Anaerobic degradation of naphthalene and 2-methylnaphthalene by strains of marine sulfate-reducing bacteria

The anaerobic biodegradation of naphthalene, an aromatic hydrocarbon in tar and petroleum, has been repeatedly observed in environments but scarcely in pure cultures. To further explore the relationships and physiology of anaerobic naphthalene-degrading microorganisms, sulfate-reducing bacteria (SRB) were enriched from a Mediterranean sediment with added naphthalene. Two strains (NaphS3, NaphS6) with oval cells were isolated which showed naphthalene-dependent sulfate reduction. According to 16S rRNA gene sequences, both strains were Deltaproteobacteria and closely related to each other and to a previously described naphthalene-degrading sulfate-reducing strain (NaphS2) from a North Sea habitat. Other close relatives were SRB able to degrade alkylbenzenes, and phylotypes enriched anaerobically with benzene. If in adaptation experiments the three naphthalene-grown strains were exposed to 2-methylnaphthalene, this compound was utilized after a pronounced lag phase, indicating that naphthalene did not induce the capacity for 2-methylnaphthalene degradation. Comparative denaturing gel electrophoresis of cells grown with naphthalene or 2-methylnaphthalene revealed a striking protein band which was only present upon growth with the latter substrate. Peptide sequences from this band perfectly matched those of a protein predicted from genomic libraries of the strains. Sequence similarity (50% identity) of the predicted protein to the large subunit of the toluene-activating enzyme (benzylsuccinate synthase) from other anaerobic bacteria indicated that the detected protein is part of an analogous 2-methylnaphthalene-activating enzyme. The absence of this protein in naphthalene-grown cells together with the adaptation experiments as well as isotopic metabolite differentiation upon growth with a mixture of d(8)-naphthalene and unlabelled 2-methylnaphthalene suggest that the marine strains do not metabolize naphthalene by initial methylation via 2-methylnaphthalene, a previously suggested mechanism. The inability to utilize 1-naphthol or 2-naphthol also excludes these compounds as free intermediates. Results leave open the possibility of naphthalene carboxylation, another previously suggested activation mechanism.     

Anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment culture

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 microM 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.     

Conversion of cis‐2‐carboxycyclohexylacetyl‐CoA in the downstream pathway of anaerobic naphthalene degradation

The cyclohexane derivative cis‐2‐(carboxymethyl)cyclohexane‐1‐carboxylic acid [(1R,2R)‐/(1S,2S)‐2‐(carboxymethyl)cyclohexane‐1‐carboxylic acid] has previously been identified as metabolite in the pathway of anaerobic degradation of naphthalene by sulfate‐reducing bacteria. We tested the corresponding CoA esters of isomers and analogues of this compound for conversion in cell free extracts of the anaerobic naphthalene degraders Desulfobacterium strain N47 and Deltaproteobacterium strain NaphS2. Conversion was only observed for the cis‐isomer, verifying that this is a true intermediate and not a dead‐end product. Mass‐spectrometric analyses confirmed that conversion is performed by an acyl‐CoA dehydrogenase and a subsequent hydratase yielding an intermediate with a tertiary hydroxyl‐group. We propose that a novel kind of ring‐opening lyase is involved in the further catabolic pathway proceeding via pimeloyl‐CoA. In contrast to degradation pathways of monocyclic aromatic compounds where ring‐cleavage is achieved via hydratases, this lyase might represent a new ring‐opening strategy for the degradation of polycyclic compounds. Conversion of the potential downstream metabolites pimeloyl‐CoA and glutaryl‐CoA was proved in cell free extracts, yielding 2,3‐dehydropimeloyl‐CoA, 3‐hydroxypimeloyl‐CoA, 3‐oxopimeloyl‐CoA, glutaconyl‐CoA, crotonyl‐CoA, 3‐hydroxybutyryl‐CoA and acetyl‐CoA as observable intermediates. This indicates a link to central metabolism via β‐oxidation, a non‐decarboxylating glutaryl‐CoA dehydrogenase and a subsequent glutaconyl‐CoA decarboxylase.     

Metabolism of naphthalene, 2-methylnaphthalene, salicylate, and benzoate by Pseudomonas PG: regulation of tangential pathways.

Naphthalene is metabolized by Pseudomonas PG through 1,2-dihydroxynaphthalene and salicylate to catechol, which is then degraded by the meta pathway. 2-Methylnaphthalene, but not 1-methylnaphthalene, also serves as a growth substrate and is metabolized by the same route, through 4-methylcatechol. The same nonspecific meta pathway enzymes appear to be induced by growth on either naphthalene or 2-methylnaphthalene. The level to which 2-hydroxymuconic semialdehyde hydrolase is induced is low and probably of no metabolic significance. Growth on salicylate or catechol, both intermediates of naphthalene degradation, or benzoate results in induction of the ortho pathway, the alternative route for catechol dissimilation. No induction of 1,2-dihydroxynaphthalene oxygenase was found in salicylate-grown cells. Anaerobic growth on a succinate-nitrate medium in the presence of various inducers indicates that cis, cis-muconate, or one of its metabolites is the inducer of the ortho pathway enzymes. The inducer or inducers of the early enzymes of naphthalene degradation and of the meta pathway enzymes must be an early intermediate of the naphthalene pathway above salicylate.     

Identification of new enzymes potentially involved in anaerobic naphthalene degradation by the sulfate-reducing enrichment culture N47

The sulfate-reducing highly enriched culture N47 is capable to anaerobically degrade naphthalene, 2-methylnaphthalene, and 2-naphthoic acid. A proteogenomic investigation was performed to elucidate the initial activation reaction of anaerobic naphthalene degradation. This lead to the identification of an alpha-subunit of a carboxylase protein that was two-fold up-regulated in naphthalene-grown cells compared to 2-methylnaphthalene-grown cells. The putative naphthalene carboxylase subunit showed 48% similarity to the anaerobic benzene carboxylase from an iron-reducing, benzene-degrading culture and 45% to alpha-subunit of phenylphosphate carboxylase of Aromatoleum aromaticum EbN1. A gene for the beta-subunit of putative naphthalene carboxylase was located nearby on the genome and was expressed with naphthalene. Similar to anaerobic benzene carboxylase, there were no genes for gamma- and delta-subunits of a putative carboxylase protein located on the genome which excludes participation in degradation of phenolic compounds. The genes identified for putative naphthalene carboxylase subunits showed only weak similarity to 4-hydroxybenzoate decarboxylase excluding ATP-independent carboxylation. Several ORFs were identified that possibly encode a 2-naphthoate-CoA ligase, which is obligate for activation before the subsequent ring reduction by naphthoyl-CoA reductase. One of these ligases was exclusively expressed on naphthalene and 2-naphthoic acid and might be the responsible naphthoate-CoA-ligase.     

Pyrene degradation by a Mycobacterium sp.: identification of ring oxidation and ring fission products

The degradation of pyrene, a polycyclic aromatic hydrocarbon containing four aromatic rings, by pure cultures of a Mycobacterium sp. was studied. Over 60% of [14C]pyrene was mineralized to CO2 after 96 h of incubation at 24 degrees C. High-pressure liquid chromatography analyses showed the presence of one major and at least six other metabolites that accounted for 95% of the total organic-extractable 14C-labeled residues. Analyses by UV, infrared, mass, and nuclear magnetic resonance spectrometry and gas chromatography identified both pyrene cis- and trans-4,5-dihydrodiols and pyrenol as initial microbial ring-oxidation products of pyrene. The major metabolite, 4-phenanthroic acid, and 4-hydroxyperinaphthenone and cinnamic and phthalic acids were identified as ring fission products. 18O2 studies showed that the formation of cis- and trans-4,5-dihydrodiols were catalyzed by dioxygenase and monooxygenase enzymes, respectively. This is the first report of the chemical pathway for the microbial catabolism of pyrene.     

Kinetics of chromate reduction during naphthalene degradation in a mixed culture

A mixed culture of Bacillus sp. K1 and Sphingomonas paucimobilis EPA 505 was exposed to chromate and naphthalene. Batch experiments showed that chromate was reduced and naphthalene was degraded by the mixed culture. Chromate reduction occurred initially at a high rate followed by a decrease in rate until chromate reduction ceased. Chromate reduction decreased in the mixed culture when a lower ratio of S. paucimobilis EPA 505 to Bacillus sp. K1 was utilized. A kinetic model incoporating a term for the cell density ratio is proposed to describe chromate reduction in the mixed culture under both chromate limited and electron donor limited conditions. The validity of the model, and its parameter values, was verified by experimental data generated under a variety of initial population compositions and a broad range of chromate concentrations. The consistent result of experimental data with model predictions implies that the model is useful for evaluating the interactions and the use of mixed culture for chromate removal. (c) 1996 John Wiley & Sons, Inc.     

Fermentative degradation of glyoxylate by a new strictly anaerobic bacterium

A new strictly anaerobic, gram-negative, nonsporeforming bacterium, Strain PerGlx1, was enriched and isolated from marine sediment samples with glyoxylate as sole carbon and energy source. The guanineplus-cytosine content of the DNA was 44.1±0.2 mol %. Glyoxylate was utilized as the only substrate and was stoichiometrically degraded to carbon dioxide, hydrogen, and glycolate. An acetyl-CoA and ADP-dependent glyoxylate converting enzyme activity, malic enzyme, and pyruvate synthase were found at activities sufficient for growth (0.25 U x mg protein^-1). These findings allow to design a new degradation pathway for glyoxylate: glyoxylate is condensed with acetyl-CoA to form malyl-CoA; the free energy of the thioester linkage in malyl-CoA is conserved by substrate level phosphorylation. Part of the electrons released during glyoxylate oxidation to CO₂ reduce a small fraction of glyoxylate to glycolate.     

Biotransformation of biphenyl by Paecilomyces lilacinus and characterization of ring cleavage products

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4'-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.     

Transformation of diphenyl ethers by Trametes versicolor and characterization of ring cleavage products

The white-rot fungi Trametes versicolor SBUG 1050, DSM 11269 and DSM 11309 are able to oxidize diphenyl ether and its halogenated derivatives 4-bromo- and 4-chlorodiphenyl ether. The products formed from diphenyl ether were 2- and 4-hydroxydiphenyl ether. Both 4-bromo- and 4-chlorodiphenyl ether were transformed to the corresponding products hydroxylated at the non-halogenated ring. Additionally, ring-cleavage products were detected by high perfomance liquid chromatography and characterized by gas chromatography/mass spectrometry and proton nuclear magnetic resonance spectroscopy. Unhalogenated diphenyl ether was degraded to 2-hydroxy-4-phenoxymuconic acid and 6-carboxy-4-phenoxy-2-pyrone. Brominated derivatives of both these compounds were formed from 4-bromodiphenyl ether, and 4-chlorodiphenyl ether was transformed in the same way to the analogous chlorinated ring cleavage products. Additionally, 4-bromo- and 4-chlorophenol were detected as intermediates from 4-bromo- and 4-chlorodiphenyl ether, respectively. In the presence of the cytochrome-P450 inhibitor 1-aminobenzotriazole, no metabolites were formed by cells of Trametes versicolor from the diphenyl ethers investigated. Cell-free supernatants of whole cultures with high laccase and manganese peroxidase activities were not able to transform the unhydroxylated diphenyl ethers used.     

2-Hydroxycyclohexanecarboxyl coenzyme A dehydrogenase, an enzyme characteristic of the anaerobic benzoate degradation pathway used by Rhodopseudomonas palustris

A gene, badH, whose predicted product is a member of the short-chain dehydrogenase/reductase family of enzymes, was recently discovered during studies of anaerobic benzoate degradation by the photoheterotrophic bacterium Rhodopseudomonas palustris. Purified histidine-tagged BadH protein catalyzed the oxidation of 2-hydroxycyclohexanecarboxyl coenzyme A (2-hydroxychc-CoA) to 2-ketocyclohexanecarboxyl-CoA. These compounds are proposed intermediates of a series of three reactions that are shared by the pathways of cyclohexanecarboxylate and benzoate degradation used by R. palustris. The 2-hydroxychc-CoA dehydrogenase activity encoded by badH was dependent on the presence of NAD(+); no activity was detected with NADP(+) as a cofactor. The dehydrogenase activity was not sensitive to oxygen. The enzyme has apparent K(m) values of 10 and 200 microM for 2-hydroxychc-CoA and NAD(+), respectively. Western blot analysis with antisera raised against purified His-BadH identified a 27-kDa protein that was present in benzoate- and cyclohexanecarboxylate-grown but not in succinate-grown R. palustris cell extracts. The active form of the enzyme is a homotetramer. badH was determined to be the first gene in an operon, termed the cyclohexanecarboxylate degradation operon, containing genes required for both benzoate and cyclohexanecarboxylate degradation. A nonpolar R. palustris badH mutant was unable to grow on benzoate or cyclohexanecarboxylate but had wild-type growth rates on succinate. Cells blocked in expression of the entire cyclohexanecarboxylate degradation operon excreted cyclohex-1-ene-1-carboxylate into the growth medium when given benzoate. This confirms that cyclohex-1-ene-1-carboxyl-CoA is an intermediate of anaerobic benzoate degradation by R. palustris. This compound had previously been shown not to be formed by Thauera aromatica, a denitrifying bacterium that degrades benzoate by a pathway that is slightly different from the R. palustris pathway. 2-Hydroxychc-CoA dehydrogenase does not participate in anaerobic benzoate degradation by T. aromatica and thus may serve as a useful indicator of an R. palustris-type benzoate degradation pathway.     

Propionic acid accumulation and degradation during restart of a full-scale anaerobic biowaste digester

The methane formation rate of 300 m(3) of sludge from a full scale biowaste reactor, that was stored without feeding for six weeks during a maintenance period, was about 60% of the methanogenic activity before maintenance. The 300 m(3) sludge was then pumped back into the biowaste reactor. On the third day, after refilling of the stored biowaste suspension, anaerobic conditions were obtained and feeding was started by addition of 36.1 m(3) of fresh biowaste suspension (=11.3 tons biowaste). The pH dropped from originally pH 7.7 to pH 7.3 and later on to pH 6.8, which was considered the minimum allowed pH for methanogenesis to recover. Maximum concentrations of acetate (1.78 gl(-1)), n-butyrate (0.57 gl(-1)) and n-valerate (0.44 gl(-1)) accumulated during the following days with feeding of 11.8 tons on day 5 and twice 6.5 tons on days 7 and 9, respectively. Thereafter, acetate, n-butyrate and n-valerate were degraded completely, whereas the concentration of propionate was still increasing. Propionic acid was the dominant fatty acid during the restart period and reached its maximum concentration of 6.2 gl(-1) 17 days after start of feeding. This high level of propionate was degraded completely in about 5 days with maximum degradation rates of 2.14 gl(-1)d(-1), and the pH of the anaerobic sludge increased from 7.1 to 7.4. During restart, the methane content of the biogas increased successively to 65%. Samples that were taken at different time intervals during the restart phase of the methane reactor showed different fatty acid degradation capabilities. After 10 days, when acetate and n-butyrate still accumulated in the methane reactor the maximum acetate degradation rate was 1.52 gl(-1)d(-1) and the n-butyrate degradation rate was 0.51 gl(-1)d(-1). Oxidation of n-valerate caused an increase of propionate, which was degraded after a lag phase of 6 days with a maximum rate of 0.6 gl(-1)d(-1). In the samples taken after 16 and 23 days, the propionate degradation rate increased to 1.42 gl(-1)d(-1) and 1.55 gl(-1)d(-1), respectively, and the lag phase for propionate degradation was reduced or had disappeared completely. The maximum propionate degradation rate was measured in the methane reactor in the fourth week after restart. The synthrophic propionate oxidizing bacteria were apparently the most suffering bacteria during sludge storage. If the propionate oxidizing bacteria could be kept active and the propionate degrading activity of the biowaste suspension of 6.16 gl(-1)d(-1) before the maintenance period could be maintained, then accumulation of 6.2 gl(-1) propionate in the methane reactor after restart could be avoided and full activity reached even earlier.     

In vitro metabolic stability of obestatin: kinetics and identification of cleavage products

The in vitro metabolic stability testing on synthetic obestatin peptides from two different species (human hOb and mouse mOb) using HPLC analysis is described. A reversed-phase C(18) column of 300A pore size was used, with a gradient system based on aqueous formic acid and acetonitrile. Electrospray ionization (ESI) ion trap mass spectrometry was used for identification of the chromatographic eluting peptide metabolic products, while UV (DAD) and fluorescence served quantitative purposes. Differences in the metabolic degradation kinetics of hOb and mOb were found in plasma, liver and kidney homogenate, with half-lives ranging between 12.6 and 138.0min. Proteolytic hydrolysis at the N-terminal Phe residue and cleavage at Pro(4)-Phe(5) were found to be two major metabolic pathways, accounting for more than 50% of the metabolic degradation. Several other labile peptide bonds were located. The influence of a standard protease inhibitor cocktail was investigated, as well as the metabolism of iodinated human obestatin in liver homogenate. Our results indicate that the major instability of obestatin peptides, as currently used in biomedical investigations, should be taken into account in the interpretation of the obtained results.     

Isolation and characterization of a new denitrifying spirillum capable of anaerobic degradation of phenol

Two kinds of phenol-degrading denitrifying bacteria, Azoarcus sp. strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively. Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under iron-limited conditions, which may have resulted in the observed change in the phenol-degrading bacteria during the enrichment process. Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions. Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a Magnetospirillum sp., a member of the alpha subclass of the class Proteobacteria, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group. Unlike previously described Magnetospirillum strains, however, this strain did not exhibit magnetotaxis. It grew on phenol only under denitrifying conditions. Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features.     

Metagenome and metaproteome analyses of microbial communities in mesophilic biogas-producing anaerobic batch fermentations indicate concerted plant carbohydrate degradation

Microbial communities in biogas batch fermentations, using straw and hay as co-substrates, were analyzed at the gene and protein level by metagenomic and metaproteomic approaches. The analysis of metagenomic data revealed that the Clostridiales and Bacteroidales orders were prevalent in the community. However, the number of sequences assigned to the Clostridiales order decreased during fermentation, whereas the number of sequences assigned to the Bacteroidales order increased. In addition, changes at the functional level were monitored and the metaproteomic analyses detected transporter proteins and flagellins, which were expressed mainly by members of the Bacteroidetes and Firmicutes phyla. A high number of sugar transporters, expressed by members of the Bacteroidetes, proved their potential to take up various glycans efficiently. Metagenome data also showed that methanogenic organisms represented less than 4% of the community, while 20–30% of the identified proteins were of archeal origin. These data suggested that methanogens were disproportionally active.     

Studies on the mechanism of ring hydrolysis in phenylacetate degradation: a metabolic branching point

The widespread, long sought-after bacterial aerobic phenylalanine/phenylacetate catabolic pathway has recently been elucidated. It proceeds via coenzyme A (CoA) thioesters and involves the epoxidation of the aromatic ring of phenylacetyl-CoA, subsequent isomerization to an uncommon seven-membered C-O-heterocycle (oxepin-CoA), and non-oxygenolytic ring cleavage. Here we characterize the hydrolytic oxepin-CoA ring cleavage catalyzed by the bifunctional fusion protein PaaZ. The enzyme consists of a C-terminal (R)-specific enoyl-CoA hydratase domain (formerly MaoC) that cleaves the ring and produces a highly reactive aldehyde and an N-terminal NADP(+)-dependent aldehyde dehydrogenase domain that oxidizes the aldehyde to 3-oxo-5,6-dehydrosuberyl-CoA. In many phenylacetate-utilizing bacteria, the genes for the pathway exist in a cluster that contains an NAD(+)-dependent aldehyde dehydrogenase in place of PaaZ, whereas the aldehyde-producing hydratase is encoded outside of the cluster. If not oxidized immediately, the reactive aldehyde condenses intramolecularly to a stable cyclic derivative that is largely prevented by PaaZ fusion in vivo. Interestingly, the derivative likely serves as the starting material for the synthesis of antibiotics (e.g. tropodithietic acid) and other tropone/tropolone related compounds as well as for ω-cycloheptyl fatty acids. Apparently, bacteria made a virtue out of the necessity of disposing the dead-end product with ring hydrolysis as a metabolic branching point.     

Mnl2, a novel component of the ER associated protein degradation pathway

In eukaryotes, membrane and soluble proteins of the secretory pathway enter the endoplasmic reticulum (ER) after synthesis in an unfolded state. Directly after entry, most proteins are modified with glycans at suitable glycosylation sites and start to fold. A protein that cannot fold properly will be degraded in a process called ER associated degradation (ERAD). Failures in ERAD, either by loss of function or by premature degradation of proteins, are a cause of severe diseases. Therefore, the search for novel ERAD components to gain better insight in this process is of high importance. Carbohydrate trimming is a relevant process in ER quality control. In this work a novel putative yeast mannosidase encoded by the open reading frame YLR057W was identified and named Mnl2. Deletion of MNL2 diminished the degradation efficiency of misfolded CPY^* in the absence of the cognate mannosidase Mnl1, indicating a specific role in ERAD.