of X CO-2 by lumichrome in spectrophotometric titrations has been re-examined in the light of a recently reported extinction coefficient of 10 500 M-1 cm-1 at pH 6, which is in agreement with 10 270 +/- 100 M-1 cm-1 determined here. The average uptake was 1.8 +/- 0.1, independent of pH in the range 6.3-9.0. The major product appears to be a dihydro-alloxazine, which can be reoxidized quantitatively to lumichrome by X Br-2 radicals or by O2. As in the case of dihydroflavins, oxidation by O2 is biphasic. As in the case of flavins, a two electron reduction of lumichrome was also observed with the disulphide monoanion of lipoamide (LS X 2-), but that reduction does not go to 100 per cent yield. Contrary to our earlier conclusions, which were based on an erroneous extinction coefficient, the combination of lumichrome radicals (2 X LcH----HLc--LcH) was of relatively little (less than or equal to 20 per cent) importance, and the behaviour of lumichrome on treatment with reducing species was rather similar to that of flavins.     

Absorption and distribution of [1-14C]dolichol intubated into rats

[1-14C]Dolichol was mixed in vitro with sunflower seed oil and intubated into rats. Radioactivity began to appear in the blood at 3 h and peaked after about 6 h. The absorbed radioactivity was rapidly cleared from the blood. At 7.5 h postintubation two thirds of the radioactivity in the serum was associated with chylomicrons and about one quarter with the high density lipoproteins. At 12 h the proportion of the radioactivity in the chylomicrons had fallen to one third and that in the high density lipoproteins had increased to one half of the total. Less than 0.02% of the dose was found in the tissues after 12 h. Liver and blood each contained about one third of the total, with smaller amounts in the lungs and spleen. The heart, testes, brain, and kidneys contained only traces of radioactivity. After 12 h most of the radioactivity in the tissues and feces was present as [1-14C]dolichol. The radioactive compounds in the urine (about 0.05% of the dose) were more polar than [1-14C]dolichol as determined by thin-layer chromatography.     

Benzopyranones with retro-amide side chains as (inhibitory) beta-lactamase substrates

3-(N-Benzylcarbamoyl)-7-carboxy-3, 4-dihydro-2H-1-benzo-pyran-2-one and its 8-carboxy analogue have been synthesized and evaluated as potential (inhibitory) substrates of beta-lactam-recognizing enzymes. These compounds are bicyclic delta-lactones with retro-amide (with respect to classical beta-lactams) side chains. They were found to be comparably effective as substrates of typical class A, C and D beta-lactamases as analogous benzopyranones bearing 'normal' amide side chains. The new 8-carboxy derivative, however, formed a much more (1000-fold) tightly-bound acyl-enzyme with a class C beta-lactamase than did its 'normal' analogue, and thus provides a structural lead to new inhibitors of this class of beta-lactamase.     

Thin-layer chromatographic analysis of lumichrome, riboflavin and indole acetic acid in cell-free culture filtrate ofPsoralea nodule bacteria grown at different pH, salinity and temperature regimes

Using thin-layer chromatography, 16 bacterial isolates from root nodules of 8 differentPsoralea species were quantitatively assessed for their exudation of the metabolites lumichrome, riboflavin and IAA in response to pH, salinity and temperature. Our data showed that the bacterial strains tested differed in their levels of secretion of the three metabolites. For example, strain AS2 produced significantly greater amounts of lumichrome at both pH 5.1 and 8.1, while strains RT1 and PI produced more lumichrome per cell at only pH 8.1. Strains API and RP2 also produced more riboflavin at pH 5.1 than at pH 8.1; conversely strain RTl secreted more riboflavin at pH 8.1 than at pH 5.1. TwoP. repens strains (RP1 and RP2) isolated from very saline environments close to the Indian Ocean produced significant levels of lumichrome and riboflavin at both low and high salinity treatments. However, strains ACI and LI (fromP. aculeata andP. laxa) even produced greater amounts of lumichrome and riboflavin at higher salinity (i.e. 34.2 mM NaCl) and probably originated from naturally saline soils. In this study, high acidity and high temperature induced the synthesis and release of high levels of IAA by bacterial cells. In contrast, there was greater strain secretion of lumichrome at lower temperature (10°C) than at high temperature (30°C). The variations in the secretion of lumichrome, riboflavin and IAA by bacterial strains exposed to different pH, salinity and temperature regimes suggest that genes encoding these metabolites are regulated differently by the imposed environmental factors. The data from this study also suggest that natural changes of pH, salinity and/or temperature in plant rhizospheres could potentially elevate the concentrations of lumichrome, riboflavin and IAA in soils. An accumulation of these molecules in the rhizosphere would have consequences for ecosystem functioning as both lumichrome and riboflavin have been reported to act as developmental signals that affect species in all three plant, animal, and microbial kingdoms.     

A note on the metabolism of 5 alpha-androst-16-en-3-one in the young boar in vivo

The metabolism of plasma 5 alpha-androst-16-en-3-one (androstenone) was studied in two young boars weighing about 100 kg in which a single dose of tritiated androstenone was injected intravenously. The peripheral blood of one boar was continuously sampled for 6 h after injection; the total radioactivity per liter of plasma increased up to 14 min after the injection, and then declined rather slowly since plasma radioactivity was still measurable 7 days after injection. The metabolic clearance rate of androstenone was calculated to be about 80 000 liters per day. This quick disappearance of plasma androstenone was probably mainly due to storage in fatty tissue and, to a lesser extent, to catabolism into 5 alpha-androst-16-en-3 alpha-ol, 5 alpha-androst-16-en-3 beta-ol and particularly into unknown more polar compounds of which there were at least three. Radioactivity was mainly eliminated in the urine in the form of the same unknown polar compounds.     

Time course of distribution to tissues of 125I-somatomedin A injected into the rat

125I-somatomedin A (SMA) was injected iv into rats. Distribution studies in rats showed concentrations of radioactivity to be high in kidney and plasma, low in brain, and intermediate in other tissues. The concentration of total and trichloracetic acid (TCA) precipitable radioactivity in rat blood and tissues fell at rapid rate. Ninety per cent of the radioactivity was in the urine in 24 hr, and only 15% of urine radioactivity was TCA precipitable. The half-life of the radioactivity in TCA-precipitable fraction from blood and that from tissues were nearly identical (about 6 hr). In both liver and kidney, TCA-precipitable radioactivity was detected in membrane and/or organellar fraction and cytosol fraction. Sephadex G-200 chromatography at neutral PHY AT NEUTRAL PH of plasma after injection of 125I-SMA revealed 3 peaks of radioactivity in higher molecular weight region than purified SMA.