Degradation of substituted indoles by an indole-degrading methanogenic consortium.

Degradation of indole by an indole-degrading methanogenic consortium enriched from sewage sludge proceeded through a two-step hydroxylation pathway yielding oxindole and isatin. The ability of this consortium to hydroxylate and subsequently degrade substituted indoles was investigated. Of the substituted indoles tested, the consortium was able to transform or degrade 3-methylindole and 3-indolyl acetate. Oxindole, 3-methyloxindole, and indoxyl were identified as metabolites of indole, 3-methylindole, and 3-indolyl acetate degradation, respectively. Isatin (indole-2,3-dione) was produced as an intermediate when the consortium was amended with oxindole, providing evidence that degradation of indole proceeded through successive hydroxylation of the 2- and 3-positions prior to ring cleavage between the C-2 and C-3 atoms on the pyrrole ring of indole. The presence of a methyl group (-CH3) at either the 1- or 2-position of indole inhibited the initial hydroxylation reaction. The substituted indole, 3-methylindole, was hydroxylated in the 2-position but not in the 3-position and could not be further metabolized through the oxindole-isatin pathway. Indoxyl (indole-3-one), the deacetylated product of 3-indolyl acetate, was not hydroxylated in the 2-position and thus was not further metabolized by the consortium. When an H atom or electron-donating group (i.e., -CH3) was present at the 3-position, hydroxylation proceeded at the 2-position, but the presence of electron-withdrawing substituent groups (i.e., -OH or -COOH) at the 3-position inhibited hydroxylation.     

Degradation of substituted indoles by an indole-degrading methanogenic consortium.

Degradation of indole by an indole-degrading methanogenic consortium enriched from sewage sludge proceeded through a two-step hydroxylation pathway yielding oxindole and isatin. The ability of this consortium to hydroxylate and subsequently degrade substituted indoles was investigated. Of the substituted indoles tested, the consortium was able to transform or degrade 3-methylindole and 3-indolyl acetate. Oxindole, 3-methyloxindole, and indoxyl were identified as metabolites of indole, 3-methylindole, and 3-indolyl acetate degradation, respectively. Isatin (indole-2,3-dione) was produced as an intermediate when the consortium was amended with oxindole, providing evidence that degradation of indole proceeded through successive hydroxylation of the 2- and 3-positions prior to ring cleavage between the C-2 and C-3 atoms on the pyrrole ring of indole. The presence of a methyl group (-CH3) at either the 1- or 2-position of indole inhibited the initial hydroxylation reaction. The substituted indole, 3-methylindole, was hydroxylated in the 2-position but not in the 3-position and could not be further metabolized through the oxindole-isatin pathway. Indoxyl (indole-3-one), the deacetylated product of 3-indolyl acetate, was not hydroxylated in the 2-position and thus was not further metabolized by the consortium. When an H atom or electron-donating group (i.e., -CH3) was present at the 3-position, hydroxylation proceeded at the 2-position, but the presence of electron-withdrawing substituent groups (i.e., -OH or -COOH) at the 3-position inhibited hydroxylation.     

Development of a defined medium for Clostridium scatologenes ATCC 25775

Aims:  To develop a defined medium for Clostridium scatologenes ATCC 25775, which produces the malodorants 3-methylindole (skatole) and 4-methylphenol ( p- cresol).
Methods and Results:  Clostridium scatologenes was cultured in anaerobic broth medium (pH 6·3) at 37°C containing ammonia, minerals and a commercial vitamin solution. Data indicate α-ketoglutarate, l- glutamate or l- glutamine is a required nutrient that can also serve as a primary carbon and energy source. When cultured in defined medium containing glutamate; glucose, fructose and betaine served as primary carbon and energy sources. l- Tryptophan, l- tyrosine, sorbitol and indole acetic acid did not enhance growth. In the absence of tryptophan, cells produced indole when grown using glucose or fructose. 4-Methylphenol was produced when growing cells were supplied with tyrosine. When supplied with tryptophan, 3-methylindole was produced by glucose- or fructose-growing cells but not from glutamate-growing cells. Cells grown in the presence of pyruvate produced indole, 3-methylindole and 4-methylphenol.
Conclusions:  Clostridium scatologenes requires α-ketoglutarate, l- glutamate, or l- glutamine for growth in defined medium. Cells produce indole when glucose or fructose is included in defined medium.
Significance and Impact of the Study:  The development of a defined medium will assist in physiology studies and genetic analysis of this strain.     

Production of indolic compounds by rumen bacteria isolated from grazing ruminants

AIM: To screen rumen bacterial cultures and fresh ruminal isolates for indole and skatole production. METHODS AND RESULTS: Culture collection strains and fresh bacterial isolates from rumen contents of sheep and dairy cows were screened for the production of indolic compounds. Clostridium aminophilum FT, Peptostreptococcus ssp. S1, Fusobacterium necrophorum D4 produced indole and Clostridium sticklandii SR produced indoleacetic acid. Fresh isolates from sheep (TrE9262 and TrE7262) and dairy cows (152R-1a, 152R-1b, 152R-3 and 152R-4) produced indole, indolepropionic acid, tryptophol and skatole from the fermentation of tryptophan and indoleacetic acid. Glucose altered the indolic compounds produced in some, but not all, isolates. TrE7262 and 152R-4 were identified as Clostridium sporogenes and 152R-1b as a new Cl. aminophilum strain. Isolates TrE9262, 152R-1a and 152R-3 were not closely related to any described species but belong to Megasphaera, Prevotella and Actinomyces genera, respectively. CONCLUSIONS: Rumen bacteria that produced a range of indolic compounds were identified. Some isolates are distinct from the previously described bacteria and may represent novel species. SIGNIFICANCE AND IMPACT OF THE STUDY: These observations will contribute to understanding skatole and indole formation in the rumen and will lead to methods that control the formation of indolic compounds in pasture-grazed ruminants.     

3-Methylindole (skatole) and indole production by mixed populations of pig fecal bacteria.

Pig fecal slurries converted added L-tryptophan either to indole without detectable intermediates or to 3-methylindole (skatole) via indole-3-acetate. The initial rate of production of 3-methylindole was greatest at pH 6.5 and less at pH 5.0 and 8.0; the initial rates of indole production were similar at pH 6.5 and 8.0. More than 80% of the tryptophan added was converted to 3-methylindole at pH 5.0; at pH 8.0 85% was converted to indole. Both pathways had similar Km values for tryptophan and similar maximum rates. Indole-3-carbinol and indole-3-acetonitrile completely inhibited the production of 3-methylindole from indole-3-acetate but had no effect on the reactions involving L-tryptophan.     

Metabolic pathways of quinoline, indole and their methylated analogs by Desulfobacterium indolicum (DSM 3383)

The transformation of quinoline, isoquinoline and 3-, 4-, 6- and 8-methylquinoline by Desulfobacterium indolicum was compared with that of the N-containing analogues indole and 1-, 2-, 3- and 7-methylindole. The metabolites were identified using high-performance liquid chromatography with UV detection, thin-layer chromatography, combined gas chromatography/mass spectrometry and proton NMR spectroscopy. All degraded compounds were initially hydroxylated at position 2 by D. indolicum. A new degradation product of quinoline was observed in the second transformation step, where 3,4-dihydro-2-quinolinone accumulated. This ring-reduced compound was further transformed into unidentified products. The transformation pathway of indole was characterized by well-known steps through oxindole, isatin, and anthranilic acid. No further transformation of the hydroxylated methyl analogues: 3- and 7-methyloxindole and 3- and 4-methyl-2-quinolinone, was observed within 162 days of incubation. These degradation products accumulated in stoichiometric amounts, while 6- and 8-methyl-2-quinolinone were further degraded to 6- and 8-methyl-3,4-dihydro-2-quinolinone in stoichiometric amounts. Isoquinoline, 2-methylquinoline and 1- and 2-methylindole were not degraded by D. indolicum. These observations indicate that a methyl group at or close to position 2 results in blockage of the microbial attack, and that transformation of hydroxyquinolines methylated at the heterocyclic ring also was blocked or sterically inhibited. An incomplete transformation of some methylated compounds was observed, e.g. for 3- and 6-methylquinoline and 3- and 7-methylindole, with residual concentrations of 0.5–4 mg/l in relation to initial concentrations of 10–15 mg/l. Received: 23 July 1996 / Received revision: 4 October 1996 / Accepted: 25 October 1996     

Gas chromatographic determination of indole and 3-methylindole (skatole) in bacterial culture media, intestinal contents and faeces

A simple, rapid and inexpensive gas chromatographic method was developed for the determination of indole and 3-methylindole (skatole) in faeces, intestinal contents and bacterial cultures. It involves a simple homogenization and extraction with chloroform. The extract is injected onto a gas chromatograph equipped with a 12.5-m fused-silica capillary column coated with BP20 and a film thickness of 0.5 μm. To simplify the chromatograms and to get a higher sensitivity a nitrogen—phosphorus-sensitive detector is applied. The detection limit for indole and 3-methylindole under the conditions employed is 20 μg/kg, which is well below the values typically found in intestinal contents (up to 100 mg/kg). Recovery for both compounds was close to 100%, and the mean coefficients of variation were 3.5% for indole and 3.0% for 3-methylindole. The method has demonstrated its practical value in the analysis of more than 50 000 samples in our laboratory. More than 100 samples can be analyzed per day.     

Dissimilation of Tryptophan and Related Indolic Compounds by Ruminal Microorganisms In Vitro

Intraruminal doses of L-tryptophan cause acute pulmonary edema and emphysema in cattle. The D and L isomers of tryptophan and 22 related indolic compounds were incubated with ruminal microorganisms in vitro. Incubation of L-[U-benzene ring-(14)C]tryptophan with ruminal microorganisms for 24 h resulted in 39% of the added radioactivity being incorporated into skatole, 7% into indole, and 4% into indoleacetate (IAA). D-Tryptophan was not degraded to any of these metabolites. The major pathway of skatole formation from L-tryptophan appeared to be by the decarboxylation of IAA. Incubation of [2-(14)C]IAA with ruminal microorganisms for 24 h resulted in 38% incorporation into skatole. L-[5-Hydroxy]tryptophan was degraded to 5-hydroxyskatole and 5-hydroxyindole, whereas 5-hydroxyindoleacetate was degraded to only 5-hydroxyskatole. Incubation of indolepyruvate, indolelactate, and indolealdehyde with ruminal microorganisms resulted in the formation of both skatole and indole. Under similar conditions, indoleacetaldehyde was converted to IAA and tryptophol. The addition of increasing concentrations of glucose (0 to 110 mM) reduced the formation of both skatole and indole from L-tryptophan and resulted in the accumulation of IAA. Antibiotics reduced the degradation of L-tryptophan to skatole and indole, with kanamycin and neomycin particularly effective in reducing the decarboxylation of IAA to skatole.     

Catabolic Pathway for the Production of Skatole and Indoleacetic Acid by the Acetogen Clostridium drakei, Clostridium scatologenes, and Swine Manure

Skatole (3-methylindole) is a malodorous chemical in stored swine manure and is implicated as a component of foul-tasting pork. Definitive evidence for the skatole pathway is lacking. Deuterium-labeled substrates were employed to resolve this pathway in the acetogenic bacterium Clostridium drakei and Clostridium scatologenes and to determine if a similar pathway is used by microorganisms present in stored swine manure. Indoleacetic acid (IAA) was synthesized from tryptophan by both bacteria, and skatole was synthesized from both IAA and tryptophan. Microorganisms in swine manure produced skatole and other oxidation products from tryptophan, but IAA yielded only skatole. A catabolic mechanism for the synthesis of skatole is proposed.     

Effects of fructooligosaccharide on conversion of L-tryptophan to skatole and indole by mixed populations of pig fecal bacteria

An in vitro study was conducted to examine the effects of fructooligosaccharide (FOS) at levels of 0.5, 1.0, and 1.5% on conversion of L-tryptophan to skatole and indole by a mixed bacterial population from the large intestines of pigs. Microbial suspensions were anaerobically incubated at 38 degrees C for 24 h. Samples were periodically removed for determination of pH and indole compounds. After 24 h incubation, microbial populations in each culture media were analyzed. Addition of 0.5, 1.0, and 1.5% FOS to the slurries with L-tryptophan significantly decreased the skatole concentration, the peak value of indole-3-acetic acid and the medium pH. The viable counts of Bifidobacterium were significantly higher as compared with the control. Addition of 1.0 and 1.5% FOS significantly decreased the rate of tryptophan degradation and the relative rate of skatole production. The relative rate of indole production was significantly increased. The viable counts of Clostridium and Escherichia coli were significantly reduced. The total viable counts of anaerobes were significantly increased. These results suggest that the reduced concentration of skatole observed in the presence of FOS may be caused by the decreased tryptophan degradation due to the increased need for amino acids in the synthesis of bacterial cellular protein, and by shifting microbial metabolism of tryptophan toward indole production at the expense of skatole, which might result from the changed microbial ecosystem and pH. Our observations open the possibility of inhibiting microbial production of skatole and decreasing the skatole concentration in backfat by feeding pigs diets containing FOS, but it remains to be demonstrated in vivo.     

Metabolism of 3-methylindole by a methanogenic consortium.

A methanogenic 3-methylindole (3-MI)-degrading consortium, enriched from wetland soil, completely mineralized 3-MI. Degradation proceeded through an initial hydroxylation reaction forming 3-methyloxindole. The consortium was unable to degrade oxindole or isatin, suggesting a new pathway for 3-MI fermentation.     

Rapid high-performance liquid chromatographic method for simultaneous determination of androstenone, skatole and indole in back fat from pigs

A rapid high-performance reversed-phase liquid chromatographic method for the simultaneous quantitative determination of the main boar taint compounds androstenone, skatole (3-methylindole) and indole, in back fat from pigs has been developed. The compounds are extracted by a simple homogenisation of adipose tissue in methanol; interfering lipids are removed by precipitation after cooling and centrifugation. Androstenone is derivatized pre-column with dansylhydrazine (5 min at ambient temperature) using BF₃ as catalyst. The compounds are separated on a 60 × 4.6 mm I.D., 3 μm Hypersil ODS column (Hewlett-Packard) using a step-gradient; total time for the separation is 15 min. Fluorescence is used for selective detection. The limit of quantitation for indole and skatole is 30 ng/g and for androstenone 200 ng/g back fat. The results for skatole obtained by the present method were compared with those of a colorimetric method, while androstenone determinations were compared with two GC—MS methods and a RIA method. The correlations observed were in the range of 0.946–0.993. The average contents of androstenone and skatole were 640 ± 700 and 78 ± 113 ng/g (n = 1162 male pigs), respectively.     

Pathway of indole metabolism by a denitrifying microbial community

The metabolism of indole in a mineral-salts medium inoculated with 9% anaerobically digested nitrate-reducing sewage sludge was studied. The sequential occurrence of four structurally-related compounds — oxindole, isatin, dioxindole, and anthranilic acid — was detected using high-performance liquid or thin-layer chromatography. Mass spectrometry and proton nuclear resonance were used to identify isatin and dioxindole isolated from the culture fluids. Prior exposure of the microorganisms to indole, oxindole, isatin, or anthranilic acid resulted in accelerated decomposition of these compounds in a pattern that was consistent with a proposed pathway for the metabolism of indole under denitrifying conditions.     

Metabolism of 3-methylindole by a methanogenic consortium.

A methanogenic 3-methylindole (3-MI)-degrading consortium, enriched from wetland soil, completely mineralized 3-MI. Degradation proceeded through an initial hydroxylation reaction forming 3-methyloxindole. The consortium was unable to degrade oxindole or isatin, suggesting a new pathway for 3-MI fermentation.     

Peroxide oxidation of indole to oxindole by chloroperoxidase catalysis.

In the presence of chloroperoxidase, indole was oxidized by H2O2 to give oxindole as the major product. Under most conditions oxindole was the only product formed, and under optimal conditions the conversion was quantitative. This reaction displayed maximal activity at pH 4.6, although appreciable activity was observed throughout the entire pH range investigated, namely pH 2.5-6.0. Enzyme saturation by indole could not be demonstrated, up to the limit of indole solubility in the buffer. The oxidation kinetics were first-order with respect to indole up to 8 mM, which was the highest concentration of indole that could be investigated. On the other hand, 2-methylindole was not affected by H2O2 and chloroperoxidase, but was a strong inhibitor of indole oxidation. The isomer 1-methylindole was a poor substrate for chloroperoxidase oxidation, and a weak inhibitor of indole oxidation. These results suggest the possibility that chloroperoxidase oxidation of the carbon atom adjacent to the nitrogen atom in part results from hydrogen-bonding of the substrate N-H group to the enzyme active site.     

Photomonomerization of pyrimidine dimers by indoles and proteins.

Model systems for the study of photoreactivation have been developed that utilize a variety of indole derivatives. These systems can split uracil cis-syn cyclobutadipyrimidine, either free or in RNA, when irradiated at wave-lengths absorbed only by the indole moiety. The ability of indole compounds to split dimers is closely related to their electronic properties. Those of high electron-donor capacity such as indole, 3-methylindole, indole-3-acetic acid, 5-hydroxytryptophan and tryptophan are good photosensitizers, with efficacy in that order. Indoles with electron-withdrawing substituents such as indole-3-carboxylic acid, indole-3-aldehyde and oxindole are inactive in the monomerization reaction. These findings support the proposed mechanism that the photosensitized monomerization occurs as a result of electron transfer from the excited indole molecules to the pyrimidine bases.Proteins containing fully exposed tryptophan residues (chicken egg white lysozyme and bovine diisopropylphosphoryltrypsin) also cause the splitting of the ¹⁴C-labeled dimers under the same conditions. In the case of lysozyme the quantum yield of monomerization is similar to that of free tryptophan. Much of the monomerization ability of lysozyme was lost after the solvent-available tryptophan had been oxidized by treatment with N-bromosuccinimide. Bovine pancreatic ribonuclease A, a protein devoid of tryptophan, failed to exhibit photosensitized monomerization of uracil dimers. The biological implication of these reactions involving a protein with an exposed tryptophan residue is discussed.Although indoles are able to split the dimers in RNA, they fail to photo-reactivate u.v.-damaged TMV-RNA. Indole-3-acetic acid, 3-methylindole and 5-hydroxytryptophan rapidly inactivate viral RNA when irradiated at 313 nm, possibly because of side reactions.     

Isolation and characteristics of a skatole-producing Lactobacillus sp. from the bovine rumen.

A bacterium that is capable of decarboxylating indoleacetic acid to skatole (3-methylindole) has been isolated from an L-tryptophan enrichment of bovine rumen fluid. The bacterium is a gram-positive, nonmotile, nonsporeforming rod. It is an obligate anaerobe, and strains predominatly produce D-(-)-lactic acid, with smaller amounts of L-(+)-lactic acid and acetic acid, from sugar. All four strains isolated gave a negative reaction to the indole test because they cannot form skatole directly from tryptophan. This is the first report of indoleacetic acid decarboxylation to skatole in pure culture and the demonstration of skatole production by a Lactobacillus species.     

Induction of Biofilm Formation in the Betaproteobacterium Burkholderia unamae CK43B Exposed to Exogenous Indole and Gallic Acid

Burkholderia unamae CK43B, a member of the Betaproteobacteria that was isolated from the rhizosphere of a Shorea balangeran sapling in a tropical peat swamp forest, produces neither indole nor extracellular polymeric substances associated with biofilm formation. When cultured in a modified Winogradsky''s medium supplemented with up to 1.7 mM indole, B. unamae CK43B maintains its planktonic state by cell swelling and effectively degrades exogenous indole. However, in medium supplemented with 1.7 mM exogenous indole and 1.0 mM gallic acid, B. unamae CK43B produced extracellular polymeric substances and formed a biofilm. The concentration indicated above of gallic acid alone had no effect on either the growth or the differentiation of B. unamae CK43B cells above a certain concentration threshold, whereas it inhibited indole degradation by B. unamae CK43B to 3-hydroxyindoxyl. In addition, coculture of B. unamae CK43B with indole-producing Escherichia coli in nutrient-rich Luria-Bertani medium supplemented with 1.0 mM gallic acid led to the formation of mixed cell aggregates. The viability and active growth of B. unamae CK43B cells in a coculture system with Escherichia coli were evidenced by fluorescence in situ hybridization. Our data thus suggest that indole facilitates intergenus communication between indole-producing gammaproteobacteria and some indole-degrading bacteria, particularly in gallic acid-rich environments.     

Production of Skatole and para-Cresol by a Rumen Lactobacillus sp

The objective of this study was to examine the substrate specificity of several ruminal strains of a Lactobacillus sp. which previously was shown to produce skatole (3-methylindole) by the decarboxylation of indoleacetic acid. A total of 13 compounds were tested for decarboxylase activity. The Lactobacillus strains produced p-cresol (4-methylphenol) by the decarboxylation of p-hydroxyphenylacetic acid, but did not produce either o-cresol or m-cresol from the corresponding hydroxyphenylacetic acid isomers. These strains also decarboxylated 5-hydroxyindoleacetic acid to 5-hydroxyskatole and 3,4-dihydroxyphenylacetic acid to methylcatechol. Skatole and p-cresol were produced in a 0.5:1 ratio, when indoleacetic acid and p-hydroxyphenylacetic acid were combined in equimolar concentrations. Competition studies with indoleacetic acid and p-hydroxyphenylacetic acid suggested that two different decarboxylating enzymes are involved in the production of skatole and p-cresol by these strains. This is the first demonstration of both skatole production and p-cresol production by a single bacterium.     

Environmental factors affecting indole metabolism under anaerobic conditions

The influence of physiological and environmental factors on the accumulation of oxindole during anaerobic indole metabolism was investigated by high-performance liquid chromatography. Under methanogenic conditions, indole was temporarily converted to oxindole in stoichiometric amounts in media inoculated with three freshwater sediments and an organic soil. In media inoculated with methanogenic sewage sludge, the modest amounts of oxindole detected at 35 degrees C reached higher concentrations and persisted longer when the incubation temperature was decreased from 35 to 15 degrees C. Also, decreasing the concentration of sewage sludge used as an inoculum from 50 to 1% caused an increase in the accumulation of oxindole from 10 to 75% of the indole added. Under denitrifying conditions, regardless of the concentration or source of the inoculum, oxindole appeared in trace amounts but did not accumulate during indole metabolism. In addition, denitrifying consortia which previously metabolized indole degraded oxindole with no lag period. Our data suggest that oxindole accumulation under methanogenic, but not under denitrifying conditions is caused by differences between relative rates of oxindole production and destruction.