Plasmid-mediated degradation of dibenzothiophene by Pseudomonas species.

The microbial transformation of dibenzothiophene (DBT) is of interest in the potential desulfurization of oil. We isolated three soil Pseudomonas species which oxidized DBT to characteristic water-soluble, sulfur-containing products. Two of our isolates harbored a 55-megadalton plasmid; growth in the presence of novobiocin resulted in both loss of the plasmid and loss of the ability to oxidize DBT. Reintroduction of the plasmid restored the ability to oxidize DBT to water-soluble products. The products resulting from the oxidation of DBT were characterized and included 3-hydroxy-2-formyl benzothiophene, 3-oxo-[3'-hydroxy-thionaphthenyl-(2)-methylene]-dihydrothionaph thene, and the hemiacetal and trans forms of 4-[2-(3-hydroxy)-thianaphthenyl]-2-oxo-3-butenoic acid. The products of DBT oxidation were inhibitory to cell growth and further DBT oxidation. DBT oxidation in our soil isolates was induced by naphthalene or salicylate and to a much lesser extent by DBT and was repressed by succinate.     

Modification of Water-Soluble Coal-Derived Products by Dibenzothiophene-Degrading Microorganisms

To study mechanisms by which microorganisms oxidize thiophenic sulfur in coal, we tested bacterial cultures for the ability to degrade dibenzothiophene (DBT), DBT-5-oxide, and DBT-sulfone and to modify water-soluble coal products derived from Illinois no. 6 and Ugljevik coals. In yeast extract medium, the majority of selected isolates degraded DBT and accumulated DBT-5-oxide in culture fluids; all but one of the cultures degraded DBT-5-oxide, and none of them degraded DBT-sulfone. Elemental analysis data indicated that the microbial cultures were able to decrease the amount of sulfur in soluble coal products derived from Illinois no. 6 and Ugljevik coals. However, these data suggested that microbially mediated sulfur removal from soluble Ugljevik coal occurred by nonspecific mechanisms. That is, extensive degradation of the carbon structure was concurrent with the loss of sulfur. This conclusion was supported by X-ray photoelectron spectroscopic data which indicated that the reduced sulfur forms in the soluble Ugljevik coal product was not oxidized by microbial treatment.     

Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Batch and Fed-Batch Cultures

The DbtS(sup+) phenotype (which confers the ability to oxidize selectively the sulfur atom of dibenzothiophene [DBT] or dibenzothiophene sulfone [DBTO(inf2)]) of Rhodococcus erythropolis N1-36 was quantitatively characterized in batch and fed-batch cultures. In flask cultures, production of the desulfurization product, monohydroxybiphenyl (OH-BP), was maximal at pH 6.0, while specific productivity (OH-BP cell(sup-1)) was maximal at pH 5.5. Quantitative measurements in fermentors (in both batch and fed-batch modes) demonstrated that DBTO(inf2) as the sole sulfur source yielded a greater amount of product than did DBT. Specifically, 100 (mu)M DBT maximally yielded (apprx=)40 (mu)M OH-BP, while 100 (mu)M DBTO(inf2) yielded (apprx=)60 (mu)M OH-BP. Neither maintaining the pH at 6.0 nor adding an additional carbon source increased the yield of OH-BP. The presence of SO(inf4)(sup2-) in growth media repressed expression of desulfurization activity, but SO(inf4)(sup2-) added to suspensions of cells grown in DBT or DBTO(inf2) did not inhibit desulfurization activity.     

Genome data mining and soil survey for the novel group 5 [NiFe]-hydrogenase to explore the diversity and ecological importance of presumptive high-affinity H(2)-oxidizing bacteria

Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H(2) possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H(2)-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H(2) oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 10(6) to 10(8) hhyL gene copies g (dry weight)(-1). Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H(2)-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H(2) oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H(2) by soil, because high-affinity H(2) oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur.     

Conservation of plasmid-encoded dibenzothiophene desulfurization genes in several rhodococci.

The cloned sulfur oxidation (desulfurization) genes (sox) for dibenzothiophene (DBT) from the prototype Rhodococcus sp. strain IGTS8 were used in Southern hybridization and PCR experiments to establish the DNA relatedness in six new rhodococcal isolates which are capable of utilizing DBT as a sole sulfur source for growth. The ability of these strains to desulfurize appears to be an exclusive property of a 4-kb gene locus on a large plasmid of ca. 150 kb in IGTS8 and ca. 100 kb in the other strains. Besides a difference in plasmid profile, IGTS8 is distinguishable from the other strains in at least the copy number of the insertion sequence IS1166, which is associated with the sox genes.     

Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells

The ability of endothelin to promote phospholipid hydrolysis has been studied in myo-[2-3H]-inositol-, [3H]-arachidonic acid- or methyl-[3H]choline chloride-prelabelled cultured vascular smooth muscle cells (VSMC) from rat and bovine thoracic aortae and human omental vessels. The biochemical responses to endothelin were comparable between the different VSMC isolates. Endothelin promoted the accumulation of glycerolphospho[3H]inositol and concomitant loss of [3H]-inositol label from phosphatidylinositol. Exposure of [3H]choline-labelled VSMC to endothelin resulted in a loss of radioactivity from phosphatidylcholine that was inversely parallelled by an increase in water-soluble [3H]-choline metabolites. In [3H]-arachidonic acid ([3H]-AA)-labelled VSMC, endothelin induced extracellular release of [3H]-AA which derived from both phosphatidylcholine and phosphatidylinositol. Half-maximally effective concentrations of endothelin for all these responses were approximately 2-7 nM and did not vary between VSMC types. Endothelin-induced release of [3H]-AA into VSMC medium-overlay was inhibited by quinacrine and nordihydroguaiaretic acid but not by neomycin or indomethacin. The data herein implicate activation of phospholipase A2 by endothelin with subsequent metabolism of arachidonic acid via the lipoxygenase pathway.     

Detection of microbial growth on polycyclic aromatic hydrocarbons in microtiter plates by using the respiration indicator WST-1

We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 [4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate], in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.     

Characterization of populations of aerobic hydrogen-oxidizing soil bacteria

Abstract Freshly isolated soil bacteria were screened for different characteristics of the H₂ metabolism without prior selection for growth on H₂. The bacteria were isolated from different grain size fractions of a neutral meadow cambisol and an acidic forest cambisol, and then tested (1) for the ability to oxidize H₂, (2) for chemolithoautotrophic growth on H₂ as sole electron donor and energy source, (3) for DNA-DNA-hybridization with two hydrogenase gene fragments from Alcaligenes eutrophus and Rhizobium leguminosarum , and (4) for reduction of 2,3,5-triphenyl-2H-tetrazoliumchloride (TTC) in the presence of H₂. Many (65–90%) of the isolates were able to reduce TTC, but only 30–65% were actually able to oxidize H₂ indicating that the TTC test was not a specific characteristic for H₂ oxidation ability. The TTC test was only reliable in pure cultures of known bacteria with optimized test conditions, here shown for Alcaligenes eutrophus, Bradyrhizobium japonicum and Nocardia opaca , but not in mixed cultures of unknown bacteria. Still less (< 30%) of the isolates were able to grow chemolithoautotrophically indicating that culturable aerobic bacteria with the ability for H₂ oxidation are more abundant than bacteria with the ability for chemolithoautotrophic growth. The DNA-DNA-hybridization test failed to detect many of the bacteria with H₂ oxidation activity, probably since the hydrogenase genes present in the isolates were too diverse to be all detected by the DNA probes applied.     

Detection of Microbial Growth on Polycyclic Aromatic Hydrocarbons in Microtiter Plates by Using the Respiration Indicator WST-1

We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate}, in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.     

Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

18O]-labeled singlet oxygen as a tool for mechanistic studies of 8-oxo-7,8-dihydroguanine oxidative damage: detection of spiroiminodihydantoin,imidazolone and oxazolone derivatives

A water-soluble [18O]-labeled endoperoxide derived from N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalene-dipropanamide (DHPN18O2) has been shown to act as a clean chemical source of [18O]-labeled molecular singlet oxygen. This allows the assessment of the singlet oxygen (1O2) reactivity toward biological targets such as DNA. The present work focuses on the qualitative identification of the main 1O2-oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine, which was achieved using high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Thus, the [18O]-labeled and unlabeled imidazolone and oxazolone, together with the diastereoisomeric spiroiminodihydantoin nucleosides, were detected as the main degradation products. In addition, a modified nucleoside that exhibits similar features as those of the oxidized guanidinohydantoin molecule was detected. Our data strongly suggest that the imidazolone and oxazolone nucleosides are generated via the rearrangement of an unstable 5-hydroperoxide intermediate. Interestingly, the combined use of appropriate tools, including isotopically labeled singlet oxygen and the high- resolution HPLC-ESI-MS/MS technique, has allowed to shed new light on the 1O2-mediated oxidation reactions of guanine DNA components.     

Oxidation of Acetate by Various Strains of Bacillus popilliae

A number of strains of Bacillus popilliae were examined for their ability to oxidize acetate. Some of these would not sporulate in vitro, and some were oligosporogenous. The ability to oxidize acetate varied widely among the strains tested. A culture derived from spores of the parent strain produced in vivo and one of the asporogenous strains derived from it failed to produce any significant levels of (14)CO(2) from [(14)C]acetate. Oligosporogenous strains derived from the same parent culture all produced (14)CO(2) from both [1-(14)C] and [2-(14)C]acetate but at relatively low rates. The highest rates of acetate oxidation were observed with three strains which did not produce spores in vitro. When cultured under appropriate conditions, one of these strains displayed a secondary growth response concomitant with a decrease in the titratable acidity and an increase in the pH of the medium. The data indicate that B. popilliae has a complete citric acid cycle but that the activity of the cycle is strongly repressed in wild-type strains under the usual conditions used for in vitro cultivation.     

Effect of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid (TDGA) on glucose and fatty acid oxidation in isolated rat soleus muscle

1. The effect of 2-tetradecylglycidic acid (TDGA), a potent, specific inhibitor of long-chain fatty acid oxidation, on fatty acid and glucose oxidation by isolated rat soleus muscle was studied. 2. TDGA inhibited [1-14C]palmitate oxidation by soleus muscle in a concentration-dependent manner. 3. TDGA inhibited the activity of soleus muscle mitochondrial carnitine palmitoyltransferase A (CPT-A). 4. Added palmitate (0.5 mM) significantly inhibited D-[U-14C]glucose oxidation and, under conditions where TDGA inhibited palmitate oxidation, the oxidation of D-[U-14C]glucose by isolated soleus muscle was significantly stimulated. 5. TDGA stimulation of glucose oxidation was reversed by octanoate, a medium-chain fatty acid whose oxidation is not inhibited by TDGA. 6. When nondiabetic rats were treated with TDGA (10 mg/kg p.o./day x 3 days), fasting plasma glucose was significantly lowered and the ability of isolated contralateral soleus muscles to oxidize palmitate was inhibited while glucose oxidation was significantly stimulated.     

Synthesis of coumarin-polyamine-based molecular probe for the detection of hydroxyl radicals generated by gamma radiation

To develop a molecular probe for detection of hydroxyl radicals in the vicinity of DNA, the coumarin-polyamine complexes, N(1),N(12)-bis[2-oxo-2H-chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane (5) and tris[2-(2-oxo-2H-chromene-3-carboxamido)ethyl]amine (7), and their hydroxylated derivatives, N(1),N(12)-bis[7-hydroxy-2-oxo-2H-chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane (6) and tris[2-(7-hydroxy-2-oxo-2H-chromene-3-carboxamido)ethyl]amine (8), have been synthesized. Using computer-generated molecular modeling, the derivatives have been docked onto DNA dodecamer d(CGCGAATTCGCG)(2), the ligand-DNA complexes have been minimized, and the free binding energies (DeltaG(binding)) and inhibition constants (K(i)) have been calculated. Compound 7 is not water-soluble at the concentrations required for the project. When aqueous solutions of 5 are irradiated with gamma rays, the relationship between induced fluorescence and dose is linear in the range of 0 to 10 Gy. The fluorescence emission spectrum of irradiated 5 is similar to that of its dihydroxy derivative 6, indicating conversion of 5 to 6, and induction of fluorescence records formation of hydroxyl radicals in aqueous solution. The dicoumarin-polyamine 5, a novel compound for the detection of hydroxyl radicals close to DNA, is a sensitive and quantitative probe with potential for applications in biological systems.     

Expression of dibenzothiophene-degradative genes in two Pseudomonas species

The genes encoding dibenzothiophene (DBT) degradation in Pseudomonas alcaligenes strain DBT2 were cloned into plasmid pC1 by other workers. This plasmid was conjugally transferred into a spontaneous variant of Pseudomonas sp. HL7b (designated HL7bR) incapable of oxidizing DBT (Dbt- phenotype). Acquisition of plasmid pC1 simultaneously restored oxidation of DBT and naphthalene to the transconjugant, although the primary DBT metabolite produced by transconjugant HL7bR(pC1) corresponded to that produced by wild-type strain DBT2 rather than that from wild-type strain HL7b. Inducers of the naphthalene pathway (naphthalene, salicylic acid, and 2-aminobenzoate) stimulated DBT oxidation in transconjugant HL7bR(pC1) when present at 0.1 mM concentrations but had no effect on wild-type strain HL7b. Higher concentrations (5 mM) of salicylic acid and naphthalene were inhibitory to DBT oxidation in all strains. DNA-DNA hybridization was not observed between plasmid pC1 and genomic DNA from strains HL7b or HL7bR, nor between authentic naphthalene-degradative genes (plasmid NAH2) and either plasmid pC1 or strain HL7b, despite the observation that the degradative genes encoded on plasmid pC1 functionally resembled broad-specificity naphthalene-degradative genes. Transconjugant HL7bR(pC1) is a mosaic of the parental types regarding DBT metabolite production, regulation, and use of carbon sources.     

Biodesulfurization of naphthothiophene and benzothiophene through selective cleavage of carbon-sulfur bonds by Rhodococcus sp. strain WU-K2R

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WU-K2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2'-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c][1,2]oxathiin S-oxide, benzo[c][1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2'-hydroxyphenyl)ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.     

Independent synthesis of aminophospholipid-linked maillard products

Phospholipid-linked glycation products are supposed to play an important role in lipid oxidation in vivo. Independent syntheses and unequivocal structural characterization are reported for the phosphatidyl ethanolamine (PE)-derived Amadori compound 4-hydroxy-4-oxo-1-[(palmitoyloxy)methyl]-9-(2,3,4,5-tetrahydrox ytetrahydro-2H-pyran-2-yl)-3,5-dioxa-8-aza-4lambda5-ph osphanon-1-yl palmitate, pyrrolecarbaldehyde 2-[[[2-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]ethoxy](hydroxy)phosph oryl]oxy]-1-[(palmitoyloxy)methyl]ethyl palmitate, the carboxymethyl (CM) derivative 7-hydroxy-7,13-dioxo-10-(palmitoyloxy)-6,8,12-trioxa-3-aza-+ ++7lambda5-phosphaoctacosan-1-oic acid, and the carboxyethyl (CE) derivative 7-hydroxy-2-methyl-7,13-dioxo-10-(palmitoyloxy)-6,8,12-trioxa++ +-3-aza-7lambda5-phosphaoctacosan-l-oic acid. With these reference compounds, a liquid chromatography-mass spectrometry (LCMS) method for the determination of such PE-linked Maillard products has been developed.     

Lignin-solubilizing ability of actinomycetes isolated from termite (Termitidae) gut

The lignocellulose-degrading abilities of 11 novel actinomycete strains isolated from termite gut were determined and compared with that of the well-characterized actinomycete, Streptomyces viridosporus T7A. Lignocellulose bioconversion was followed by (i) monitoring the degradation of [14C]lignin- and [14C]cellulose-labeled phloem of Abies concolor to 14CO2 and 14C-labeled water-soluble products, (ii) determining lignocellulose, lignin, and carbohydrate losses resulting from growth on a lignocellulose substrate prepared from corn stalks (Zea mays), and (iii) quantifying production of a water-soluble lignin degradation intermediate (acid-precipitable polymeric lignin). The actinomycetes were all Streptomyces strains and could be placed into three groups, including a group of five strains that appear superior to S. viridosporus T7A in lignocellulose-degrading ability, three strains of approximately equal ability, and three strains of lesser ability. Strain A2 was clearly the superior and most effective lignocellulose decomposer of those tested. Of the assays used, total lignocellulose weight loss was most useful in determining overall bioconversion ability but not in identifying the best lignin-solubilizing strains. A screening procedure based on 14CO2 evolution from [14C-lignin]lignocellulose combined with measurement of acid-precipitable polymeric lignin yield was the most effective in identifying lignin-solubilizing strains. For the termite gut strains, the pH of the medium showed no increase after 3 weeks of growth on lignocellulose. This is markedly different from the pattern observed with S. viridosporus T7A, which raises the medium pH considerably. Production of extracellular peroxidases by the 11 strains and S. viridosporus T7A was followed for 5 days in liquid cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lignin-solubilizing ability of actinomycetes isolated from termite (Termitidae) gut.

The lignocellulose-degrading abilities of 11 novel actinomycete strains isolated from termite gut were determined and compared with that of the well-characterized actinomycete, Streptomyces viridosporus T7A. Lignocellulose bioconversion was followed by (i) monitoring the degradation of [14C]lignin- and [14C]cellulose-labeled phloem of Abies concolor to 14CO2 and 14C-labeled water-soluble products, (ii) determining lignocellulose, lignin, and carbohydrate losses resulting from growth on a lignocellulose substrate prepared from corn stalks (Zea mays), and (iii) quantifying production of a water-soluble lignin degradation intermediate (acid-precipitable polymeric lignin). The actinomycetes were all Streptomyces strains and could be placed into three groups, including a group of five strains that appear superior to S. viridosporus T7A in lignocellulose-degrading ability, three strains of approximately equal ability, and three strains of lesser ability. Strain A2 was clearly the superior and most effective lignocellulose decomposer of those tested. Of the assays used, total lignocellulose weight loss was most useful in determining overall bioconversion ability but not in identifying the best lignin-solubilizing strains. A screening procedure based on 14CO2 evolution from [14C-lignin]lignocellulose combined with measurement of acid-precipitable polymeric lignin yield was the most effective in identifying lignin-solubilizing strains. For the termite gut strains, the pH of the medium showed no increase after 3 weeks of growth on lignocellulose. This is markedly different from the pattern observed with S. viridosporus T7A, which raises the medium pH considerably. Production of extracellular peroxidases by the 11 strains and S. viridosporus T7A was followed for 5 days in liquid cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of hydroxylamines to nitroxide spin labels in living cells

In the presence of oxygen, cells can oxidize hydroxylamines, which are the products of the reduction of nitroxides in cells, back to nitroxides. Lipid-soluble hydroxylamines are oxidized much more rapidly than water-soluble ones, and most of this oxidation is inactivated by heat or trichloroacetic acid, indicating that the principal mechanism is enzyme-linked. The rates of oxidation of some lipophilic hydroxylamines are comparable to the rates of reduction of the corresponding nitroxides. Hydroxylamines formed by reduction of aqueous soluble nitroxides are not oxidized by cells, except for slight oxidation of some pyrrolidine derivatives. The latter is due to autoxidation. The kinetics of oxidation of reduced lipid-soluble nitroxides are all first-order with respect to hydroxylamines, regardless of the position of the nitroxide group along the carbon backbone, indicating that the oxidation occurs within the membrane. The oxidation of hydroxylamines in cells in inhibited by cyanide but not by antimycin A or SKF-525A. We also describe an effective method to oxidize hydroxylamines and follow this reaction; the method is based on the use of perdeuterated [15N]Tempone.