indigo-carmine and indole

Polygonum tinctorium (P. tinctorium) is an indigo plant that is cultivated for a specific metabolite that it produces i.e., indoxyl β-D-glucoside (indican). In this study, flavin-containing monooxygenase (PtFMO) from P. tinctorium was cloned. When recombinant PtFMO was expressed in E. coli in the presence of tryptophan, indigo production was observed. Furthermore, we measured the activity of PtFMO using the membrane fraction from E. coli and found that it could produce indigo using indole as a substrate. The co-expression of PtFMO with indoxyl β-D-glucoside synthase (PtIGS), which catalyzes the glucosylation of indoxyl, brought about the formation of indican in E. coli. The results showed that indican was synthesized by sequential reactions of PtFMO and PtIGS. In three-week-old P. tinctorium specimens, the first leaves demonstrated higher levels of PtFMO expression than the subsequent leaves. This result coincided with that of our prior study on PtIGS expression level. Our study provides evidence that PtFMO might contribute to indican biosynthesis.

Topics: Amino Acid Sequence; Coloring Agents; Escherichia coli; Indican; Indigo Carmine; Indoles; Oxidation-Reduction; Oxygenases; Polygonum

Indigo is currently produced by a century-old petrochemical-based process, therefore it is highly attractive to develop a more environmentally benign and efficient biotechnological process to produce this timeless dye. Flavin-containing monooxygenases (FMOs) are able to oxidize a wide variety of substrates. In this paper we show that the bacterial mFMO can be adapted to improve its ability to convert indole into indigo. The improvement was achieved by a combination of computational and structure-inspired enzyme redesign. We showed that the thermostability and the

Topics: Escherichia coli; Indigo Carmine; Indoles; Mixed Function Oxygenases; Oxidation-Reduction

Indole is a typical nitrogen-containing aromatic pollutant in coking wastewater, and it can be used for the microbial production of indigo, one of the oldest dyestuffs. In this study, the activated sludge system bioaugmented with two indigo-producing bacterial strains, wild strain Comamonas sp. MQ and recombinant Escherichia coli (ND_IND), was constructed to investigate indigo bioproduction from indole. During the operation, the bioaugmentation could promote the production of indigo, especially in early stages, and the indigo yields gradually increased from 17.5 ± 0.4 to 44.3 ± 0.5 mg/L with the increase of influent indole (80 to 282 mg/L). Illumina MiSeq sequencing revealed that the microbial community could have a noticeable shift driven by the bioaugmentation and high indole pressure. The indigenous bacteria could be more responsible for indigo production, and the dominant genera Comamonas, Diaphorobacter, Paracoccus, Aquamicrobium, Pseudomonas, and Truepera could be the key functional taxa. Based on FAPROTAX (Functional Annotation of Prokaryotic Taxa) analysis, the nitrogen metabolism-related functional groups could play important roles in indole biotransformation and indigo biosynthesis. This study should provide insights into microbial production of indigo by microbial communities.

Topics: Biotransformation; Comamonas; Escherichia coli; Indigo Carmine; Indoles; Microbiota; Sewage

In this study, two phenol-degrading bacterial strains, designated as PI1 and PI2, were isolated from activated sludge for the production of indigoids from indole. According to the 16S ribosomal RNA (rRNA) gene sequence analysis, strains PI1 and PI2 were identified as Pseudomonas sp. and Acinetobacter sp., respectively. Liquid chromatography/time-of-flight/mass spectrometry (LC/TOF/MS) was applied to analyze the metabolites during the biotransformation of indole by the phenol-degrading strains. The results indicated that both strains could catalyze the formation of four indigoids with the same prominent molecular ion (M-H)(-) peak at m/z 261.067 and molecular formula of C16H10N2O2, including indigo and a purple product, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one. Isatin and 7-hydroxyindole were detected as the intermediates. Thus, the possible pathways for the production of indigoids from indole were proposed. Subsequently, the optimal conditions for the production of indigo from indole were determined using response surface methodology, and 11.82 ± 0.30 and 17.19 ± 0.49 mg/L indigo were produced by strains PI1 and PI2, respectively. The present study should provide potential candidates for microbial production of indigoids.

Topics: Acinetobacter; Analysis of Variance; Biodegradation, Environmental; Indigo Carmine; Indoles; Mass Spectrometry; Metabolome; Molecular Sequence Data; Phenol; Phylogeny; Pseudomonas

Biosynthesis of the popular dyestuff indigo from indole has been comprehensively studied using pure cultures, but less has been done to characterize the indigo production by microbial communities. In our previous studies, a wild strain Comamonas sp. MQ was isolated from activated sludge and the recombinant Escherichia coli nagAc carrying the naphthalene dioxygenase gene (nag) from strain MQ was constructed, both of which were capable of producing indigo from indole. Herein, three activated sludge systems, G1 (non-augmented control), G2 (augmented with Comamonas sp. MQ), and G3 (augmented with recombinant E. coli nagAc), were constructed to investigate indigo production. After 132-day operation, G3 produced the highest yields of indigo (99.5 ± 3.0 mg/l), followed by G2 (27.3 ± 1.3 mg/l) and G1 (19.2 ± 1.2 mg/l). The microbial community dynamics and activities associated with indigo production were analyzed by Illumina Miseq sequencing of 16S rRNA gene amplicons. The inoculated strain MQ survived for at least 30 days, whereas E. coli nagAc was undetectable shortly after inoculation. Quantitative real-time PCR analysis suggested the abundance of naphthalene dioxygenase gene (nagAc) from both inoculated strains was strongly correlated with indigo yields in early stages (0-30 days) (P < 0.001) but not in later stages (30-132 days) (P > 0.10) of operation. Based on detrended correspondence analysis (DCA) and dissimilarity test results, the communities underwent a noticeable shift during the operation. Among the four major genera (> 1% on average), the commonly reported indigo-producing populations Comamonas and Pseudomonas showed no positive relationship with indigo yields (P > 0.05) based on Pearson correlation test, while Alcaligenes and Aquamicrobium, rarely reported for indigo production, were positively correlated with indigo yields (P < 0.05). This study should provide new insights into our understanding of indigo bio-production by microbial communities.

Topics: Bacteria; Indigo Carmine; Indoles; Microbial Viability; RNA, Ribosomal, 16S; Sequence Analysis, RNA; Sewage

Indole, a derivative of the amino acid tryptophan, is a toxic signaling molecule, which can inhibit bacterial growth. To overcome indole-induced toxicity, many bacteria have developed enzymatic defense systems to convert indole to non-toxic, water-insoluble indigo. We previously demonstrated that, like other aromatic compound-degrading bacteria, Acinetobacter baumannii can also convert indole to indigo. However, no work has been published investigating this mechanism. Here, we have shown that the growth of wild-type A. baumannii is severely inhibited in the presence of 3.5 mM indole. However, at lower concentrations, growth is stable, implying that the bacteria may be utilizing a survival mechanism to oxidize indole. To this end, we have identified a flavoprotein oxygenase encoded by the iifC gene of A. baumannii. Further, our results suggest that expressing this recombinant oxygenase protein in Escherichia coli can drive indole oxidation to indigo in vitro. Genome analysis shows that the iif operon is exclusively present in the genomes of A. baumannii and Pseudomonas syringae pv. actinidiae. Quantitative PCR and Western blot analysis also indicate that the iif operon is activated by indole through the AraC-like transcriptional regulator IifR. Taken together, these data suggest that this species of bacteria utilizes a novel indole-detoxification mechanism that is modulated by IifC, a protein that appears to be, at least to some extent, regulated by IifR.

Topics: Acinetobacter baumannii; Amino Acid Sequence; Bacterial Proteins; Blotting, Western; Dioxygenases; Dose-Response Relationship, Drug; Flavoproteins; Gene Expression Regulation, Bacterial; Indigo Carmine; Indoles; Microbial Viability; Molecular Sequence Data; Mutation; Operon; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid

Biotransformation of indole to indigo in liquid-liquid biphasic systems was performed in Escherichia coli cells expressing phenol hydroxylase. It was suggested that indole could inhibit the cell growth even at low concentration of 0.1 g/L. The critical Log P for strain PH_(IND) was about 5.0. Three different solvents, i.e., decane, dodecane, and dioctyl phthalate, were selected as organic phase in biphasic media. The results showed that dodecane gave the highest yield of indigo (176.4 mg/L), which was more than that of single phase (90.5 mg/L). The optimal conditions for biotransformation evaluated by response surface methodology were as follows: 540.26 mg/L of indole concentration, 42.27 % of organic phase ratio, and 200 r/min of stirrer speed; under these conditions, the maximal production of indigo was 243.51 mg/L. This study proved that the potential application of strain PH_(IND) in the biotransformation of indole to indigo using liquid-liquid biphasic systems.

Topics: Biotransformation; Escherichia coli; Indigo Carmine; Indoles; Mixed Function Oxygenases

Optimization of indigo production process from indole using a newly isolated phenol-degrading bacterial strain was performed by Plackett-Burman design and response surface methodology. The strain designated as QM was identified as Pseudomonas sp. according to 16S rDNA analysis. Spectrum analysis of indole biotransformation products revealed the presence of indigo and a by-product indirubin. To improve indigo yield, Plackett-Burman design was used to select significant factors from 8 viriables. Then response surface methodology based on a 2(3) central composite design was used to further optimize the transformation process. Under the optimal conditons, strain QM can produce 27.20 mg/l indigo after 24 h cultivation at 30 °C, which was 151.3% higher than that from the initial conversion condition. The results indicated that Pseudomonas sp. QM should be a potential candidate for indigo industial production.

Topics: Biotransformation; Culture Media; DNA, Ribosomal; Indigo Carmine; Indoles; Phenol; Phylogeny; Pseudomonas

A betaproteobacterium within the family Rhodocyclaceae previously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and β-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed in Escherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of the E. coli constructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene.

Topics: Cloning, Molecular; Cluster Analysis; Dioxygenases; Escherichia coli; Gene Expression; Indigo Carmine; Indoles; Metagenome; Molecular Sequence Data; Phenanthrenes; Phylogeny; Polycyclic Aromatic Hydrocarbons; Polymerase Chain Reaction; Pyrenes; Recombinant Proteins; Rhodocyclaceae; Sequence Analysis, DNA; Sequence Homology; Soil Microbiology

The styAB genes from Pseudomonas putida CA-3, which encode styrene monooxygenase, were subjected to three rounds of in vitro evolution using error-prone polymerase chain reaction with a view to improving the rate of styrene oxide and indene oxide formation. Improvements in styrene monooxygenase activity were monitored using an indole to indigo conversion assay. Each round of random mutagenesis generated variants improved in indigo formation with third round variants improved nine- to 12-fold over the wild type enzyme. Each round of in vitro evolution resulted in two to three amino acid substitutions in styrene monooxygenase. While the majority of mutations occurred in styA (oxygenase), mutations were also observed in styB (reductase). A mutation resulting in the substitution of valine with isoleucine at amino acid residue 303 occurred near the styrene and flavin adenine dinucleotide binding site of styrene monooxygenase. One mutation caused a shift in the reading frame in styA and resulted in a StyA variant that is 19 amino acids longer than the wild-type protein. Whole cells expressing the best styrene monooxygenase variants (round 3) exhibited eight- and 12-fold improvements in styrene and indene oxidation rates compared to the wild-type enzyme. In all cases, a single enantiomer, (S)-styrene oxide, was formed from styrene while (1S,2R)-indene oxide was the predominant enantiomer (e.e. 97%) formed from indene. The average yield of styrene oxide and indene oxide from their respective alkene substrates was 65% and 90%, respectively.

Topics: Biotransformation; Cloning, Molecular; Directed Molecular Evolution; Epoxy Compounds; Escherichia coli; Indenes; Indigo Carmine; Indoles; Models, Molecular; Mutagenesis; Oxygenases; Pseudomonas putida; Stereoisomerism; Styrene

Toluene dioxygenase (TDO) is ubiquitous in nature and has a broad substrate range, including benzene, toluene, ethylbenzene and xylenes (BTEX). Pseudomonas putida F1 (PpF1) induced on toluene is known to produce indigo from indole through the activity of TDO. In this work, a spectrophotometric assay previously developed to measure indole to indigo production rates was modified to characterize the effects of various ethanol concentrations on toluene aerobic biodegradation activity and assess catabolite repression of TDO. Indigo production rate by cells induced on toluene alone was 0.0012 +/- 0.0006 OD(610) min(-1). The presence of ethanol did not fully repress TDO activity when toluene was also available as a carbon source. However, indigo production rates by PpF1 grown on ethanol:toluene mixtures (3:1 w/w) decreased by approximately 50%. Overall, the proposed spectrophotometric assay is a simple approach to quantify TDO activity, and demonstrates how the presence of ethanol in groundwater contaminated with reformulated gasoline is likely to interfere with naturally occurring microorganisms from fully expressing their aerobic catabolic potential towards hydrocarbons bioremediation.

Topics: Aerobiosis; Biodegradation, Environmental; Enzyme Assays; Ethanol; Indigo Carmine; Indoles; Oxygenases; Pseudomonas putida; Spectrophotometry; Toluene

Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.

Topics: Amino Acid Substitution; Bacterial Proteins; Biocatalysis; Carbon; Chlorzoxazone; Coumarins; Cytochrome P-450 Enzyme System; Enzyme Stability; Escherichia coli; Heme; Humans; Indigo Carmine; Indoles; Kinetics; Molecular Dynamics Simulation; NADPH-Ferrihemoprotein Reductase; Nitrophenols; Oxazines; Oxidation-Reduction; Phenacetin; Protein Engineering; Recombinant Proteins; Transformation, Genetic; Umbelliferones

A technique developed to determine naphthalene dioxygenase (NDO) activity was optimized and used to study the biotransformation of indole to indigo by Pseudomonas sp. J26 whole cells. The maximum production of indigo was achieved at 25 degrees C using 2.5 mM indole when J26 was grown in the complex medium JPP, while indole concentrations higher than 4 mM proved toxic for cells. The maximum rate of indigo production was 0.56 nmol min(-1) mg dry biomass(-1), obtaining 75.5 microM of indigo after 8 h of incubation, while a maximal concentration (138.1 microM) of indigo was obtained after 20 h.

Topics: Argentina; Culture Media; Geologic Sediments; Indigo Carmine; Indoles; Naphthalenes; Pseudomonas; Temperature

Oxygenases are useful for the production of many industrially important molecules. Screening of an effluent treatment plant (ETP) sludge metagenomic library identified two clones encoding proteins, B1 and B2, with similarity to putative flavin monooxygenases from Mesorhizobium loti and Sphingomonas wittichi, respectively. The deduced amino acid sequences show only 20% identity, but both have a paired Rossman fold and a flavin monooxygenase (FMO) motif. B1 and B2 appear to be members of the flavin-containing monooxygenase and the Baeyer-Villiger monooxygenases subfamilies, respectively. When expressed in Escherichia coli, the two clones produced activities that oxidized indole to a mixture of indigo and indirubin pigments. These results suggest that B1 and B2 have potential as a biocatalyst in indigo/indirubin production.

Topics: Amino Acid Sequence; Biotransformation; Clone Cells; Gene Library; Indigo Carmine; Indoles; Metagenomics; Mixed Function Oxygenases; Molecular Sequence Data; Phylogeny; Pigments, Biological; Sewage; Waste Disposal, Fluid; Water Purification

Cytochrome P450 (P450) 2A6 is able to catalyze indole hydroxylation to form the blue dye indigo. The wild-type P450 2A6 enzyme was randomly mutated throughout the whole open reading frame and screened using 4-chloroindole hydroxylation, a substituted indole selected from 30 indole compounds for enhanced color development. Mutants with up to 5-fold increases of catalytic efficiency (k(cat)/K(m)) and 2-fold increases in k(cat) were selected after two rounds of screening. Important residues located both in (e.g., Thr305) and outside the active site (e.g., Ser224) were identified. The study utilized a better substrate for "indigo assay" to obtain new information on the structure-functional relationship of P450 2A6 that was not revealed by previous mutagenesis studies with this enzyme.

Topics: Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP2A6; Databases, Protein; Escherichia coli; Humans; Hydroxylation; Indigo Carmine; Indoles; Kinetics; Membranes; Models, Chemical; Models, Molecular; Mutagenesis; Sequence Analysis, Protein

Purple urine bag syndrome (PUBS) is considered as an uncommon phenomenon. This cohort study aimed to figure out the causative factors and the potential risks of PUBS.. We recruited 84 patients with long-term urinary catheterization from seven nursing institutions and home care centre, then compared the differences of demographic, clinical, urine test and bacteriological data between the patients with and without PUBS.. The 2-month period prevalence of PUBS in chronic catheterized patients was 16.7%. PUBS-affecting patients had significantly higher proportion of female gender (P = 0.034), self oral intake (P = 0.036) and eating self-prepared food (P = 0.007). Otherwise, no statistical associations were found. Five different bacterial species were isolated from the urine of PUBS-affecting patients. No sequelae were found after forward follow-up for 6 months. We further discuss the associations between certain factors and PUBS according to literature review and the results of our study.. PUBS is not as rare as we thought before. The causative factors of PUBS have not been clearly characterized. It may be the combination of several factors that cause the PUBS. Female gender and food content were found to be associated factors of PUBS in our study. Asymptomatic PUBS is unnecessary to be treated by antibiotics.

Topics: Aged; Aged, 80 and over; Color; Constipation; Female; Humans; Hydrogen-Ion Concentration; Indigo Carmine; Indoles; Male; Middle Aged; Syndrome; Urinary Catheterization; Urinary Tract Infections

Topics: Aged, 80 and over; Bacteria; Female; Humans; Indigo Carmine; Indoles; Intestines; Liver; Syndrome; Tryptophanase; Urine

Cytochrome P450 BM-3 with the mutations A74G, F87V, and L188Q could catalyze indole to produce indigo and indirubin. To further enhance this capability, site-directed and random mutageneses on the monooxygenase domain of P450 BM-3 mutant (A74G/F87V/L188Q; 3X) were performed. The mutant libraries created by error-prone polymerase chain reaction were screened using a colorimetric colony-based method on agar plates followed by a spectroscopic assay involving in absorption of indigo at 670 nm and NADPH at 340 nm in microtiter plate. Three mutants (K434R/3X, E435D/3X, and D168N/ A225V/K440N/3X) exhibited higher hydroxylation activity toward indole in comparison to parent enzyme. Moreover, using saturation site-directed mutagenesis at amino acid positions 168, 225, 434, 435, and 440, two P450 BM-3 variants (D168H/3X, E435T/3X) with an up to sixfold increase in catalytic efficiency (kcat/Km) were identified, and the mutant D168H/3X acquired higher regioselectivity resulting in more indigo (dimerized 3hydroxy-indole) compared to parent mutant (93 vs 72%).

Topics: Bacillus megaterium; Bacterial Proteins; Base Sequence; Catalytic Domain; Cytochrome P-450 Enzyme System; Directed Molecular Evolution; DNA, Bacterial; Hydroxylation; Indigo Carmine; Indoles; Kinetics; Mixed Function Oxygenases; Models, Molecular; Mutagenesis; Mutagenesis, Site-Directed; NADPH-Ferrihemoprotein Reductase; Protein Conformation; Recombinant Proteins

Rhodococcus opacus strain B-4, which has recently been isolated as an organic solvent-tolerant bacterium, has a high hydrophobicity and exhibits a high affinity for hydrocarbons. This bacterium was able to survive for at least 5 days in organic solvents, including n-tetradecane, oleyl alcohol, and bis(2-ethylhexyl) phthalate (BEHP), which contained water less than 1% (w/v). The biocatalytic ability of R. opacus B-4 was demonstrated in the essentially nonaqueous BEHP using indigo production from indole as a model conversion. By the catabolism of oleic acid for NADH regeneration, indigo production increased up to 71.6 microg ml(-1) by 24 h.

Topics: Alkanes; Anti-Bacterial Agents; Biotransformation; Culture Media; Diethylhexyl Phthalate; Drug Resistance, Bacterial; Fatty Alcohols; Fermentation; Indigo Carmine; Indoles; Industrial Microbiology; Microbial Viability; NAD; Oleic Acid; Rhodococcus; Solvents

Broad-substrate-range monooygenase enzymes, including toluene-4-monooxygenase (T4MO), can catalyze the oxidation of indole. The indole oxidation products can then condense to form the industrially important dye indigo. Site-directed mutagenesis of T4MO resulted in the creation of T4MO isoforms with altered pigment production phenotypes. High-pressure liquid chromatography, thin-layer chromatography, and nuclear magnetic resonance analysis of the indole oxidation products generated by the mutant T4MO isoforms revealed that the phenotypic differences were primarily due to changes in the regiospecificity of indole oxidation. Most of the mutations described in this study changed the ratio of the primary indole oxidation products formed (indoxyl, 2-oxindole, and isatin), but some mutations, particularly those involving amino acid G103 of tmoA, allowed for the formation of additional products, including 7-hydroxyindole and novel indigoid pigments. For example, mutant G103L converted 17% of added indole to 7-hydroxyindole and 29% to indigoid pigments including indigo and indirubin and two other structurally related pigments. The double mutant G103L:A107G converted 47% of indole to 7-hydroxyindole, but no detectable indigoid pigments were formed, similar to the product distribution observed with the toluene-2-monooxygenase (T2MO) of Burkholderia cepacia G4. These results demonstrate that modification of the tmoA active site can change the products produced by the enzyme and lead to the production of novel pigments and other indole oxidation products with potential commercial and medicinal utility.

Topics: Binding Sites; Catalytic Domain; Escherichia coli; Indigo Carmine; Indoles; Mutation; Oxidation-Reduction; Oxygenases; Pigments, Biological; Protein Engineering; Protein Isoforms

The aim of this study was to further characterize a bacterial culture (VUN 10,010) capable of benzo[a]pyrene cometabolism.. The bacterial culture, previously characterized as a pure culture of Stenotrophomonas maltophilia (VUN 10,010), was found to also contain another bacterial species (Mycobacterium sp. strain 1B), capable of degrading a similar range of PAH substrates. Analysis of its 16S rRNA gene sequence and growth characteristics revealed the strain to be a fast-growing Mycobacterium sp., closely related to other previously isolated PAH and xenobiotic-degrading mycobacterial strains. Comparison of the PAH-degrading characteristics of Mycobacterium sp. strain 1B with those of S. maltophilia indicated some similarities (ability to degrade phenanthrene and pyrene), but some differences were also noted (S. maltophilia able to degrade fluorene, but not fluoranthene, whereas Mycobacterium sp. strain 1B can degrade fluoranthene, but not fluorene). Unlike the S. maltophilia culture, there was no evidence of benzo[a]pyrene degradation by Mycobacterium sp. strain 1B, even in the presence of other PAHs (ie pyrene) as co-metabolic substrates. Growth of Mycobacterium sp. strain 1B on other organic carbon sources was also limited compared with the S. maltophilia culture.. This study isolated a Mycobacterium strain from a bacterial culture capable of benzo[a]pyrene cometabolism. The Mycobacterium strain displays different PAH-degrading characteristics to those described previously for the PAH-degrading bacterial culture. It is unclear what role the two bacterial strains play in benzo[a]pyrene cometabolism, as the Mycobacterium strain does not appear to have endogenous benzo[a]pyrene degrading ability.. This study describes the isolation and characterization of a novel PAH-degrading Mycobacterium strain from a PAH-degrading culture. Further studies utilizing this strain alone, and in combination with other members of the consortium, will provide insight into the diverse roles different bacteria may play in PAH degradation in mixed cultures and in the environment.

Topics: Anti-Infective Agents; Base Sequence; Biodegradation, Environmental; Coloring Agents; Culture Media; Fatty Acids; Fluorenes; Indigo Carmine; Indoles; Molecular Weight; Mycobacterium; Phenanthrenes; Polycyclic Aromatic Hydrocarbons; Pyrenes; RNA, Bacterial; RNA, Ribosomal, 16S; Salicylic Acid

Acinetobacter sp. strain ST-550 produces indigo from indole in the presence of a large volume of diphenylmethane and a high level of indole. Particular proteins increased remarkably in strain ST-550 grown in the two-phase culture system for indigo production. One of the proteins showed a N-terminal amino acid sequence that was identical to that of the largest subunit of phenol hydroxylase (MopN) from A. calcoaceticus NCBI8250. The indigo-producing activity was strongly induced when ST-550 was grown with phenol as a sole carbon source. Genes coding for the multicomponent phenol hydroxylase were cloned, based on the homology with mopKLMOP from A. calcoaceticus NCBI8250. Escherichia coli carrying the genes produced indigo from indole. E. coli JA300 and its cyclohexane-resistant mutant, OST3410, carrying the hydroxylase genes and the NADH regeneration system were grown in the two-phase culture system for indigo production. The OST3410 recombinant produced 52 microg indigo ml(-1) of medium in the presence of diphenylmethane. This productivity was 4.3-fold higher than that of the JA300 recombinant.

Topics: Acinetobacter calcoaceticus; Alkanes; Bacterial Proteins; Benzhydryl Compounds; Cyclohexanes; Cyclooctanes; Drug Resistance, Bacterial; Escherichia coli; Formate Dehydrogenases; Hydrophobic and Hydrophilic Interactions; Indigo Carmine; Indoles; Mixed Function Oxygenases; Multienzyme Complexes; NAD; Octanes; Operon; Phenol; Recombinant Fusion Proteins; Solvents; Water

To isolate and characterize the phorate [O,O-diethyl-S-(ethylthio)methyl phosphoradiothioate] degrading bacteria from agricultural soil, and their assessment for multifarious biological activities of environmental and agronomic significance.. Based on their morphological and biochemical characteristics, the selected isolates PS-1, PS-2 and PS-3 were presumptively identified as Rhizobium, Pseudomonas and Proteus species, respectively. The HPLC analysis of phorate in bioaugmented soil revealed its complete disappearance within 40 days. The degradation isotherms of the isolates PS-1, PS-2 and PS-3 suggested time-dependent disappearance of phorate following the first-order rate kinetics at the corresponding rate constants of 0.04, 0.05 and 0.04 d-1. Besides, the isolates concurrently exhibited substantial phosphate solubilization, indole acetic acid (IAA) and siderophore production, as well as limited biocontrol activity against fungal phytopathogens.. The data revealed the potential of isolates for collateral plant growth promotion, biocontrol and bioremediation. The selected strains may serve as an important bioresource for development of effective super-bioinoculants.

Topics: Coloring Agents; Dioxygenases; Escherichia coli; Indigo Carmine; Indoles; Multienzyme Complexes; Oxygenases; Recombinant Proteins

Directed enzyme evolution of 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC ) from Pseudomonas azelaica HBP1 resulted in an enzyme variant (HbpA(ind)) that hydroxylates indole and indole derivatives such as hydroxyindoles and 5-bromoindole. The wild-type protein does not catalyze these reactions. HbpA(ind) contains amino acid substitutions D222V and V368A. The activity for indole hydroxylation was increased 18-fold in this variant. Concomitantly, the K(d) value for indole decreased from 1.5 mm to 78 microm. Investigation of the major reaction products of HbpA(ind) with indole revealed hydroxylation at the carbons of the pyrrole ring of the substrate. Subsequent enzyme-independent condensation and oxidation of the reaction products led to the formation of indigo and indirubin. The activity of the HbpA(ind) mutant monooxygenase for the natural substrate 2-hydroxybiphenyl was six times lower than that of the wild-type enzyme. In HbpA(ind), there was significantly increased uncoupling of NADH oxidation from 2-hydroxybiphenyl hydroxylation, which could be attributed to the substitution D222V. The position of Asp(222) in HbpA, the chemical properties of this residue, and the effects of its substitution indicate that Asp(222) is involved in substrate activation in HbpA.

Topics: Catalysis; Hydroxylation; Indigo Carmine; Indoles; Mixed Function Oxygenases; NAD; Oxidation-Reduction; Structure-Activity Relationship

On complex medium Escherichia coli strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of Ralstonia eutropha HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods. A 1,251-bp open reading frame (bec) was cloned and sequenced. The deduced amino acid sequence of bec showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Cloning, Molecular; Cupriavidus necator; Escherichia coli; Indigo Carmine; Indoles; Mixed Function Oxygenases; Molecular Sequence Data; Plasmids; Recombinant Proteins; Sequence Analysis, DNA; Spectrometry, Fluorescence; Tryptophanase

Cytochrome P450 (P450) 2A6 mutants from randomized libraries generated in the substrate recognition sequence (SRS) regions were screened in Escherichia coli on the basis of indole metabolism. SRS 3 and 4 libraries yielded colonies that produced indigo at least as well as wild-type (WT) P450 2A6, and some colonies were consistently more blue upon replating. One mutant, F209T, showed indole 3-hydroxylation WT. The double mutant L240C/N297Q consistently produced very blue colonies. Five mutants yielded mixtures of pigments from indole different than WT, as judged by visible spectra and HPLC of products. When bacteria expressing the mutants were grown in the presence of each of 26 substituted indoles, a variety of patterns of formation of different dyes was seen with several of the mutants. This approach has potential value in understanding P450 2A6 function and generating new dyestuffs and other products.

Topics: Aryl Hydrocarbon Hydroxylases; Cell Membrane; Chromatography, High Pressure Liquid; Coloring Agents; Coumarins; Cytochrome P-450 CYP2A6; Cytochrome P-450 Enzyme System; Escherichia coli; Humans; Indigo Carmine; Indoles; Kinetics; Mixed Function Oxygenases; Mutagenesis; Oxidation-Reduction; Spectrophotometry; Umbelliferones

The development of bicistronic systems for coexpression of recombinant human cytochrome P450 enzymes (P450s) with their redox partner, NADPH-cytochrome P450 reductase (NPR), has enabled P450 activity to be reconstituted within bacterial cells. During expression of recombinant P450 2E1 and some other forms, we observed the formation of a blue pigment in bacterial cultures. The pigment was extracted from cultures and shown to comigrate with standard indigo on TLC. UV-visible spectroscopy and mass spectrometric analysis provided further support for identification of the pigment as indigo. Indigo is known to form following the spontaneous oxidation of 3-hydroxyindole. Accordingly, we speculated that indole, formed as a breakdown product of tryptophan in bacteria, was hydroxylated by the P450 system, leading to indigo formation. Bacterial membranes containing recombinant P450 2E1 and human NPR were incubated in vitro with indole and shown to catalyze formation of a blue pigment in a time- and cofactor-dependent manner. These studies suggest potential applications of mammalian P450 enzymes in industrial indigo production or in the development of novel colorimetric assays based on indole hydroxylation.

Topics: Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System; Escherichia coli; Genes; Humans; In Vitro Techniques; Indigo Carmine; Indoles; NADPH-Ferrihemoprotein Reductase; Pigments, Biological; Recombinant Proteins

Pigments produced by Escherichia coli containing a cloned piece of DNA from Rhodococcus sp. ATCC 21145 were extracted in chloroform and separated into blue and pink components. Evidence from TLC, NMR spectroscopy, absorption spectrum analysis and solubility behaviour suggested that the blue pigment was indigo and the pink pigment was indirubin, a structural isomer of indigo. The proposed pathway for pigment production on LB agar involves the conversion of tryptophan to indole by tryptophanase of E. coli and the oxidation of indole to indigo by the product of the cloned Rhodococcus DNA insert.

Topics: Chromatography, Thin Layer; Cloning, Molecular; Culture Media; DNA, Bacterial; Escherichia coli; Genetic Vectors; Indigo Carmine; Indoles; Magnetic Resonance Spectroscopy; Pigments, Biological; Plasmids; Rhodococcus; RNA, Bacterial; Solubility; Transformation, Bacterial; Tryptophan; Tryptophanase