Degradation of caffeine and related methylxanthines bySerratia marcescens isolated from soil under coffee cultivation

A strain of Serratia marcescens showing the ability to degrade caffeine and other methylxanthines was isolated from soil under coffee cultivation. Growth was observed only with xanthines methylated at the 7 position (caffeine, 1,3,7-dimethylxanthine; paraxanthine, 1,7-dimethylxanthine; theobromine, 3,7-dimethylxanthine and 7-methylxanthine). Paraxanthine and theobromine were released in liquid medium when caffeine was used as the sole source of carbon and nitrogen. When paraxanthine or theobromine were used, 3-methylxanthine, 7-methylxanthine, and xanthine were detected in the liquid medium. Serratia marcescens did not grow with theophylline (1,3-dimethylxanthine), 1-methylxanthine, and 3-methylxanthine, and poor growth was observed with xanthine. Methyluric acid formation from methylxanthines was tested in cell-free extracts by measuring dehydrogenase reduction of tetrazolium salt in native-polyacrylamide gel electrophoresis gel. Activity was observed for all methylxanthines, even those with which no bacterial growth was observed. Our results suggest that in this strain of S. marcescens caffeine is degraded to theobromine (3,7-dimethylxanthine) and/or paraxanthine (1,7-dimethylxanthine), and subsequently to 7-methylxanthine and xanthine. Methyluric acid formation could not be confirmed. Correspondence to: Paulo Mazzafera.     

Degradation of lawsone by Pseudomonas putida L2

From humus obtained from Stuttgart, a bacterium was isolated with lawsone (2-hydroxy-1,4-naphthoquinone) as selective source of carbon. This bacterium is capable of utilizing lawsone as sole source of carbon and energy. Morphological and physiological characteristics of the bacterium were examined and it was identified as a strain of Pseudomonas putida. The organism is referred to as Pseudomonas putida L2. The degradation of lawsone by Pseudomonas putida L2 was investigated. Salicylic acid and catechol were isolated and identified as metabolites. In lawsone-induced cells of Pseudomonas putida L2, salicylic acid is converted to catechol by salicylate 1-monooxygenase. Catechol 1,2-dioxygenase catalyses ortho-fission of catechol which is then metabolized via the beta-ketoadipate pathway. Formation of cis,cis-muconate and beta-ketoadipate was demonstrated by enzyme assays. Salicylate 1-monooxygenase and catechol 1,2-dioxygenase are induced sequentially. The enzymes of the beta-ketoadipate pathway are also inducible. Naphthoquinone hydroxylase, however, was demonstrated in induced and non-induced cells. This constitutive enzyme enables Pseudomonas putida L2 to degrade various 1,4-naphthoquinones in experiments with resting cells.     

Simultaneous determination of 16 purine derivatives in urinary calculi by gradient reversed-phase high-performance liquid chromatography with UV detection

A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection has been developed for the analysis of purines in urinary calculi. The method using gradient of methanol concentration and pH was able to separate 16 compounds: uric acid, 2,8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol and oxypurinol as well as 10 methyl derivatives of uric acid or xanthine (1-, 3-, 7- and 9-methyluric acid, 1,3-, 1,7- and 3,7-dimethyluric acid, 1-, 3- and 7-methylxanthine). Limits of detection for individual compounds ranged from 0.006 to 0.035 mg purine/g of the stone weight and precision (CV%) was 0.5-2.4%. The method enabled us to detect in human uric acid stones admixtures of nine other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methylated purines (1-, 3- and 7-methyluric acid, 1,3-dimethyluric acid, 3- and 7-methylxanthine) originating from the metabolism of methylxanthines (caffeine, theophylline and theobromine). The method allows simultaneous quantitation of all known purine constituents of urinary stones, including methylated purines, and may be used as a reference one for diagnosing disorders of purine metabolism and research on the pathogenesis of urolithiasis.     

Two Distinct Pathways for Metabolism of Theophylline and Caffeine Are Coexpressed in Pseudomonas putida CBB5

Pseudomonas putida CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1- and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1- and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1- and 3-methylxanthines to 1,3-dimethyluric acid and 1- and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methylxanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria.Caffeine (1,3,7-trimethylxanthine) and related methylxanthines are widely distributed in many plant species. Caffeine is also a major human dietary ingredient that can be found in common beverages and food products, such as coffee, tea, and chocolates. In pharmaceuticals, caffeine is used generally as a cardiac, neurological, and respiratory stimulant, as well as a diuretic (3). Hence, caffeine and related methylxanthines enter soil and water easily through decomposed plant materials and other means, such as effluents from coffee- and tea-processing facilities. Therefore, it is not surprising that microorganisms capable of degrading caffeine have been isolated from various natural environments, with or without enrichment procedures (3, 10). Bacteria use oxidative and N-demethylating pathways for catabolism of caffeine. Oxidation of caffeine by a Rhodococcus sp.-Klebsiella sp. mixed-culture consortium at the C-8 position to form 1,3,7-trimethyluric acid (TMU) has been reported (8). An 85-kDa, flavin-containing caffeine oxidase was purified from this consortium (9). Also, Mohapatra et al. (12) purified a 65-kDa caffeine oxidase from Alcaligenes sp. strain CF8. Cells of a caffeine-degrading Pseudomonas putida strain (ATCC 700097) isolated from domestic wastewater (13) showed a fourfold increase in a cytochrome P450 absorption spectrum signal compared to cells grown on glucose. Recently, we reported a novel non-NAD(P)⁺-dependent heterotrimeric caffeine dehydrogenase from Pseudomonas sp. strain CBB1 (20). This enzyme oxidized caffeine to TMU stoichiometrically and hydrolytically, without producing hydrogen peroxide. Further metabolism of TMU has not been elucidated.Several caffeine-degrading bacteria metabolize caffeine via the N-demethylating pathway and produce theobromine (3,7-dimethylxanthine) or paraxanthine (1,7-dimethylxanthine) as the initial product. Theophylline (1,3-dimethylxanthine) has not been reported to be a metabolite in bacterial degradation of caffeine. Subsequent N demethylation of theobromine or paraxanthine to xanthine is via 7-methyxanthine. Xanthine is further oxidized to uric acid by xanthine dehydrogenase/oxidase (3, 10). Although the identities of metabolites and the sequence of metabolite formation for caffeine N demethylation are well established, there is very little information on the number and nature of N-demethylases involved in this pathway.The lack of adequate information on the metabolism and enzymology of theophylline, caffeine, and related methylxanthines prompted us to investigate the degradation of these compounds in detail. We isolated a unique caffeine-degrading bacterium, P. putida CBB5, from soil via enrichment with caffeine as the sole source of carbon and nitrogen. Here we describe a detailed study of the metabolism of theophylline, caffeine, and related di- and monomethylxanthines by CBB5. Our results indicate that CBB5 initially N demethylated caffeine to produce theobromine (major product) and paraxanthine (minor product) before the pathways converged to 7-methylxanthine and xanthine. Surprisingly, CBB5 was also capable of utilizing theophylline as a sole carbon and nitrogen source. CBB5 N demethylated theophylline to 1-methylxanthine and 3-methylxanthine, which were further N demethylated to xanthine. Theophylline N-demethylase activity was detected in cell extracts prepared from theophylline-grown CBB5 cells. 1-Methylxanthine and 3-methylxanthine were detected as products of this NAD(P)H-dependent reaction. To our knowledge, this is the first report of a theophylline degradation pathway in bacteria and coexpression of distinct caffeine and theophylline degradation pathways.     

一株甲醛降解菌的筛选及降解特性的研究

【目的】以甲醛为唯一碳源与能源,以期从印染厂采集的活性污泥中筛选出快速降解甲醛的菌株。【方法】采用传统微生物纯培养方法和形态学特征、生理生化试验,结合16S rRNA基因序列分析以及甲醛关键脱氢酶(FDH)对筛选的菌株W1进行系统研究,并利用正交设计法研究不同因素处理对菌株W1降解甲醛特性的影响。【结果】分类结果显示鉴定菌株W1属于恶臭假单胞菌(Pseudomonas putida),通过单因素试验和正交试验考察培养条件对菌株降解甲醛的影响,得出菌株W1降解甲醛的最适条件为:甲醛浓度为500 mg/L,温度30°C,pH 6.0,摇床转速为200 r/min,接种量为3%。【结论】在最适条件下菌株W1具有较强的降解甲醛能力,在24 h其甲醛降解率达98%。     

A common pathway of sulfide oxidation by sulfate-reducing bacteria

Abstract Pseudomonas putida strain DMB capable of growing on 3,4-dimethylbenzoic acid as the only C and energy source was isolated by enrichment techniques. It does not utilize for growth or cooxidize the other dimethylbenzoate isomers tested. 3,4-Dimethylsalicylic acid, 3,4-dimethylphenol and 3,4-dimethylcatechol were isolated and identified by nuclear magnetic resonance and mass spectra in the reaction mixture of P. putida washed cells. The detection of the two first metabolites suggests that the initial step in the degradation of 3,4-dimethylbenzoic acid is the formation of 3,4-dimethylcyclohexa-3,5-diene-1, 2-diol-1-carboxylic acid which underwent an acid-catalyzed dehydration yielding 3,4-dimethylsalicylic acid and 3,4-dimethylphenol. Further degradation proceeds through 3,4-dimethylcatechol via the meta pathway.     

Dimerization of N-methyltransferases involved in caffeine biosynthesis

Caffeine is synthesized from the precursor xanthosine through three methylation and one nucleoside removal steps. Methylation is catalyzed by N-methyltransferases, designated as CaXMT1, CaMXMT1 and CaDXMT1, which, respectively, convert xanthosine into 7-methylxanthosine, 7-methylxanthine into 3,7-dimethylxanthine, and 3,7-dimethylxanthine into 1,3,7-trimethylxanthine (caffeine). In the present study, we examined their cytological and biochemical properties using fusion proteins with fluorescent proteins. All three enzymes were found to localize in cytosol as visualized by green fluorescence protein fusions. The possibility of dimer formation among these enzyme proteins was examined in vivo by transient expression of bimolecular fluorescence complementation of yellow fluorescent protein (YFP) using onion epidermal cell layers. Results showed that each enzyme protein formed a homo-dimer in cytosol as seen by a clear reconstituted YFP fluorescence. In addition, each enzyme also formed a hetero-dimer with each of the other two enzymes in cytosol. The biological significance of dimerization among structurally resembling methyltransferases involved in caffeine biosynthesis is discussed.     

Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene.

The well-characterized plasmid-encoded naphthalene degradation pathway in Pseudomonas putida PpG7(NAH7) was used to investigate the role of the NAH plasmid-encoded pathway in mineralizing phenanthrene and anthracene. Three Pseudomonas strains, designated 5R, DFC49, and DFC50, were recovered from a polynuclear aromatic hydrocarbon-degrading inoculum developed from a manufactured gas plant soil slurry reactor. Plasmids pKA1, pKA2, and pKA3, approximately 100 kb in size, were isolated from these strains and characterized. These plasmids have homologous regions of upper and lower NAH7 plasmid catabolic genes. By conjugation experiments, these plasmids, including NAH7, have been shown to encode the genotype for mineralization of [9-14C]phenanthrene and [U-14C]anthracene, as well as [1-14C]naphthalene. One strain, Pseudomonas fluorescens 5RL, which has the complete lower pathway inactivated by transposon insertion in nahG, accumulated a metabolite from phenanthrene and anthracene degradation. This is the first direct evidence to indicate that the NAH plasmid-encoded catabolic genes are involved in degradation of polynuclear aromatic hydrocarbons other than naphthalene.     

Plasmid-determined cadmium resistance in Pseudomonas putida GAM-1 isolated from soil.

Cadmium-resistant Pseudomonas putida GAM-1, which was able to grow in concentrations of CdCl2 as high as 7 mM, was isolated from soil in a rice paddy. This bacterium harbored a DNA plasmid of about 52 kilobases. The plasmid (pGU100) transformed Escherichia coli C600 to cadmium resistance. A cadmium-resistant transformant of E. coli C600 contained a plasmid corresponding to that seen in P. putida GAM-1. The transformant did not take up cadmium as well as P. putida GAM-1 did.     

Analogs of caffeine: antagonists with selectivity for A2 adenosine receptors

Several analogs of caffeine have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. Among these analogs, 1-propargyl-3,7-dimethylxanthine was about 4- to 7-fold and 7-propyl-1,3-dimethylxanthine about 3- to 4-fold more potent than caffeine at A2 receptors of PC12 cells and platelets. At A1 receptors of fat cells, both compounds were about 2-fold less potent than caffeine. These caffeine analogs have an A1/A2 selectivity ratio of about 10-20 and are the first selective A2 receptor antagonists yet reported. The results may provide the basis for the further development of highly potent and highly selective A2 adenosine receptor antagonists.     

Isolation and characterization of a unicellular manganese-oxidizing bacterium from a freshwater lake in northwestern Russia

A unicellular manganese-oxidizing bacterium (strain L7), isolated from Lake Ladoga, is identified as "Siderocapsa" sp. according to its morphology. However, this bacterium belongs to the phylogenetic cluster of Pseudomonas putida. The physiological characteristics (utilization of sugars, polyatomic alcohols, organic acids, and phenolic substrates as carbon and energy sources) also indicate the similarity of strain L7 to representatives of the genus Pseudomonas. The growing culture oxidizes Mn(II); the rate of oxidation depends on the type of added organic substrate. Carbonate requirement for this process indicates mixotrophic metabolism. The relatedness of the isolated bacterium to the representatives of the genus Pseudonomas and their phenotypic similarity provide a basis for considering strain L7 not as "Siderocapsa" sp., but as a new species, Pseudomonas siderocapsa sp. nov., of the P. putida cluster.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds

The caffeine-sensitive Ca2+ release pathway in skeletal muscle was identified and characterized by studying the release of 45Ca2+ from heavy sarcoplasmic reticulum (SR) vesicles and by incorporating the vesicles or the purified Ca2+ release channel protein complex into planar lipid bilayers. First-order rate constants for 45Ca2+ efflux of 1 s-1 were obtained in the presence of 1-10 microM free Ca2+ or 2 X 10(-9) M free Ca2+ plus 20 mM caffeine. Caffeine- and Ca2+-induced 45Ca2+ release were potentiated by ATP and Mg.ATP, and were both inhibited by Mg2+. Dimethylxanthines were similarly (3,9-dimethylxanthine) or more (1,7-, 1,3-, and 3,7-dimethylxanthine) effective than caffeine in increasing the 45Ca2+ efflux rate. 1,9-Dimethylxanthine and 1,3-dimethyluracil (which lacks the imidazole ring) did not appreciably stimulate 45Ca2+ efflux. Recordings of calcium ion currents through single channels showed that the Ca2+- and ATP-gated SR Ca2+ release channel is activated by addition of caffeine to the cis (cytoplasmic) and not the trans (lumenal) side of the channel in the bilayer. The single channel measurements further revealed that caffeine activated Ca2+ release by increasing the number and duration of open channel events without a change of unit conductance (107 pS in 50 mM Ca2+ trans). These results suggest that caffeine exerts its Ca2+ releasing effects in muscle by activating the high-conductance, ligand-gated Ca2+ release channel of sarcoplasmic reticulum.     

Carbon and hydrogen stable isotope fractionation during aerobic bacterial degradation of aromatic hydrocarbons

13C/(12)C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.     

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene.

A bacterium, Pseudomonas sp. strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated. The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite. Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp. clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium. Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene. Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp. clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source. All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives. Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found.     

D-泛酸高产菌株C21的筛选及鉴定

从10个植物根际土样中分离出315株根际微生物,然后采用泛酸测定平板初筛和摇瓶复筛的方法,从中筛选到7株D-泛酸高产菌株,其中从菜豆根际土壤分离到的菌株C21的D-泛酸产量最高,达到24.1mg/L。最后通过形态和生理生化特性以及16SrDNA鉴定,确定菌株C21为恶臭假单胞菌(Pseudomonas putida)。     

Microbial Transformation of Terpenoids. I. Indentification of Metabolites Produced by a Pseudomonad from Citronellal and Citral

A bacterium capable of utilizing citronellal or citral as the sole source of carbon and energy has been isolated from soil by the enrichment culture technique. It metabolizes citronellal to citronellic acid (65%), citronellol (0.6%), dihydrocitronellol (0.6%), menthol (0.75%), and 3,7-dimethyl-1,7-octane diol (1.7%). The metabolites of citral were geranic acid (62%), 6-methyl-5-heptanoic acid (0.5%), 3-methyl-2-butenoic acid (1%), and 1-hydroxy-3, 7-dimethyl-6-octen-2-one (0.75%).     

Microbial metabolism of quinoline and related compounds. II. Degradation of quinoline by Pseudomonas fluorescens 3, Pseudomonas putida 86 and Rhodococcus spec. B1

Quinoline catabolism was investigated with different bacterial strains, able to use quinoline as sole source of carbon, nitrogen and energy. Some degradation products of quinoline were isolated from the culture fluids and identified. With Pseudomonas fluorescens and Pseudomonas putida we found 2-oxo-1,2-dihydroquinoline, 8-hydroxy-2-oxo-1,2-dihydroquinoline, 8-hydroxycoumarin and 2,3-dihydroxyphenylpropionic acid as intermediates. With a Rhodococcus strain 2-oxo-1,2-dihydroquinoline, 6-hydroxy-2-oxo-1,2-dihydroquinoline, a red meta-cleavage product and a blue fluorescent compound were isolated. The red compound was identified as 5-hydroxy-6-(3-carboxy-3-oxopropenyl)-1H-2-pyridone. From this the blue fluorescent azacoumarin 2H-pyrano-2-one-[3,2b]-5H-6-pyridone is formed by chemical decomposition. Therefore it can be considered a by-product of quinoline-degradation in Rhodococcus spec. With the present results two different degradation pathways for quinoline in different microorganisms are proposed.     

Factors influencing the ability of Pseudomonas putida strains epI and II to degrade the organophosphate ethoprophos

Two strains of Pseudomonas putida (epI and epII), isolated previously from ethoprophos-treated soil, were able to degrade ethoprophos (10 mg 1(-1)) in a mineral salts medium plus nitrogen (MSMN) in less than 50 h with a concurrent population growth. Addition of glucose or succinate to MSMN did not influence the degrading ability of Ps. putida epI, but increased the lag phase before rapid degradation commenced with Ps. putida epII. The degrading ability of the two isolates was lost when the pesticide provided the sole source of phosphorus. Degradation of ethoprophos was most rapid when bacterial cultures were incubated at 25 and 37 degrees C. Pseudomonas putida epI was capable of completely degrading ethoprophos at a slow rate at 5 degrees C, compared with Ps. putida epII which could not completely degrade ethoprophos at the same time. Pseudomonas putida epI was capable of degrading ethoprophos when only 60 cells ml(-1) were used as initial inoculum. In contrast, Ps. putida epII was able to totally degrade ethoprophos when inoculum densities of 600 cells ml(-1) or higher were used. In general, longer lag phases accompanied the lower inoculum levels. Both isolates rapidly degraded ethoprophos in MSMN at pHs ranging from 5.5 to 7.6, but not at pH 5 or below.     

Biotransformation of limonene by Pseudomonas putida

From a study of three fungal and 15 bacterial strains, it was observed that Pseudomonas putida MTCC 1072 oxidized limonene with the highest efficiency of. Fermentation of limonene by P. putida MTCC 1072 was conducted for 120 h at 30 degrees C at a fixed pH of 5.0. Major bioconversion products were isolated and characterized by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy, and by elemental analysis. The bioconversion products were identified as perillyl alcohol and p-menth-1-ene-6,8-diol, and under optimum conditions the yields were 36% and 44%, respectively (a rate kinetic model indicated corresponding limiting yields of 44% and 56%). No further degradation of the products was observed using this bacteria.