Degradation of anthracene by Mycobacterium sp. strain LB501T proceeds via a novel pathway,through o-phthalic acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Degradation of Phenanthrene and Anthracene by Cell Suspensions of Mycobacterium sp. Strain PYR-1

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, ¹H and ¹³C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2′-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.     

Responses of Mycobacterium sp. LB501T to the low bioavailability of solid anthracene

Several recent reports have indicated that some bacteria may have adapted to the low bioavailability of hydrophobic environmental chemicals and that generalizations about the bioavailability of compounds such as polycyclic aromatic hydrocarbons (PAHs) may be inappropriate. Experimental evidence and theoretical considerations show that the utilization of PAHs requires bioavailability-enhancing mechanisms of the bacteria such as: (1) high-affinity uptake systems, (2) adhesion to the solid substrate, and (3) biosurfactant excretion. We examined possible specific physiological responses of anthracene-degrading Mycobacterium sp. LB501T to poorly water-soluble anthracene in batch cultures, using solid anthracene as a sole carbon source. Mycobacterium sp. LB501T exhibited a high specific affinity for anthracene (a(o)A=32,500 l g(-1) protein h(-1)) and grew as a confluent biofilm on solid anthracene present as sole carbon source. No biofilm formation on anthracene was observed when excess glucose was provided as an additional substrate. This difference could be attributed to a modification of the cell surface of the bacterium. Anthracene-grown cells were significantly more hydrophobic and more negatively charged than glucose-grown cells. In adhesion experiments, anthracene-grown cells adhered 1.5- to 8.0-fold better to hydrophobic Teflon and up to 70-fold better to anthracene surfaces than glucose-grown cells. However, no production of biosurfactants was observed. Our results thus indicate that attachment and biofilm formation may be a specific response of Mycobacterium sp. LB501T to optimize substrate bioavailability.     

Metabolism of 2-Chloro-4-Nitroaniline via Novel Aerobic Degradation Pathway by Rhodococcus sp. Strain MB-P1

2-chloro-4-nitroaniline (2-C-4-NA) is used as an intermediate in the manufacture of dyes, pharmaceuticals, corrosion inhibitor and also used in the synthesis of niclosamide, a molluscicide. It is marked as a black-listed substance due to its poor biodegradability. We report biodegradation of 2-C-4-NA and its pathway characterization by Rhodococcus sp. strain MB-P1 under aerobic conditions. The strain MB-P1 utilizes 2-C-4-NA as the sole carbon, nitrogen, and energy source. In the growth medium, the degradation of 2-C-4-NA occurs with the release of nitrite ions, chloride ions, and ammonia. During the resting cell studies, the 2-C-4-NA-induced cells of strain MB-P1 transformed 2-C-4-NA stoichiometrically to 4-amino-3-chlorophenol (4-A-3-CP), which subsequently gets transformed to 6-chlorohydroxyquinol (6-CHQ) metabolite. Enzyme assays by cell-free lysates prepared from 2-C-4-NA-induced MB-P1 cells, demonstrated that the first enzyme in the 2-C-4-NA degradation pathway is a flavin-dependent monooxygenase that catalyzes the stoichiometric removal of nitro group and production of 4-A-3-CP. Oxygen uptake studies on 4-A-3-CP and related anilines by 2-C-4-NA-induced MB-P1 cells demonstrated the involvement of aniline dioxygenase in the second step of 2-C-4-NA degradation. This is the first report showing 2-C-4-NA degradation and elucidation of corresponding metabolic pathway by an aerobic bacterium.     

Characterization of multiple-substrate utilization by anthracene-degrading Mycobacterium frederiksbergense LB501T

Stable carbon isotope analysis of biomass and analyses of phospholipid fatty acids (PLFA), glycolipid fatty acids (GLFA), and mycolic acids were used to characterize mixed-substrate utilization by Mycobacterium frederiksbergense LB501T under various substrate regimens. The distinct (13)C contents of anthracene and glucose as representatives of typical hydrophobic pollutants and naturally occurring organic compounds, respectively, were monitored during formation into biomass and used to quantify the relative contributions of the two carbon sources to biomass formation. Moreover, the influence of mixed-substrate utilization on PLFA, GLFA, and mycolic acid profiles and cell surface hydrophobicity was investigated. Results revealed that M. frederiksbergense LB501T degrades anthracene and forms biomass from it even in the presence of more readily available dissolved glucose. The relative ratios of straight-chain saturated PLFA to the corresponding unsaturated PLFA and the total fraction of saturated cyclopropyl-branched PLFA of M. frederiksbergense LB501T depended on the carbon source and the various rates of addition of mixed substrates, whereas no such trend was observed with GLFA. Higher proportions of anthracene in the carbon source mixture led to higher cell surface hydrophobicities and more-hydrophobic mycolic acids, which in turn appeared to be valuable indicators for substrate utilization by M. frederiksbergense LB501T. The capability of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria to utilize readily available substrates besides the poorly available PAHs favors the buildup of PAH-degrading biomass. Feeding of supplementary carbon substrates may therefore promote bioremediation, provided that it sustains the pollutant-degrading population rather than other members of the microbial community.     

Characterization of Multiple-Substrate Utilization by Anthracene-Degrading Mycobacterium frederiksbergense LB501T

Stable carbon isotope analysis of biomass and analyses of phospholipid fatty acids (PLFA), glycolipid fatty acids (GLFA), and mycolic acids were used to characterize mixed-substrate utilization by Mycobacterium frederiksbergense LB501T under various substrate regimens. The distinct ¹³C contents of anthracene and glucose as representatives of typical hydrophobic pollutants and naturally occurring organic compounds, respectively, were monitored during formation into biomass and used to quantify the relative contributions of the two carbon sources to biomass formation. Moreover, the influence of mixed-substrate utilization on PLFA, GLFA, and mycolic acid profiles and cell surface hydrophobicity was investigated. Results revealed that M. frederiksbergense LB501T degrades anthracene and forms biomass from it even in the presence of more readily available dissolved glucose. The relative ratios of straight-chain saturated PLFA to the corresponding unsaturated PLFA and the total fraction of saturated cyclopropyl-branched PLFA of M. frederiksbergense LB501T depended on the carbon source and the various rates of addition of mixed substrates, whereas no such trend was observed with GLFA. Higher proportions of anthracene in the carbon source mixture led to higher cell surface hydrophobicities and more-hydrophobic mycolic acids, which in turn appeared to be valuable indicators for substrate utilization by M. frederiksbergense LB501T. The capability of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria to utilize readily available substrates besides the poorly available PAHs favors the buildup of PAH-degrading biomass. Feeding of supplementary carbon substrates may therefore promote bioremediation, provided that it sustains the pollutant-degrading population rather than other members of the microbial community.     

Influence of the growth substrate on ester-linked phospho- and glycolipid fatty acids of PAH-degrading Mycobacterium sp. LB501T

The influences of poorly water-soluble anthracene on ester-linked phospholipid fatty acid (PLFA) and glycolipid fatty acid (GLFA) profiles of Mycobacterium sp. LB501T were studied. Bacteria were cultivated on either anthracene or glucose (one culture with successively amended small doses of this substrate and one with excess concentrations) to distinguish between influences of the chemical structure and the bioavailability of the growth substrate. Results revealed that GLFA and PLFA profiles of M. sp. LB501T depended on the availability and the structure of the carbon source. Fatty acid profiles obtained with anthracene differed from those obtained with excess glucose. They were interpreted as a specific adaptation to this poorly bioavailable polycyclic aromatic hydrocarbon (PAH). In contrast, profiles obtained with low glucose concentrations showed clear signs of starvation stress. Stable carbon isotopic ratios (delta13C) of GLFA and PLFA of M. sp. LB501T were analysed to characterize the 13C-fractionation during the biosynthesis of individual fatty acids and to evaluate their value as markers for substrate usage. Although the delta13C values of PLFA and GLFA showed differential isotope fractionation during anthracene- and glucose-degradation, they were sufficiently distinct to be used as signatures of bacterial substrate usage.     

Chlorobenzene Degradation via Ortho-Cleavage Pathway by Newly Isolated Microbacterium sp. Strain TAS1CB from a Petrochemical-Contaminated Site

Chlorobenzene (CB), a dense nonaqeuous phase liquid (DNAPL), is categorized as a priority pollutant by the US EPA. It enters into ecosystems via solid and liquid waste discharge. Bioremediation is a key technique to remediate such contaminated sites. The present study aimed to isolate a chlorobenzene-degrading bacterium, determine the metabolic pathway for chlorobenzene degradation, and characterize biosurfactant production. Microbacterium sp. strain TAS1CB was isolated from contaminated sites and identified by 16S rRNA gene sequencing. Cells possessing positive chemotaxis for CB indicated their ability to degrade CB. Cells degraded CB via production of chlorobenzene dioxygenase, which converted CB to chlorocatechol. Chlorobenzene dioxygenase production was higher at 7 pH and 30°C. Intermediate metabolite analysis by UV scanning, HPLC, and GC-MS analysis revealed production of chlorocatechol and cis-cis muconate. Thus, Microbacterium was able to degrade CB via an ortho-cleavage pathway. In addition to chlorobenzene dioxygenase production, cells also produced biosurfactant which pseudosolubilized CB and increased degradation rate. Chemical characterization showed it to be a glycolipid-type biosurfactant. A phytotoxity study showed 60% of toxicity decreased after 72 hrs of degradation by isolate.     

Characterization of a Novel Angular Dioxygenase from Fluorene-Degrading Sphingomonas sp. Strain LB126

In this study, the genes involved in the initial attack on fluorene by Sphingomonas sp. strain LB126 were investigated. The α and β subunits of a dioxygenase complex (FlnA1-FlnA2), showing 63 and 51% sequence identity, respectively, to the subunits of an angular dioxygenase from the gram-positive dibenzofuran degrader Terrabacter sp. strain DBF63, were identified. When overexpressed in Escherichia coli, FlnA1-FlnA2 was responsible for the angular oxidation of fluorene, 9-hydroxyfluorene, 9-fluorenone, dibenzofuran, and dibenzo-p-dioxin. Moreover, FlnA1-FlnA2 was able to oxidize polycyclic aromatic hydrocarbons and heteroaromatics, some of which were not oxidized by the dioxygenase from Terrabacter sp. strain DBF63. The quantification of resulting oxidation products showed that fluorene and phenanthrene were the preferred substrates of FlnA1-FlnA2.     

Prothrombin Activation by Platelet-associated Prothrombinase Proceeds through the Prethrombin-2 Pathway via a Concerted Mechanism

The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30–40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released.     

Degradation of Polycarbonate by a Polyester-Degrading Strain, Amycolatopsis sp. Strain HT-6

Amycolatopsis sp. strain HT-6, a poly(tetramethylene succinate) (PTMS)-degrading actinomycete, was observed to degrade poly(tetramethylene carbonate) (PTMC). In a liquid culture with 150 mg of PTMC film, 59% degradation was achieved, but with a low yield of cell growth. On the other hand, PTMS copolymerized with a small amount of PTMC, forming a copolyester carbonate (PEC) that was completely and rapidly degraded with a high yield of cell growth.     

Degradation of 2,3-Diethyl-5-Methylpyrazine by a Newly Discovered Bacterium, Mycobacterium sp. Strain DM-11

A bacterium was isolated from the waste gas treatment plant at a fishmeal processing company on the basis of its capacity to use 2,3-diethyl-5-methylpyrazine (DM) as a sole carbon and energy source. The strain, designated strain DM-11, grew optimally at 25°C and had a doubling time of 29.2 h. The strain did not grow on complex media like tryptic soy broth, Luria-Bertani broth, or nutrient broth or on simple carbon sources like glucose, acetate, oxoglutarate, succinate, or citrate. Only on Löwenstein-Jensen medium was growth observed. The 16S rRNA gene sequence of strain DM-11 showed the highest similarity (96.2%) to Mycobacterium poriferae strain ATCC 35087^T. Therefore, strain DM-11 merits recognition as a novel species within the genus Mycobacterium. DM also served as a sole nitrogen source for the growth of strain DM-11. The degradation of DM by strain DM-11 requires molecular oxygen. The first intermediate was identified as 5,6-diethyl-2-hydroxy-3-methylpyrazine (DHM). Its disappearance was accompanied by the release of ammonium into the culture medium. No other metabolite was detected. We conclude that ring fission occurred directly after the formation of DHM and ammonium was eliminated after ring cleavage. Molecular oxygen was essential for the degradation of DHM. The expression of enzymes involved in the degradation of DM and DHM was regulated. Only cells induced by DM or DHM converted these compounds. Strain DM-11 also grew on 2-ethyl-5(6)-methylpyrazine (EMP) and 2,3,5-trimethylpyrazine (TMP) as a sole carbon, nitrogen, and energy source. In addition, the strain converted many pyrazines found in the waste gases of food industries cometabolically.     

Regiospecificity of Dioxygenation of Di- to Pentachlorobiphenyls and Their Degradation to Chlorobenzoates by the bph-Encoded Catabolic Pathway of Burkholderia sp. Strain LB400

Burkholderia sp. strain LB400 is one of the most potent aerobic polychlorobiphenyl (PCB)-degrading microorganisms that have been characterized. Its PCB-dioxygenating activity originates predominantly or exclusively from the biphenyl dioxygenase encoded by its bph gene cluster. Analysis of the dioxygenation products of several di- to pentachlorinated biphenyls formed by this enzyme revealed a complex dependence of the regiospecificity and the yield of dioxygenation on the substitution patterns of both the oxidized and the nonoxidized rings. No dioxygenolytic attack involving chlorinated meta or para carbons was observed. Therefore, the ability of the enzyme to hydroxylate chlorinated carbons appears to be limited to the ortho position. However, it is not limited to monochlorinated rings, as evidenced by dioxygenation of the 2,4-disubstituted ring at carbons 2 and 3. This site of attack is strikingly different from that of the 2,5-dichlorinated ring, which has been shown to be dihydroxylated at positions 3 and 4 (J. D. Haddock, J. R. Horton, and D. T. Gibson, J. Bacteriol. 177:20–26, 1995). These results demonstrate that a second substituent of ortho-chlorinated rings crucially influences the site of dioxygenation at this ring and thereby determines whether or not the initial chlorobiphenyl oxidation product is further metabolized through the bph-encoded pathway. The 2,4-dichlorinated ring can alternatively be attacked at carbons 5 and 6. The preferred site crucially depends on the substitution pattern of the other ring. The formation of more than a single dioxygenation product was found predominantly with congeners that contain two chlorinated rings, both of which are similarly prone to dioxygenation or one is substituted only at carbon 3.     

Novel Partial Reductive Pathway for 4-Chloronitrobenzene and Nitrobenzene Degradation in Comamonas sp. Strain CNB-1

Comamonas sp. strain CNB-1 grows on 4-chloronitrobenzene (4-CNB) and nitrobenzene as sole carbon and nitrogen sources. In this study, two genetic segments, cnbB-orf2-cnbA and cnbR-orf1-cnbCaCbDEFGHI, located on a newly isolated plasmid, pCNB1 (ca. 89 kb), and involved in 4-CNB/nitrobenzene degradation, were characterized. Seven genes (cnbA, cnbB, cnbCa, cnbCb, cnbD, cnbG, and cnbH) were cloned and functionally expressed in recombinant Escherichia coli, and they were identified as encoding 4-CNB nitroreductase (CnbA), 1-hydroxylaminobenzene mutase (CnbB), 2-aminophenol 1,6-dioxygenase (CnbCab), 2-amino-5-chloromuconic semialdehyde dehydrogenase (CnbD), 2-hydroxy-5-chloromuconic acid (2H5CM) tautomerase, and 2-amino-5-chloromuconic acid (2A5CM) deaminase (CnbH). In particular, the 2A5CM deaminase showed significant identities (31 to 38%) to subunit A of Asp-tRNA^Asn/Glu-tRNA^Gln amidotransferase and not to the previously identified deaminases for nitroaromatic compound degradation. Genetic cloning and expression of cnbH in Escherichia coli revealed that CnbH catalyzed the conversion of 2A5CM into 2H5CM and ammonium. Four other genes (cnbR, cnbE, cnbF, and cnbI) were tentatively identified according to their high sequence identities to other functionally identified genes. It was proposed that CnbH might represent a novel type of deaminase and be involved in a novel partial reductive pathway for chloronitrobenzene or nitrobenzene degradation.     

Degradation of phenanthrene-analogue azaarenes by Mycobacterium gilvum strain LB307T under aerobic conditions

A polycyclic aromatic hydrocarbon degrading Mycobacterium gilvum, strain LB307T, was able to degrade the azaarenes 5,6-benzoquinoline, 7,8-benzoquinoline, and phenanthridine (nitrogen-containing heterocyclic aromatic hydrocarbons) under aerobic conditions. The strain was able to use 5,6-benzoquinoline as sole sources of carbon, nitrogen, and energy. However, inhibition of degradation and growth was observed with increasing substrate concentration. During degradation, metabolites built up transiently. One of the metabolites detected during 5,6-benzoquinoline degradation is suggested to be 2-oxo-5,6-benzoquinoline. This is the first report on bacterial degradation of phenanthrene-analogue azaarenes.     

Novel Intermediates of Acenaphthylene Degradation by Rhizobium sp. Strain CU-A1: Evidence for Naphthalene-1,8-Dicarboxylic Acid Metabolism

The acenaphthylene-degrading bacterium Rhizobium sp. strain CU-A1 was isolated from petroleum-contaminated soil in Thailand. This strain was able to degrade 600 mg/liter acenaphthylene completely within three days. To elucidate the pathway for degradation of acenaphthylene, strain CU-A1 was mutagenized by transposon Tn5 in order to obtain mutant strains deficient in acenaphthylene degradation. Metabolites produced from Tn5-induced mutant strains B1, B5, and A53 were purified by thin-layer chromatography and silica gel column chromatography and characterized by mass spectrometry. The results suggested that this strain cleaved the fused five-membered ring of acenaphthylene to form naphthalene-1,8-dicarboxylic acid via acenaphthenequinone. One carboxyl group of naphthalene-1,8-dicarboxylic acid was removed to form 1-naphthoic acid which was transformed into salicylic acid before metabolization to gentisic acid. This work is the first report of complete acenaphthylene degradation by a bacterial strain.