Degradation of catechin by Bradyrhizobium japonicum

Rhizobia utilize phenolic substances as sole carbonsource. Bradyrhizobium japonicum utilizescatechin, a unit of condensed tannin as carbonsource. To establish the degradative pathway ofcatechin, the products of catechin degradation wereisolated by paper chromatography and TLC andidentified by HPLC, UV, IR and NMR spectra. B.japonicum cleaves catechin through catechinoxygenase. Phloroglucinolcarboxylic acid andprotocatechuic acid were identified as the initialproducts of degradation. Phloroglucinolcarboxylicacid is further decarboxylated to phloroglucinolwhich is dehydroxylated to resorcinol. Resorcinolis hydroxylated to hydroxyquinol. Protocatechuicacid and hydroxyquinol undergo intradiol cleavagethrough protocatechuate 3,4-dioxygenase andhydroxyquinol 1,2-dioxygenase to form-carboxy cis, cis-muconic acidand maleylacetate respectively. The enzymes ofcatechin degradative pathway are inducible. Estimation of all the enzymes involved in thecatabolism of catechin reveals the existence of acatechin degradative pathway in B. japonicum.     

Utilization of catechin byRhizobium sp.

Rhizobium sp. isolated fromLeucaena leucocephala utilized catechin as sole carbon source. Optimum growth occurred at 2 to 5 mM. From replacement cultures containing catechin, phloroglucinolcarboxylic acid, phloroglucinol, resorcinol, protocatechuate, catechol and hydroxyquinol were separated by chromatography. Rothera's test confirmed theortho ring fission of the end products of catechin.     

Degradation of nitrocellulose by fungi

Three lignocellulolytic fungi, Trametes versicolor, Pleurotus ostreatus, and Coprinus cinereus, and two cellulolytic fungi Trichoderma reesei andChaetomium elatum were tested for their ability to degrade nitrocellulose. They were provided with different carbon and nitrogen sources in liquid cultures. Nitrocellulose (N content above 12%) was added as nitrogen source (in solution in acetone) alongside amino acids or as sole N source. Either starch or carboxy-methyl cellulose were provided as carbon sources. After 28 days of growth the highest decrease of nitrocellulose was observed with Chaetomium elatum when up to 43% was degraded in a medium containing nitrocellulose as the only nitrogen source. Coprinus cinereus caused a 37% decrease of nitrocellulose when provided with amino acids and starch as co-substrate. In cultures of Trametes versicolor, Pleurotus ostreatus andTrichoderma reesei, only 10%–22% decrease of nitrocellulose was measured in all media. In the presence of nitrocellulose with N content below 12% supplied as 3 mm pellets as the only carbon source, or with nitrocellulose with carboxy-methyl cellulose, the release of nitrite and nitrate from liquid cultures of Chaetomium elatum was measured. Between 6 and 9 days of growth in these media, an increase in both nitrite and nitrate was observed with a loss in weight of nitrocellulose up to 6% achieved after 34 days. The physical nature of the NC pellets may have reduced the rate of degradation in comparison with supplying NC in solution in the cultures.     

Degradation of tannic acid and purification and characterization of tannase from Enterococcus faecalis

Tannins, present in various foods, feeds and forages, have anti-nutritional activity; however, presence of tannase in microorganisms inhabiting rumen and gastrointestinal tract of animals results in detoxification of these tannins. The present investigation was carried out to study the degradation profile of tannins by Enterococcus faecalis and to purify tannase. E. faecalis was observed to degrade tannic acid (1.0% in minimal media) to gallic acid, pyrogallol and resorcinol. Tannase from E. faecalis was purified up to 18.7 folds, with a recovery of 41.7%, using ammonium sulphate precipitation, followed by DEAE-cellulose and Sephadex G-150. The 45 kDa protein had an optimum activity at 40 °C and pH 6.0 at substrate concentration of 0.25 mM methyl gallate.     

Acceptability of catechin and its oxidative condensation products to the rose aphid, Macrosiphum rosae

The rose aphid showed a significant level of nonpreference for concentrations of 0.3 mg/ml catechin and above, whether in a simple base diet of sucrose or a complex artificial diet containing in addition to other components a total amino acid content equivalent to that found in the sap pressed from the pedicels of rose-buds. Similar concentrations of catechin were found in the pressed sap from pedicels of nonpreferred buds in the field. A number of factors influenced absolute values for deterrence: increased concentrations of sucrose and especially amino acids lowered the deterrence of catechin; early instars showed nonpreference for low concentrations of catechin that were nondeterrent to the later instars. A possible explanation for such variation was provided by discovery that the aphids were able to alter the phenolic composition of diets during feeding, converting catechin into condensation products that were either phagostimulant or nondeterrent.

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Résumé M. rosae n'a pas présenté de dissuation significative en présence de catéchine à des concentrations voisines de 0,3 mg/ml, soit dans un aliment simple à base de sucrose, soit dans un aliment complexe contenant, entre autres, des acides aminés avec une concentration totale correspondant à celle obtenue à partir du jus de tisses de rosier écrasés. Des concentrations voisines de catéchine ont été obtenues à partir du jus provenant de tisus écrasés pour lequel ce puceron ne montrait aucune préférence dans la nature. Un certain nombre de facteurs influent sur la valeur absolue du pouvoir dissuasif: l'augmentation de la concentration en sucrose et surtout en acides aminés réduit cet effet dissuasif; les stades jeunes n'ont pas présenté de préférence pour les concentrations faibles en catéchine que avaient semblé attractive pour les stades âgés.Une hypothèse plausible pour expliquer de telles variations a été élaborée à partir de l'observation de la modification, par le puceron, au cours de sa succion, de la composition phénolique de l'aliment, avec transformation de la catéchine en polymères oxydés attractifs. Il est possible que tout ce qui augmente l'activité oxydase polyphénolique de la salive des insectes, réduise ou inverse l'effet de la tenuer originelle de la catéchine.

     

Cloning of Acinetobacter calcoaceticus chromosomal region involved in catechin degradation

Acinetobacter calcoaceticus utilizes catechin as sole carbon source. The chromosomal region involved in catechin catabolism was cloned in Escherichia coli DH5alpha from the genomic DNA of A. calcoaceticus. A recombinant E. coli containing 9.2 kb DNA fragment of A. calcoaceticus inserted in pUC19 showed a halo zone around the colony in plate assays, indicating the catechin utilizing ability of the clone. Enzyme assays revealed the expression of the cloned DNA fragment of A. calcoaceticus. High performance thin layer chromatography confirmed protocatechuic acid and phloroglucinol carboxylic acid as cleavage products of catechin in A. calcoaceticus and the catechin degrading ability of the clones. A. calcoaceticus followed the beta-ketoadipate pathway for catechin degradation. The sub-clone (pASCI) of this insert was sequenced and analyzed. The sequence showed three major ORFs but only ORF 2 showed similarities to other aromatic oxygenases and the sequence of ORF 2 was submitted to GenBank (AF369935).     

Isolation of L8 and L8S8 forms of ribulose bisphosphate carboxylase/oxygenase from Chromatium vinosum

The enzyme ribulose bisphosphate carboxylase/oxygenase has been purified from Chromatium vinosum. When an extract is subjected to centrifugation at 35,000xg in the presence of polyethylene glycol (PEG)-6000 and the supernatant is treated with 50 mM Mg²⁺ and the precipitate is then fractionated by vertical centrifugation into a reoriented sucrose gradient followed by chromatography on diethylaminoethyl (DEAE)-Sephadex A50, the resultant enzyme contains large (L) and small (S) subunits. Alternatively, centrifugation of extracts at 175,000xg in the presence of PEG-6000 followed by fractionation with Mg²⁺, density gradient centrifugation, and chromatography on DEAE-Sephadex A50 yields an enzyme free of small subunits. The two forms have comparable carboxylase and oxygenase activities and have compositions and molecular weights corresponding to L₈ and L₈S₈ enzymes. The amino acid compositions of L and S subunits are reported. The L₈S₈ enzyme from spinach cannot be similarly dissociated by centrifugation at 175,000xg in the presence of PEG-6000.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine-tetraacetate - MOPS 3-(N-morpholino)propanesulfonic acid - PEG polyethylene glycol - RuBisCO d-ribulose 1,5-bisphosphate caboxylase/oxygenase - RnBP d-ribulose 1,5-bisphosphate - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Dedicated to Professor G. Drews on occasion of his 60th birthday     

Production and partial purification of naringinase by Penicillium decumbens PTCC 5248

Penicillium decumbens PTCC 5248 produced naringinase when grown in a medium contained naringin as a source of carbon. Rhamnose also induced production of naringinase. Prunin disappeared as the time of enzymatic reaction increased. On fractionation with isopropanol 24-fold purification was achieved. Optimum pH and temperature for naringinase activity were determined to be 4.5 and 55 °C respectively. The K_m value of the enzyme with respect to naringin was found to be 1.7 mM. Citric acid, glucose, Ca²⁺, Mg²⁺, Zn²⁺ all inhibited naringinase activity.     

Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates

The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial species were belonging to the genera Acidithiobacillus, Leptospirillum, Sulfobacillus, and Sphingomonas, accounting for 6.3, 66.7, 18.8, and 8.3%, respectively; the sole archaeal species was Ferroplasma sp. (100%). Quantitative RT-PCR revealed that the 16S rRNA gene copy numbers (per gram of concentrates) of bacteria and archaea were 4.59 × 10⁹ and 6.68 × 10⁵, respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor _Fx, sor _SA, and sor _SB were identified from metagenomic DNAs of the bioreactors. The sor _Fx is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor _SA and sor _SB showed no significant identity to any genes in GenBank databases. The sor _SB was cloned and expressed in Escherichia coli, and SOR activity was determined. Quantitative RT-PCR determination of the gene densities of sor _SA and sor _SB were 1,000 times higher than archaeal 16S rRNA gene copy numbers, indicating that these genes were mostly impossible from archaea. Furthermore, with primers specific to the sor _SB gene, this gene was PCR-amplified from the newly isolated Acidithiobacillus sp. strain SM-1. So far as we know, this is the first time to determine SOR activity originating from bacteria and to document SOR gene in bioleaching reactors and Acidithiobacillus species.     

Leptospira interrogans requires a functional heme oxygenase to scavenge iron from hemoglobin

The transposon TnSC189 was used to construct a mutant in the putative heme oxygenase gene hemO (LB186) of Leptospira interrogans. Unlike its parent strain, the mutant grew poorly in medium in which hemoglobin was the sole iron source. The putative heme oxygenase was over expressed in a His-tagged form, purified and was demonstrated to degrade heme in vitro. Unexpectedly, it was also found that the L. interrogans growth rate was significantly increased when medium was supplemented with hemoglobin, but only if ferrous iron sources were absent. This result was mirrored in the expression of some iron-related genes and suggests the presence of regulatory mechanisms detecting Fe²⁺ and hemoglobin. This is the first demonstration of a functional heme oxygenase from a spirochete.     

Tabtoxin-resistant protein: overexpression, purification, and characterization

One of the self-protection mechanisms in Pseudomonas syringae pv. tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr). In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels. The purified recombinant tabtoxin-resistant protein (TTR) had an apparent molecular mass of about 21 kDa on SDS-PAGE as well as by mass spectroscopy and had a pI of 6.6 on isoelectric focusing-PAGE. Spectral analysis showed that TTR possesses a maximum fluorescence wavelength (lambda(max)) of 325 nm upon excitation at 282 nm and a positive band with a maximum at 195 nm and a broad negative band with a minimum at 215 nm in the far-UV CD spectrum. The spectrophotometric assay demonstrated the strong detoxification activity of TTR. These results are the first report of the characterization of the purified tabtoxin-resistant protein. Its capacity to detoxify tabtoxinine-beta-lactam shows that it must be one of the self-protection mechanisms in pv. tabaci.     

Phenol Degradation by Immobilized Cells of Arthrobacter citreus

An aerobic microorganism with an ability to utilize phenol as carbon and energy source was isolated from a hydrocarbon contamination site by employing selective enrichment culture technique. The isolate was identified as Arthrobacter citreus based on morphological, physiological and biochemical tests. This mesophilic organism showed optimal growth at 25°C and at pH of 7.0. The phenol utilization studies with Arthrobacter citreus showed that the complete assimilation occurred in 24 hours. The organism metabolized phenol up to 22 mM concentrations whereas higher levels were inhibitory. Thin layer chromatography, UV spectral and enzyme analysis were suggestive of catechol, as a key intermediate of phenol metabolism. The enzyme activities of phenol hydroxylase and catechol 2,3-dioxygenase in cell free extracts of Arthrobacter citreus were indicative of operation of a meta-cleavage pathway for phenol degradation. The organism had additional ability to degrade catechol, cresols and naphthol. The degradation rates of phenol by alginate and agar immobilized cells in batch fermentations showed continuous phenol metabolism for a period of eight days.     

Ribulose bisphosphate carboxylase/oxygenase from Thiocapsa roseopersicina

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified 80-fold from malate-grown Thiocapsa roseopersicina by salting out the enzyme from the high-speed supernatant between 68–95% saturation with respect to (NH₄)₂SO₄, gelfiltration through Sephadex G-100, and DEAE-cellulose chromatography followed by sedimentation into a 14–34% glycerol gradient. The specific activity of enzyme for the carboxylase reaction was 2.45 mol RuBP-dependent CO₂ fixed/min · mg protein (at pH 8.0 and 30° C) and for the oxygenase reaction was 0.23 mol RuBP-dependent O₂ consumed/min · mg protein (at pH 8.6, and 25° C). The enzyme, which was ultracentrifugally homogeneous in the presence of 4 and 10% v/v glycerol, was stable for at least one year at-80° C in the presence of 10% glycerol. S_{20, w} values obtained in the presence of 4 and 10% glycerol were 19.3 and 16.2, respectively. The enzyme contained both large (53,000-daltons) and mixed small subunits (15,000- and 13,500-daltons).Borate-dependent inactivation of the enzyme by 2,3-butadione, which was greatly reduced in the presence of the product 3-phosphoglycerate, suggested that one or more arginines are at the active site.Abbreviations DTT dithiotreitol - RuBP d-ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate - TCA trichloroacetic acid - TEMBDG buffer (pH 8.0 at 25°C) containing 20 mM Tris, 1 mM disodium EDTA · 2 H₂O, 10 mM MgCl₂·6 H₂O, 50 mM NaHCO₃, 0.1 mM DTT and 10% glycerol (v/v)     

Mechanism of suppression inDrosophila: Regulation of tryptophan oxygenase by thesu(s) + allele

The suppressor gene,su(s)², inDrosophila melanogaster restores the production of red and brown eye pigments for some purple and vermilion mutant alleles, respectively. We showed previously that the product of thesu(s)⁺ allele caused inhibition of the sepiapterin synthase A produced by the purple mutant but did not affect the wild-type enzyme. Suppression was accomplished by removingsu(s)⁺ from the genome. We now report that the tryptophan oxygenase, produced by suppressible vermilion alleles, is also inhibited by extracts fromsu(s)⁺ flies. The inhibition of the vermilion enzyme can be reduced or eliminated, respectively, by prior storage of the extract at 4 or –20°C or by boiling, whereas the wild-type enzyme is not affected by extracts ofsu(s)⁺ flies. Also, when the suppressible vermilion strain is raised on certain diets, brown eye pigment production occurs. This epigenetic suppression was reduced by the presence of an extra copy ofsu(s)⁺ in the genome. These data support a posttranslational mechanism for regulation of enzyme activity in which the activity of the mutant enzyme is reduced by the product of thesu(s)⁺ allele. How thesu(s)⁺ gene product can distinguish between the normal and the mutant forms of these two enzymes is discussed, along with other mechanisms for suppression that are currently under investigation.This work was supported in part by a grant from the KOSEF, Korea Science and Engineering Foundation, and the National Science Foundation under the U.S.-East Asia Cooperative Science Program as well as the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc.     

The inhibition of ribulose 1,5-bisphosphate carboxylase oxygenase by several 2-carboxyhexitol 1- and 6-phosphates

The condensation of D-fructose 6-phosphate or 1-phosphate with cyanide has been used to synthesize 2-carboxyhexitol 6-phosphates and 1-phosphates. The products have been characterized in terms of their action on ribulose bisphosphate carboxylase/oxygenase. The reaction of D-fructose 6-phosphate with cyanide is four times as fast (at 22°C) at pH 7.5 than at pH 11.5 and the primary products of condensation are more easily isolated by anion exchange chromatography. Two minor chromatographic peaks (I and II) for diastereomeric 2-carboxyhexitol 6-phosphates are isolated in addition to two major peaks, III and IV, which are lactones. The lactones are those of 2-C-carboxy-D-glucitol 6-phosphate (CG6P) in peak III and 2-C-carboxy-D-mannitol 6-phosphate (CM6P) in peak IV, as established after dephosphorylation by the relative rates of oxidation by periodate and by gas chromatographic retention times of the acetates. Analogous methodology has been used to synthesize the diastereomeric 2-carboxy-hexitol 1-phosphates (CG1P and CM1P) and their lactones from D-fructose 1-phosphate. The four carboxylates inhibit ribulose bisphosphate carboxylase/oxygenase from spinach or Pseudomonas oxalaticus in the following decreasing order of potency: CG6P, CM6P, CG1P, CM1P. The inhibition pattern suggests that the binding of the 5-phosphate moiety of the intermediate in the reaction catalyzed by ribulose bisphosphate carboxylase/oxygenase may be stronger by an order of magnitude than the binding of the 1-phosphate group.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase expression in melon plants infected with Colletotrichum lagenarium

Ribulose-1,5-bisphosphate carboxylase/oxygelase (RuBPCase) was studied in melon leaves infected by Colletotrichum lagenarium, a fungal pathogen of melons. Electrophoretic analysis of melon leaf proteins indicated a strong effect of infection on RuBPCase, the subunits of which gradually disappeared during the different stages of infection. Enzyme activity also declined 4 d after inoculation and its content, measured by immunoelectrophoresis, decreased to a similar extent. Synthesis of the large and small subunits of RuBPCase was followed by in-vivo pulse-labeling experiments. A drastic decrease in the rate of RuBPCase-subunit synthesis occurred 3 d after inoculation and preceded the appearance of disease symptoms. There was an apparent coordination of the synthesis of the two subunits under these conditions.Abbreviations LS (SS) Large (small) subunit of RuBPCase - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Localization and partial characterization of a sex-specific enzyme in homothallic and heterothallic mucorales

A study was made of some late reactions in the trisporic acid biosynthetic pathway in Mucor mucedo. Trisporic acids induce sexual reproduction in several Mucorales.Two enzymes involved in these reactions, a NADP-dependent dehydrogenase and an esterase, appeared to be highly specific for the minus mating type.The synthesis of these enzymes is stimulated by trisporic acids, indicating a positive control of these hormones upon their own synthesis.The dehydrogenase was histochemically shown to be concentrated in the zygophores of Mucor mucedominus. In the homothallic Zygorhynchus moelleri the copulating main branch (which is known to have a minus character) appeared to be the major site of dehydrogenase activity.