scholarly journals Stable Isotopic Studies of n-Alkane Metabolism by a Sulfate-Reducing Bacterial Enrichment Culture

2005 ◽  
Vol 71 (12) ◽  
pp. 8174-8182 ◽  
Author(s):  
Irene A. Davidova ◽  
Lisa M. Gieg ◽  
Mark Nanny ◽  
Kevin G. Kropp ◽  
Joseph M. Suflita
Keyword(s):  
Enrichment Culture ◽  
Initial Step ◽  
Gas Chromatography Mass Spectrometry ◽  
Resonance Spectroscopy ◽  
Sulfate Reducing ◽  
Cell Extracts ◽  
Stable Isotopic ◽  
Isotopic Studies ◽  
Fumarate Addition ◽  
Metabolism Analysis

ABSTRACT Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n-alkanes (C6 to C12) and 1-13C-labeled n-hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d 14-hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n-hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1-13C-labeled n-hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that 13C nuclei from 1-13C-labeled n-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that 13C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177:235-243, 2002) for metabolism of deuterated n-hexane by a denitrifying bacterium.

scholarly journals Anaerobic Oxidation of n-Dodecane by an Addition Reaction in a Sulfate-Reducing Bacterial Enrichment Culture

2000 ◽  
Vol 66 (12) ◽  
pp. 5393-5398 ◽  
Author(s):  
Kevin G. Kropp ◽  
Irene A. Davidova ◽  
Joseph M. Suflita
Keyword(s):  
Addition Reaction ◽  
Enrichment Culture ◽  
Methyl Esters ◽  
Gas Chromatography Mass Spectrometry ◽  
Chromatographic Retention ◽  
Sulfate Reducing ◽  
Acid Metabolites ◽  
Fumarate Addition ◽  
Deuterium Retention ◽  
Fragmentation Patterns

ABSTRACT We identified trace metabolites produced during the anaerobic biodegradation of H26- and D26-n-dodecane by an enrichment culture that mineralizes these compounds in a sulfate-dependent fashion. The metabolites are dodecylsuccinic acids that, in the case of the perdeuterated substrate, retain all of the deuterium atoms. The deuterium retention and the gas chromatography-mass spectrometry fragmentation patterns of the derivatized metabolites suggest that they are formed by C—H or C—D addition across the double bond of fumarate. As trimethylsilyl esters, two nearly coeluting metabolites of equal abundance with nearly identical mass spectra were detected from each of H26- and D26-dodecane, but as methyl esters, only a single metabolite peak was detected for each parent substrate. An authentic standard of protonatedn-dodecylsuccinic acid that was synthesized and derivatized by the two methods had the same fragmentation patterns as the metabolites of H26-dodecane. However, the standard gave only a single peak for each ester type and gas chromatographic retention times different from those of the derivatized metabolites. This suggests that the succinyl moiety in the dodecylsuccinic acid metabolites is attached not at the terminal methyl group of the alkane but at a subterminal position. The detection of two equally abundant trimethylsilyl-esterified metabolites in culture extracts suggests that the analysis is resolving diastereomers which have the succinyl moiety located at the same subterminal carbon in two different absolute configurations. Alternatively, there may be more than one methylene group in the alkane that undergoes the proposed fumarate addition reaction, giving at least two structural isomers in equal amounts.

scholarly journals Anaerobic 1-Alkene Metabolism by the Alkane- and Alkene-Degrading Sulfate Reducer Desulfatibacillum aliphaticivorans Strain CV2803T

2007 ◽  
Vol 73 (24) ◽  
pp. 7882-7890 ◽  
Author(s):  
Vincent Grossi ◽  
Cristiana Cravo-Laureau ◽  
Alain Méou ◽  
Danielle Raphel ◽  
Frédéric Garzino ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Inorganic Carbon ◽  
Direct Evidence ◽  
Gas Chromatography Mass Spectrometry ◽  
Sulfate Reducing ◽  
Branched Fatty Acids ◽  
Initial Activation ◽  
Fumarate Addition ◽  
Chain Lengths ◽  
Methyl Branched Fatty Acids

ABSTRACT The alkane- and alkene-degrading, marine sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803T, known to oxidize n-alkanes anaerobically by fumarate addition at C-2, was investigated for its 1-alkene metabolism. The total cellular fatty acids of this strain were predominantly C-(even number) (C-even) when it was grown on C-even 1-alkenes and predominantly C-(odd number) (C-odd) when it was grown on C-odd 1-alkenes. Detailed analyses of those fatty acids by gas chromatography-mass spectrometry after 6- to 10-week incubations allowed the identification of saturated 2- and 4-ethyl-, 2- and 4-methyl-, and monounsaturated 4-methyl-branched fatty acids with chain lengths that correlated with those of the 1-alkene. The growth of D. aliphaticivorans on (per)deuterated 1-alkenes provided direct evidence of the anaerobic transformation of these alkenes into the corresponding 1-alcohols and into linear as well as 10- and 4-methyl-branched fatty acids. Experiments performed with [13C]bicarbonate indicated that the initial activation of 1-alkene by the addition of inorganic carbon does not occur. These results demonstrate that D. aliphaticivorans metabolizes 1-alkene by the oxidation of the double bond at C-1 and by the subterminal addition of organic carbon at both ends of the molecule [C-2 and C-(ω-1)]. The detection of ethyl-branched fatty acids from unlabeled 1-alkenes further suggests that carbon addition also occurs at C-3. Alkylsuccinates were not observed as potential initial intermediates in alkene metabolism. Based on our observations, the first pathways for anaerobic 1-alkene metabolism in an anaerobic bacterium are proposed. Those pathways indicate that diverse initial reactions of 1-alkene activation can occur simultaneously in the same strain of sulfate-reducing bacterium.

scholarly journals Anaerobic Naphthalene Degradation by a Sulfate-Reducing Enrichment Culture

2000 ◽  
Vol 66 (7) ◽  
pp. 2743-2747 ◽  
Author(s):  
Rainer U. Meckenstock ◽  
Eva Annweiler ◽  
Walter Michaelis ◽  
Hans H. Richnow ◽  
Bernhard Schink
Keyword(s):  
Mass Spectra ◽  
Phenylacetic Acid ◽  
Enrichment Culture ◽  
Fission Products ◽  
Substrate Utilization ◽  
Gas Chromatography Mass Spectrometry ◽  
Ring Cleavage ◽  
Sulfate Reducing ◽  
Naphthalene Degradation ◽  
Naphthoic Acid

ABSTRACT Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3,4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-13C]naphthalene or deuterated D8-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [13C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, 13C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.

scholarly journals Comparison of Mechanisms of Alkane Metabolism under Sulfate-Reducing Conditions among Two Bacterial Isolates and a Bacterial Consortium

2006 ◽  
Vol 72 (6) ◽  
pp. 4274-4282 ◽  
Author(s):  
Amy V. Callaghan ◽  
Lisa M. Gieg ◽  
Kevin G. Kropp ◽  
Joseph M. Suflita ◽  
Lily Y. Young
Keyword(s):  
Bacterial Consortium ◽  
Gas Chromatography Mass Spectrometry ◽  
Degradation Mechanisms ◽  
Bacterial Isolates ◽  
Alkane Degradation ◽  
Tetradecanoic Acid ◽  
Mass Spectral ◽  
Sulfate Reducing ◽  
Reducing Conditions ◽  
Fumarate Addition

ABSTRACT Recent studies have demonstrated that fumarate addition and carboxylation are two possible mechanisms of anaerobic alkane degradation. In the present study, we surveyed metabolites formed during growth on hexadecane by the sulfate-reducing isolates AK-01 and Hxd3 and by a mixed sulfate-reducing consortium. The cultures were incubated with either protonated or fully deuterated hexadecane; the sulfate-reducing consortium was also incubated with [1,2-13C2]hexadecane. All cultures were extracted, silylated, and analyzed by gas chromatography-mass spectrometry. We detected a suite of metabolites that support a fumarate addition mechanism for hexadecane degradation by AK-01, including methylpentadecylsuccinic acid, 4-methyloctadecanoic acid, 4-methyloctadec-2,3-enoic acid, 2-methylhexadecanoic acid, and tetradecanoic acid. By using d 34-hexadecane, mass spectral evidence strongly supporting a carbon skeleton rearrangement of the first intermediate, methylpentadecylsuccinic acid, was demonstrated for AK-01. Evidence indicating hexadecane carboxylation was not found in AK-01 extracts but was observed in Hxd3 extracts. In the mixed sulfate-reducing culture, however, metabolites consistent with both fumarate addition and carboxylation mechanisms of hexadecane degradation were detected, which demonstrates that multiple alkane degradation pathways can occur simultaneously within distinct anaerobic communities. Collectively, these findings underscore that fumarate addition and carboxylation are important alkane degradation mechanisms that may be widespread among phylogenetically and/or physiologically distinct microorganisms.

scholarly journals Anaerobic Degradation of 2-Methylnaphthalene by a Sulfate-Reducing Enrichment Culture

2000 ◽  
Vol 66 (12) ◽  
pp. 5329-5333 ◽  
Author(s):  
Eva Annweiler ◽  
Arne Materna ◽  
Michael Safinowski ◽  
Andreas Kappler ◽  
Hans H. Richnow ◽  
...  
Keyword(s):  
Succinic Acid ◽  
Anaerobic Degradation ◽  
Specific Activity ◽  
Degradation Products ◽  
Enrichment Culture ◽  
Toluene Degradation ◽  
Sulfate Reducing ◽  
Naphthalene Degradation ◽  
Naphthoic Acid ◽  
Fumarate Addition

ABSTRACT Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 μM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 � 0.003 nmol min−1 mg of protein−1 only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.

scholarly journals Anaerobic degradation of phenanthrene by a sulfate-reducing enrichment culture

2018 ◽  
Vol 20 (10) ◽  
pp. 3589-3600 ◽  
Author(s):  
Anne M. Himmelberg ◽  
Thomas Brüls ◽  
Zahra Farmani ◽  
Philip Weyrauch ◽  
Gabriele Barthel ◽  
...  
Keyword(s):  
Anaerobic Degradation ◽  
Enrichment Culture ◽  
Sulfate Reducing

scholarly journals PL1.2 Multiparametric assessment of factors influencing 2 HG accumulation in diffuse brain gliomas

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii1-iii2
Author(s):  
L Nichelli ◽  
F Branzoli ◽  
R Valabregue ◽  
L Capelle ◽  
C Ottolenghi ◽  
...  
Keyword(s):  
Tumor Volume ◽  
Treated Patient ◽  
Gas Chromatography Mass Spectrometry ◽  
Resonance Spectroscopy ◽  
Genetic Profile ◽  
Radiological Change ◽  
Cellular Density ◽  
Anti Cancer ◽  
Hg Accumulation

Abstract BACKGROUND 2-hydroxyglutarate (2HG) can be detected non-invasively in IDH-mutant gliomas by in vivo magnetic resonance spectroscopy (MRS). We investigated factors affecting 2 HG accumulation and explored the prognostic value of 2 HG detection in IDH mutant gliomas and 2 HG variations during anti-cancer therapies. MATERIAL AND METHODS We prospectively scanned by MEGA-PRESS 70 glioma patients (24 before surgery and 46 IDH mutant operated glioma) and measured 2HG. CRLB cut-off was 50%. We followed up 9 IDH mutant patients during radiotherapy and chemotherapy.We analyzed radiological parameters (tumor and cystic/necrotic volumes, fractions of VOI filled with tumor, spectroscopic profile, “infiltrative” versus “expansive” morphology, contrast-enhancement) and genetic profile (IDH1, IDH2, 1p19q codeletion). 2HG concentrations in plasma, urine, and surgical obtained samples were measured by gas chromatography-mass spectrometry (GC-MS). RESULTS MEGA-PRESS sequence detected 2HG with a sensitivity of 95% in untreated patients, and of 69% in pre-treated patient. Positive predictive value was 100% in both groups. 2HG was lower in pre-treated IDH mutant gliomas (1.1 versus 2.3 mM, P=0.02) and decreased rapidly during radiotherapy and chemotherapy before any radiological change. 2HG detection was positively correlated with tumor volume (P=0.02), choline measurements (r=0.58 P<0.0001), cellular density (measured by restricted diffusivity, Pearson r -0.40 P=0.01), “expansive” presentation, mutated reads/total reads ratio by NGS and was inversely correlated with Myo-inositol (Pearson R -0.29 P=0.03) and cystic/necrotic areas (P=0.04). 2HG by MRS positively correlated with urine 2HG (r=0.80 P=0.003). 2 HG was higher in IDH2 mutant (4.7 versus 2.4 Mm, P=0.02) and lower in non R132H IDH1 mutant (1.12 mM P=0.004). Among IDH mutant glioma patients, 2 HG detection was associated with longer survival (HR 0.09; 95%CI 0.018–0.52). CONCLUSION Tumor volume, cellular density, previous radio- and chemotherapy and genetic features determine 2 HG detection in IDH mutant gliomas. 2 HG detection is associated with better outcome and can be reliably monitored during anti-cancer treatments.

scholarly journals Identification and Sequencing of β-Myrcene Catabolism Genes from Pseudomonas sp. Strain M1

1999 ◽  
Vol 65 (7) ◽  
pp. 2871-2876 ◽  
Author(s):  
Sandra Iurescia ◽  
Andrea M. Marconi ◽  
Daniela Tofani ◽  
Augusto Gambacorta ◽  
Annalisa Paternò ◽  
...  
Keyword(s):  
Genomic Library ◽  
Enrichment Culture ◽  
Open Reading Frames ◽  
Gas Chromatography Mass Spectrometry ◽  
Complete Agreement ◽  
Wild Type ◽  
Genomic Insert ◽  
The One ◽  
Acyl Coenzyme A ◽  
Reading Frames

ABSTRACT The M1 strain, able to grow on β-myrcene as the sole carbon and energy source, was isolated by an enrichment culture and identified as a Pseudomonas sp. One β-myrcene-negative mutant, called N22, obtained by transposon mutagenesis, accumulated (E)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrcen-8-ol) as a unique β-myrcene biotransformation product. This compound was identified by gas chromatography-mass spectrometry. We cloned and sequenced the DNA regions flanking the transposon and used these fragments to identify the M1 genomic library clones containing the wild-type copy of the interrupted gene. One of the selected cosmids, containing a 22-kb genomic insert, was able to complement the N22 mutant for growth on β-myrcene. A 5,370-bp-long sequence spanning the region interrupted by the transposon in the mutant was determined. We identified four open reading frames, named myrA,myrB, myrC, and myrD, which can potentially code for an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA hydratase, respectively. myrA, myrB, andmyrC are likely organized in an operon, since they are separated by only 19 and 36 nucleotides (nt), respectively, and no promoter-like sequences have been found in these regions. ThemyrD gene starts 224 nt upstream of myrA and is divergently transcribed. The myrB sequence was found to be completely identical to the one flanking the transposon in the mutant. Therefore, we could ascertain that the transposon had been inserted inside the myrB gene, in complete agreement with the accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by the mutant. Based on sequence and biotransformation data, we propose a pathway for β-myrcene catabolism in Pseudomonas sp. strain M1.

scholarly journals Poly-3-Hydroxybutyrate in Legionella pneumophila, an Energy Source for Survival in Low-Nutrient Environments

1999 ◽  
Vol 65 (2) ◽  
pp. 822-827 ◽  
Author(s):  
Brian W. James ◽  
W. Stuart Mauchline ◽  
P. Julian Dennis ◽  
C. William Keevil ◽  
Robin Wait
Keyword(s):  
Legionella Pneumophila ◽  
Tap Water ◽  
Water Environment ◽  
Nile Red ◽  
Dry Weight ◽  
Gas Chromatography Mass Spectrometry ◽  
Resonance Spectroscopy ◽  
Term Survival ◽  
Reliable Determination ◽  
Nonculturable Cells

ABSTRACT Chloroform-soluble material was extracted from two strains ofL. pneumophila serogroup 1 following growth in continuous culture. The purified material was identified as poly-3-hydroxybutyrate (PHB) by nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry. PHB yields of up to 16% of cell dry weight were extracted from culture samples. The PHB was located in electron-dense intracellular inclusions, which fluoresced bright yellow when stained with the lipophilic dye Nile red. A Nile red spectrofluorometric assay provided a more accurate and reliable determination of the PHB content. PHB accumulation increased threefold during iron-limited culture and was inversely related to the concentration of iron metabolized. Chemostat-grown cells survived in a culturable state for at least 600 days when incubated at 24°C in a low-nutrient tap water environment. Nile red spectrofluorometry and flow cytometry demonstrated that PHB reserves were utilized during starvation. PHB utilization, as revealed by the decline in mean cellular fluorescence and cell complexity, correlated with loss of culturability. Fluorescence microscopy provided visual evidence of PHB utilization, with a marked reduction in the number of Nile red-stained granules during starvation. Heat shock treatment failed to resuscitate nonculturable cells. This study demonstrates that L. pneumophila accumulates significant intracellular reserves of PHB, which promote its long-term survival under conditions of starvation.

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