Aerobic Degradation of N-Methyl-4-Nitroaniline (MNA) by Pseudomonas sp. Strain FK357 Isolated from Soil

N-Methyl-4-nitroaniline (MNA) is used as an additive to lower the melting temperature of energetic materials in the synthesis of insensitive explosives. Although the biotransformation of MNA under anaerobic condition has been reported, its aerobic microbial degradation has not been documented yet. A soil microcosms study showed the efficient aerobic degradation of MNA by the inhabitant soil microorganisms. An aerobic bacterium, Pseudomonas sp. strain FK357, able to utilize MNA as the sole carbon, nitrogen, and energy source, was isolated from soil microcosms. HPLC and GC-MS analysis of the samples obtained from growth and resting cell studies showed the formation of 4-nitroaniline (4-NA), 4-aminophenol (4-AP), and 1, 2, 4-benzenetriol (BT) as major metabolic intermediates in the MNA degradation pathway. Enzymatic assay carried out on cell-free lysates of MNA grown cells confirmed N-demethylation reaction is the first step of MNA degradation with the formation of 4-NA and formaldehyde products. Flavin-dependent transformation of 4-NA to 4-AP in cell extracts demonstrated that the second step of MNA degradation is a monooxygenation. Furthermore, conversion of 4-AP to BT by MNA grown cells indicates the involvement of oxidative deamination (release of NH₂ substituent) reaction in third step of MNA degradation. Subsequent degradation of BT occurs by the action of benzenetriol 1, 2-dioxygenase as reported for the degradation of 4-nitrophenol. This is the first report on aerobic degradation of MNA by a single bacterium along with elucidation of metabolic pathway.     

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.     

A Novel Transformation of Polychlorinated Biphenyls by Rhodococcus sp. Strain RHA1

We have characterized a biphenyl degrader, Rhodococcus sp. strain RHA1. Biphenyl-grown cells of strain RHA1 efficiently transformed 45 components in the 62 major peaks of a polychlorinated biphenyl (PCB) mixture of Kanechlors 200, 300, 400, and 500 within 3 days, which includes mono- to octachlorobiphenyls. Among the intermediate metabolites of PCB transformation, di- and trichlorobenzoic acids were identified. The gradual decrease of these chlorobenzoic acids during incubation indicated that these chlorobenzoic acids would also be degraded by this strain. The effect of the position of chlorine substitution was determined by using PCB mixtures that have chlorine substitutions mainly at either the ortho or the meta position. This strain transformed both types of congeners, and strong PCB transformation activity of RHA1 was indicated. RHA1 accumulated 4-chlorobenzoic acid temporally during the transformation of 4-chlorobiphenyl. The release of most chloride in the course of 2,2(prm1)-dichlorobiphenyl degradation was observed. These results suggested that RHA1 would break down at least some PCB congeners into smaller molecules to a considerable extent.     

Monocyclic Aromatic Hydrocarbon Degradation by Rhodococcus sp. Strain DK17

Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.     

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.     

Metabolism of 2-Methylpropene (Isobutylene) by the Aerobic Bacterium Mycobacterium sp. Strain ELW1

An aerobic bacterium (Mycobacterium sp. strain ELW1) that utilizes 2-methylpropene (isobutylene) as a sole source of carbon and energy was isolated and characterized. Strain ELW1 grew on 2-methylpropene (growth rate = 0.05 h⁻¹) with a yield of 0.38 mg (dry weight) mg 2-methylpropene⁻¹. Strain ELW1 also grew more slowly on both cis- and trans-2-butene but did not grow on any other C₂ to C₅ straight-chain, branched, or chlorinated alkenes tested. Resting 2-methylpropene-grown cells consumed ethene, propene, and 1-butene without a lag phase. Epoxyethane accumulated as the only detected product of ethene oxidation. Both alkene consumption and epoxyethane production were fully inhibited in cells exposed to 1-octyne, suggesting that alkene oxidation is initiated by an alkyne-sensitive, epoxide-generating monooxygenase. Kinetic analyses indicated that 1,2-epoxy-2-methylpropane is rapidly consumed during 2-methylpropene degradation, while 2-methyl-2-propen-1-ol is not a significant metabolite of 2-methylpropene catabolism. Degradation of 1,2-epoxy-2-methylpropane by 2-methylpropene-grown cells led to the accumulation and further degradation of 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate, two sequential metabolites previously identified in the aerobic microbial metabolism of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA). Growth of strain ELW1 on 2-methylpropene, 1,2-epoxy-2-methylpropane, 2-methyl-1,2-propanediol, and 2-hydroxyisobutyrate was fully inhibited when cobalt ions were omitted from the growth medium, while growth on 3-hydroxybutyrate and other substrates was unaffected by the absence of added cobalt ions. Our results suggest that, like aerobic MTBE- and TBA-metabolizing bacteria, strain ELW1 utilizes a cobalt/cobalamin-dependent mutase to transform 2-hydroxyisobutyrate. Our results have been interpreted in terms of their impact on our understanding of the microbial metabolism of alkenes and ether oxygenates.     

Degradation of 2-Methylnaphthalene by Pseudomonas sp. Strain NGK1

Pseudomonas sp. strain NGK1, a soil bacterium isolated by naphthalene enrichment from biological waste effluent treatment, capable of utilizing 2-methylnaphthalene as sole source of carbon and energy. To deduce the pathway for biodegradation of 2-methylnaphthalene, metabolites were isolated from the spent medium and identified by thin-layer chromatography and high-performance liquid chromatography. The characterization of purified metabolites, oxygen uptake studies, and enzyme activities revealed that the strain degrades 2-methylnaphthalene through more than one pathway. The growth of the bacterium, utilization of 2-methylnaphthalene, and 4-methylsalicylate accumulation by Pseudomonas sp. strain NGK1 were studied at various incubation periods. Received: 20 March 2001 / Accepted: 25 April 2001     

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.     

A Two-Component para-Nitrophenol Monooxygenase Initiates a Novel 2-Chloro-4-Nitrophenol Catabolism Pathway in Rhodococcus imtechensis RKJ300

Rhodococcus imtechensis RKJ300 (DSM 45091) grows on 2-chloro-4-nitrophenol (2C4NP) and para-nitrophenol (PNP) as the sole carbon and nitrogen sources. In this study, by genetic and biochemical analyses, a novel 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with hydroxyquinol (hydroxy-1,4-hydroquinone or 1,2,4-benzenetriol [BT]) as the ring cleavage substrate. Real-time quantitative PCR analysis indicated that the pnp cluster located in three operons is likely involved in the catabolism of both 2C4NP and PNP. The oxygenase component (PnpA1) and reductase component (PnpA2) of the two-component PNP monooxygenase were expressed and purified to homogeneity, respectively. The identification of chlorohydroquinone (CHQ) and BT during 2C4NP degradation catalyzed by PnpA1A2 indicated that PnpA1A2 catalyzes the sequential denitration and dechlorination of 2C4NP to BT and catalyzes the conversion of PNP to BT. Genetic analyses revealed that pnpA1 plays an essential role in both 2C4NP and PNP degradations by gene knockout and complementation. In addition to catalyzing the oxidation of CHQ to BT, PnpA1A2 was also found to be able to catalyze the hydroxylation of hydroquinone (HQ) to BT, revealing the probable fate of HQ that remains unclear in PNP catabolism by Gram-positive bacteria. This study fills a gap in our knowledge of the 2C4NP degradation mechanism in Gram-positive bacteria and also enhances our understanding of the genetic and biochemical diversity of 2C4NP catabolism.     

Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil

Aims:  The aim of this study is to isolate and characterize organisms capable of utilizing high concentration atrazine from the contaminated sites.
Methods and Results:  A selective enrichment was used for isolating atrazine-degrading organisms from the contaminated sites resulting in isolation of an efficient atrazine-degrading organism designated as strain MB-P1. On the basis of 16S rRNA gene sequencing, total cellular fatty acid analysis and physiological and biochemical tests, strain MB-P1 was identified as a member of genus Rhodococcus . High performance liquid chromatography was performed to identify the atrazine degradation intermediates demonstrating that the degradation proceeds via formation of 'de-ethylatrazine' and 'de-isopropylatrazine'. Further, plasmid curing by SDS method showed atrazine-degrading gene(s) to be plasmid-encoded.
Conclusions:  We have successfully isolated a Rhodococcus sp. strain MB-P1 which is capable of utilizing atrazine as sole source of carbon and energy at very high concentrations of 1000 ppm. The pathway for degradation of atrazine has also been determined. The metabolic gene(s) responsible for atrazine degradation was found to be plasmid-encoded.
Significance and Impact of the Study:  Rhodococcus sp. strain MB-P1 could be used as an ideal model system for in-situ degradation and restoration of ecological niches which are heavily contaminated with atrazine.     

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.     

Chloromethane Metabolism by Methylobacterium sp. Strain CM4

Methylobacterium sp. strain CM4 metabolized chloromethane quantitatively with a molar yield of 2.8 g of whole-cell protein/mol of C. This value was similar to that observed after growth with methanol (2.9 g of protein/mol of C) and about three times larger than the yield with formate (0.94 g of protein/mol of C). Chloromethane dehalogenation activity was inducible. MiniTn5 transposon insertion mutants with altered growth characteristics with chloromethane and other C₁ compounds were isolated and characterized. Nine of these were unable to grow with chloromethane but were able to grow with methanol, methylamine, or formate. Seventy-three transposon mutants that were defective in the utilization of either methanol, methylamine, methanol plus methylamine, or formate could still grow with chloromethane. Based on the protein yield data and the properties of the transposon mutants, we propose a pathway for chloromethane metabolism that depends on methyltransferase and dehydrogenase activities.     

Metabolism of Naphthalene, 1-Naphthol, Indene, and Indole by Rhodococcus sp. Strain NCIMB 12038

The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.     

Cometabolic Degradation of Trichloroethene by Rhodococcus sp. Strain L4 Immobilized on Plant Materials Rich in Essential Oils

The cometabolic degradation of trichloroethene (TCE) by Rhodococcus sp. L4 was limited by the loss of enzyme activity during TCE transformation. This problem was overcome by repeated addition of inducing substrates, such as cumene, limonene, or cumin aldehyde, to the cells. Alternatively, Rhodococcus sp. L4 was immobilized on plant materials which contain those inducers in their essential oils. Cumin seeds were the most suitable immobilizing material, and the immobilized cells tolerated up to 68 μM TCE and degraded TCE continuously. The activity of immobilized cells, which had been inactivated partially during TCE degradation, could be reactivated by incubation in mineral salts medium without TCE. These findings demonstrate that immobilization of Rhodococcus sp. L4 on plant materials rich in essential oils is a promising method for efficient cometabolic degradation of TCE.Various bacteria have been reported to degrade trichloroethene (TCE) aerobically via cometabolic degradation with broad-substrate-specificity enzymes (2). However, TCE cometabolic degradation is considered an unsustainable process due to cytotoxicity, inhibition, or inactivation of TCE-degrading enzymes. These phenomena have been observed in studies using both whole cells and purified enzymes, including soluble methane monooxygenases from Methylosinus trichosporium OB3b (9) and Nitrosomonas europaea (13), toluene 2-monooxygenase from Burkholderia cepacia G4 (19, 27), toluene dioxygenase (TDO) from Pseudomonas putida F1 (15, 18), and butane-oxidizing bacteria, i.e., Pseudomonas butanovora, Mycobacterium vaccae, and Nocardioides sp. CF8 (11). Nevertheless, the addition of an inducer or growth substrate can maintain TCE cometabolic degradation. For example, the TCE-degrading activity of P. putida F1 toluene dioxygenase was restored after adding benzene, cumene, or toluene to displace TCE and its reactive intermediates from the enzyme active site (18). Arp et al. (2) suggested that the rate of enzyme maintenance and recovery depended on the extent of inactivation and the balance of TCE and inducer/growth substrate concentrations.Plant essential oils and their components, such as citral, limonene, cumene, and cumin aldehyde, have been found to induce TCE degradation in Rhodococcus sp. L4 (24). However, the removal of TCE by this bacterium was effective only for a short period. The impacts of TCE on Rhodococcus spp. and their enzymes have not been studied in detail, even though many bacteria of this genus exhibited high TCE-degrading activities (i.e., Rhodococcus erythropolis JE 77, R. erythropolis BD2, Rhodococcus sp. Sm-1, and Rhodococcus sp. Wrink) (5, 6, 7, 16). This study therefore investigated the changes in TCE-degrading activity of Rhodococcus sp. L4 cells and TDO during exposure to TCE. Two enzyme maintenance approaches were evaluated, namely, repeated addition of essential oil components to the system and immobilization of the bacterial cells on plant material rich in essential oils. Immobilized microorganisms are generally capable of degrading pollutants at a higher initial concentration and for a longer period than those of free cells (21, 23), possibly because the microbial cells are protected from environmental stress and toxic compounds (3). In this study, the plant materials were used to provide a solid surface for bacterial attachment and a continuous source of essential oils for inducing TCE-degrading enzymes. Our results show that the repeated addition of limonene, cumene, or cumin aldehyde enhances TCE degradation and that bacteria immobilized on cumin seeds are able to maintain their TCE-degrading activity.