Pathway and Molecular Mechanisms for Malachite Green Biodegradation in Exiguobacterium sp. MG2

Malachite green (MG), N-methylated diaminotriphenylmethane, is one of the most common dyes in textile industry and has also been used as an effective antifungal agent. However, due to its negative impact on the environment and carcinogenic effects to mammalian cells, there is a significant interest in developing microbial agents to degrade this type of recalcitrant molecules. Here, an Exiguobacterium sp. MG2 was isolated from a river in Yunnan Province of China as one of the best malachite green degraders. This strain had a high decolorization capability even at the concentration of 2500 mg/l and maintained its stable activity within the pH range from 5.0 to 9.0. High-pressure liquid chromatography, liquid chromatography-mass spectrometry and gas chromatography–mass spectrometry were employed to detect the catabolic pathway of MG. Six intermediate products were identified and a potential biodegradation pathway was proposed. This pathway involves a series of reactions of N-demethylation, reduction, benzene ring-removal, and oxidation, which eventually converted N-methylated diaminotriphenylmethane into N, N-dimethylaniline that is the key precursor to MG. Furthermore, our molecular biology experiments suggested that both triphenylmethane reductase gene tmr and cytochrome P450 participated in MG degradation, consistent with their roles in the proposed pathway. Collectively, our investigation is the first report on a biodegradation pathway of triphenylmethane dye MG in bacteria.     

Comparison of p-Nitrophenol Biodegradation in Field and Laboratory Test Systems

Acclimation of microbial communities exposed to p-nitrophenol (PNP) was measured in laboratory test systems and in a freshwater pond. Laboratory tests were conducted in shake flasks with water, shake flasks with water and sediment, eco-cores, and two sizes of microcosm. The sediment and water samples used in the laboratory experiments were obtained from the pond. After a 6-day acclimation period, PNP was biodegraded rapidly in the pond. When the pond was treated with PNP a second time, biodegradation began immediately. The acclimation periods in laboratory test systems that contained sediment were similar to that in the pond. The acclimation period was threefold longer in shake flasks without sediment. PNP was biodegraded more slowly by microbial communities acclimated in the laboratory than it was in the pond, and the rate of biodegradation varied with the type of test. The number of bacteria able to mineralize PNP increased by 3 orders of magnitude in the pond during the acclimation period. Similar increases accompanied acclimation in the laboratory systems.     

Oxidative Pathway for the Biodegradation of Nitrobenzene by Comamonas sp. Strain JS765

Previous studies have shown that the biodegradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 proceeds by the reduction of nitrobenzene through nitrosobenzene and hydroxylaminobenzene, followed by rearrangement to 2-aminophenol, which then undergoes meta ring cleavage. We report here the isolation of a Comamonas sp. that uses an oxidative pathway for the complete mineralization of nitrobenzene. The isolate, designated strain JS765, uses nitrobenzene as a sole source of carbon, nitrogen, and energy. Nitrobenzene-grown cells oxidized nitrobenzene, with the stoichiometric release of nitrite. Extracts of nitrobenzene-grown JS765 showed high levels of catechol 2,3-dioxygenase activity that were not abolished by heating the cell extracts to 60(deg)C for 10 min. The ring cleavage product had an absorbance maximum at 375 nm, consistent with that of 2-hydroxymuconic semialdehyde. Both NAD-dependent dehydrogenase and NAD-independent hydrolase activities towards 2-hydroxymuconic semialdehyde were induced in extracts of nitrobenzene-grown cells. Catechol accumulated in the reaction mixture when cells preincubated with 3-chlorocatechol were incubated with nitrobenzene. Conversion of nitrobenzene to catechol by induced cells in the presence of 3-chlorocatechol and (sup18)O(inf2) demonstrated the simultaneous incorporation of two atoms of oxygen, which indicated that the initial reaction was dioxygenation. The results indicate that the catabolic pathway involves an initial dioxygenase attack on nitrobenzene with the release of nitrite and formation of catechol, which is subsequently degraded by a meta cleavage pathway.     

Biodegradation of 2,4-Dichlorophenol through a Distal meta-Fission Pathway

Alcaligenes eutrophus JMP222, a derivative of A. eutrophus JMP134 which has lost plasmid pJP4 (encoding the tfd genes for the ortho fission pathway), was induced for the meta fission pathway when grown on o-cresol. Resting cell suspensions, grown on o-cresol, oxidized 2,4-dichlorophenol (2,4-DCP), a degradation product of 2,4-dichlorophenoxyacetic acid, to 3,5-dichlorocatechol. Further degradation of 3,5-dichlorocatechol was observed by the production of a yellow ring fission product with liberation of chloride. Oxidation of 2,4-DCP (305 (mu)M) in 47 hs resulted in 69% dehalogenation through this pathway. The ring fission product was characterized as 2-hydroxy-3,5-dichloro-6-oxo-hexa-2,4-dienoic acid by gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared spectroscopy. These data indicate that 2,4-DCP is degraded through a distal meta ring fission pathway, in contrast to either a suicidal proximal fission or the standard ortho fission pathway.     

Biodegradation of p-toluenesulphonamide by a Pseudomonas sp.

A bacterium capable of utilising p-toluenesulphonamide was isolated from activated sludge. The isolated strain designated PTSA was identified as a Pseudomonas sp. using chemotaxonomic and genetic studies. Pseudomonas PTSA grew on p-toluenesulphonamide in a chemostat with approximately 90% release of sulphate and 80% release of ammonium. The isolate was also able to grow on 4-carboxybenzenesulphonamide and 3,4-dihydroxybenzoate but did not grow on p-toluenesulphonate. The transient appearance of 4-hydroxymethylbenzenesulphonamide and 4-carboxybenzenesulphonamide during p-toluenesulphonamide degradation proves oxidation of the methyl group is the initial attack in the biodegradation pathway. Both metabolites of p-toluenesulphonamide degradation were identified by high-performance liquid chromatography-mass spectrometry. 4-Carboxybenzenesulphonamide is probably converted into 3,4-dihydroxybenzoate and amidosulphurous acid. The latter is a chemically unstable compound in aqueous solutions and immediately converted into sulphite and ammonium. Both sulphite and ammonium were formed during degradation of 4-carboxybenzenesulphonamide.     

Occurrence of two different forms of protocatechuate 3,4-dioxygenase in a Moraxella sp

Two alternative forms of protocatechuate 3,4-dioxygenase (PCase) have been purified from Moraxella sp. strain GU2, a bacterium that is able to grow on guaiacol or various other phenolic compounds as the sole source of carbon and energy. One of these forms (PCase-P) was induced by protocatechuate and had an apparent molecular weight of 220,000. The second form (PCase-G) was induced by guaiacol or other phenolic compounds, such as 2-ethoxyphenol or 4-hydroxybenzoate. It appeared to be smaller (Mr 158,000), and its turnover number was about double that of the former enzyme. Both dioxygenases had similar properties and were built from the association of equal amounts of nonidentical subunits, alpha and beta, which were estimated to have molecular weights of 29,500 and 25,500, respectively. The (alpha beta)3 and (alpha beta)4 structures were suggested for PCases G and P, respectively. On the basis of two-dimensional gel electrophoresis, the alpha and beta polypeptides of PCase-G differed from those of PCase-P. Amino acid analysis supported this conclusion. Both PCases, however, had several other properties in common. It is proposed that both isoenzymes were generated from different sets of alpha and beta subunits, and the significance of these data is discussed.     

Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp.

Previous studies of the biodegradation of nonpolar nitroaromatic compounds have suggested that microorganisms can reduce the nitro groups but cannot cleave the aromatic ring. We report here the initial steps in a pathway for complete biodegradation of 2,4-dinitrotoluene (DNT) by a Pseudomonas sp. isolated from a four-member consortium enriched with DNT. The Pseudomonas sp. degraded DNT as the sole source of carbon and energy under aerobic conditions with stoichiometric release of nitrite. During induction of the enzymes required for growth on DNT, 4-methyl-5-nitrocatechol (MNC) accumulated transiently in the culture fluid when cells grown on acetate were transferred to medium containing DNT as the sole carbon and energy source. Conversion of DNT to MNC in the presence of 18O2 revealed the simultaneous incorporation of two atoms of molecular oxygen, which demonstrated that the reaction was catalyzed by a dioxygenase. Fully induced cells degraded MNC rapidly with stoichiometric release of nitrite. The results indicate an initial dioxygenase attack at the 4,5 position of DNT with the concomitant release of nitrite. Subsequent reactions lead to complete biodegradation and removal of the second nitro group as nitrite.     

Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp.

Previous studies of the biodegradation of nonpolar nitroaromatic compounds have suggested that microorganisms can reduce the nitro groups but cannot cleave the aromatic ring. We report here the initial steps in a pathway for complete biodegradation of 2,4-dinitrotoluene (DNT) by a Pseudomonas sp. isolated from a four-member consortium enriched with DNT. The Pseudomonas sp. degraded DNT as the sole source of carbon and energy under aerobic conditions with stoichiometric release of nitrite. During induction of the enzymes required for growth on DNT, 4-methyl-5-nitrocatechol (MNC) accumulated transiently in the culture fluid when cells grown on acetate were transferred to medium containing DNT as the sole carbon and energy source. Conversion of DNT to MNC in the presence of 18O2 revealed the simultaneous incorporation of two atoms of molecular oxygen, which demonstrated that the reaction was catalyzed by a dioxygenase. Fully induced cells degraded MNC rapidly with stoichiometric release of nitrite. The results indicate an initial dioxygenase attack at the 4,5 position of DNT with the concomitant release of nitrite. Subsequent reactions lead to complete biodegradation and removal of the second nitro group as nitrite.     

Dibenzothiophene Biodegradation by a Pseudomonas sp. in Model Solutions

The presence of a fatty acid and an n-alkane may affect the biodegradation rate of aromatic sulphur compounds such as dibenzothiophene (DBT). A fatty acid (hexadecanoic acid) may form micellar structures favouring DBT bioavailability. n-Alkanes, such as n-dodecane or n-hexadecane, form a film around the aromatic sulphur molecule as a consequence of solvation, thus increasing DBT bioavailability. The mass-transfer rate from the solid to the aqueous phase controls the DBT biodegradation rate when DBT is the only carbon source. Diffusional coassimilation and microbial hydrophobic effects are rate-limiting steps in DBT biodegradation in the presence of aliphatic compounds. Diffusion depends on the DBT concentration in n-alkane, while cometabolism is associated with different n-alkane biodegradation rates. Through the definition of biodesulphurization selectivity and biodesulphurization efficiency, our investigations have shown that a selective aerobic biodesulphurization process is possible by using an unselective biocatalyst, such as a Pseudomonas sp.     

Green fluorescent protein as a visual marker in a p-nitrophenol degrading Moraxella sp.

The green fluorescent protein gene (gfp) was introduced into a p-nitrophenol-metabolizing strain of Moraxella sp. by chromosomal integration. The gfp-marked transformants, designated Moraxella sp. strains G21 and G25, exhibited green fluorescence under UV light. Molecular characterization by PCR and Southern hybridization showed the presence of gfp in both transformants. Both transformants and the parent strain degraded 720 μM of p-nitrophenol with nitrite release within 4 h after inoculation in minimal medium supplemented with yeast extract. Transformants degraded up to 1440 μM p-nitrophenol and mineralized about 60% of 720 μM p-nitrophenol, both in broth and in soil, to the same extent as the parent strain. Insertion of gfp did not adversely affect the expression of p-nitrophenol-degrading genes in the transformants. Survival studies indicated that individual green fluorescent colonies of transformants can be detected up to 2 weeks after inoculation in soil. These marked strains could be of value in studies on microbial survival in the environment.     

Occurrence of two different forms of protocatechuate 3,4-dioxygenase in a Moraxella sp.

Two alternative forms of protocatechuate 3,4-dioxygenase (PCase) have been purified from Moraxella sp. strain GU2, a bacterium that is able to grow on guaiacol or various other phenolic compounds as the sole source of carbon and energy. One of these forms (PCase-P) was induced by protocatechuate and had an apparent molecular weight of 220,000. The second form (PCase-G) was induced by guaiacol or other phenolic compounds, such as 2-ethoxyphenol or 4-hydroxybenzoate. It appeared to be smaller (Mr 158,000), and its turnover number was about double that of the former enzyme. Both dioxygenases had similar properties and were built from the association of equal amounts of nonidentical subunits, alpha and beta, which were estimated to have molecular weights of 29,500 and 25,500, respectively. The (alpha beta)3 and (alpha beta)4 structures were suggested for PCases G and P, respectively. On the basis of two-dimensional gel electrophoresis, the alpha and beta polypeptides of PCase-G differed from those of PCase-P. Amino acid analysis supported this conclusion. Both PCases, however, had several other properties in common. It is proposed that both isoenzymes were generated from different sets of alpha and beta subunits, and the significance of these data is discussed.     

Biodegradation of Xanthan Gum by Bacillus sp

Strains tentatively identified as Bacillus sp. were isolated from sewage sludge and soil and shown to elaborate extracellular enzymes that degrade the extracellular polysaccharide (xanthan gum, polysaccharide B-1459) of Xanthomonas campestris NRRL B-1459. Enzyme production by one strain was greatly enhanced when the strain was incubated in a mixed culture. Products of degradation were identified as d-glucuronic acid, d-mannose, pyruvylated mannose, 6-O-acetyl d-mannose, and a (1-->4)-linked glucan. These products correlate with the known structure of the gum. The complexity of the product mixture indicated that the xanthanase was a mixture of carbohydrases. The xanthanase complexes were similar to one another in temperature stability, pH and temperature optima, degree of substrate degradation, and enzymolysis products. Differences in pH stability, salt tolerance, recoverability, and yields of enzyme were observed.     

Biodegradation of triiodophenol by cell-free extracts of a pentachlorophenol-degrading Flavobacterium sp

Pentachlorophenol (PCP) degrading Flavobacterium sp. ATCC 39723 was found to degrade other polyhalogenated phenolic compounds, including triiodophenol, tribromophenol, and trichlorophenol. Each compound was able to induce the degradation of the other compounds. A PCP Flavobacterium sp. mutant, F-2, was unable to degrade any of the halogenated compounds. The results suggest that all of the polyhalogenated phenols were degraded by the same enzyme system. This observation led us to exploit the sensitive leuco crystal violet assay, which measures the iodide released from triiodophenol. Cell free extracts from PCP-induced cells were able to release iodide from triiodophenol. The reaction required NADPH and oxygen.     

Biodegradation of diphenyl ethers by a copper-resistant mutant ofErwinia sp.

Summary A bacterium tentatively identified as anErwinia sp. was isolated from sewage by enrichment on methanol and lignin. Several mutants developed from this strain were studied for their ability to degrade aromatic ethers. Different concentrations of the chemicals were incubated with the organisms and the degradation was estimated by high-performance liquid chromatography (HPLC). Among these mutants, one isolate,Erwinia sp. strain CU3614, showed resistance to copper ions (>20 mM CuSO₄) and the ability to degrade 4-hydroxydiphenyl ether (4-HDPE), 4-chlorodiphenyl ether (4-CDPE), 4-nitrodiphenyl ether (4-NDPE) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) in the presence of copper ions. Increased concentrations of copper in the medium resulted in higher degradation of 4-HDPE. Further studies with copper-sensitive mutants obtained fromErwinia sp. CU3614 by Tn5 transposon-induced mutagenesis showed a corresponding decrease in the ability to degrade 4-HDPE. These results suggest the presence of a copper-associated activity in the biotransformation of aromatic ethers.     

Novel transformations of i-cholesterol and 6 beta-methoxy-i-cholesterol by Moraxella sp

A soil microorganism was isolated by the enrichment culture technique using cholesterol as the sole source of carbon. The organism has been identified as belonging to the genus Moraxella. With this organism two novel biotransformations of sterols were observed viz. (1) isomerization of 3 alpha,5 alpha-cyclocholestan-6 beta-ol (i-cholesterol) to cholesterol, (2) demethylation of 6 beta-methoxy-3 alpha,5 alpha-cyclocholestane (6 beta-methoxy-i-cholesterol) to i-cholesterol with subsequent isomerization to cholesterol. The enzymes responsible for these transformations were shown to be inducible. The pH optimum of the partially purified i-cholesterol isomerase was found to be 8.4. The apparent Km value for i-cholesterol was 1.43 microM. A plausible mechanism for the i-cholesterol isomerization has been discussed.     

Purification and properties of cytochrome P-450 from Moraxella sp

A cytochrome P-450 has been purified to homogeneity from a Moraxella species that is able to grow on guaiacol as the sole source of carbon and energy. The pure cytochrome was a monomeric protein of about 52 kDa, with no catalytic activity towards guaiacol. The difference in mM extinction coefficients between 450 and 490 nm in the CO-difference spectrum was 89.5 mM-1.cm-1. The typical shift of the Soret band from 415 to 390 nm that is attributed to the high-spin state of the cytochrome was observed in the presence of guaiacol and other 2-alkoxyphenols with up to 5 carbons in the side chain. It was also obtained with anisole. The maximum difference in mM extinction coefficients between 390 and 420 nm in the P-450 + ligand minus P-450 spectrum was 65 mM-1.cm-1 in all instances. The dissociation constants of the complexes formed between the pure protein and various O-alkoxyphenols were measured, and ranged from 0.1 microM (guaiacol) to 24 microM (2-butoxyphenol). The dissociation constants were 1 microM for anisole, and over 90 microM for phenol. Catechol induced no spectral change in cytochrome P-450 and appeared to be a weak inhibitor of guaiacol binding. The same spectral shift as induced by guaiacol was observed at high P-450 concentration over 1 microM in the absence of any added ligand and disappeared after dilution. The reduction of pure P-450 by dithionite was immediate, but became very slow, and was complete after 10 min or more at 25 degrees C in the presence of guaiacol. This effect was also obtained with the 2 isomers, 3- and 4-methoxyphenols, and with metyrapone, an inhibitor of guaiacol binding that induced the low-spin state. Preliminary experiments using the crude cell lysate or a reconstructed system with purified P-450 and a protein fraction indicated NADH-dependent guaiacol degradation. This was in agreement with the former hypothesis of Moraxella P-450 acting as a monooxygenase in the demethylation of guaiacol. However, cis, cis-muconate rather than catechol was obtained from the substrate, most likely a consequence of the potent catechol 1,2-dioxygenase activity present in the non-purified protein fractions used.     

Degradation of naphthalene-2,6- and naphthalene-1,6-disulfonic acid by a Moraxella sp

A naphthalene-2,6-disulfonic acid (2,6NDS)-degrading Moraxella strain was isolated from an industrial sewage plant. This culture could also be adapted to naphthalene-1,6-disulfonic acid as growth substrate. Regioselective 1,2-dioxygenation effected desulfonation and catabolism to 5-sulfosalicylic acid (5SS), which also could be used as the sole carbon source. 5SS-grown cells exhibited high gentisate 1,2-dioxygenase activity. Neither 5SS- nor gentisate-grown cells oxidized 2,6NDS; therefore, 2,6NDS or an early metabolite must serve as an inducer of the initial catabolic enzyme(s).