Microbial Degradation of Indole and Its Derivatives

Indole and its derivatives, including 3-methylindole and 4-chloroindole, are environmental pollutants that are present worldwide. Microbial degradation of indole and its derivatives can occur in several aerobic and anaerobic pathways; these pathways involve different known and characterized genes. In thisminireview, we summarize and explain themicrobial degradation of indole, indole3-acetic acid, 4-chloroindole, and methylindole.


Introduction
Indole and its derivatives comprise a major group of heterocyclic aromatic compounds which are widely used for the synthesis of pharmaceuticals, dyes, and industrial solvents [1].Indole is used as a perfume fixative, a synthetic flavor, and a chemical intermediate for synthesis of a plant growth regulator, indole-3-acetic acid [1,2].2-Methylindole is used for dye manufacturing, including cyanine dyes and cationic diazo dyes [3].The indole ring is also present as a core building block and key functional group in many pharmaceuticals, alkaloids, and hormones [4].
Indole and its derivatives are also present in many natural products: indole occurs naturally in Robinia pseudoacacia, the jasmines, certain citrus plants, and the wood of Celtis reticulata [1].Indole is also present in coal tar [5], fuel oil [6], and cigarette smoke [7,8].Indole is one of the main degradation products of microbial metabolism of L-tryptophan, an essential amino acid present in most proteins [9,10].More than 85 species of Gram-positive and Gram-negative bacteria can produce indole [11].3-Methylindole is commonly found in feces and sewage and is well known for its unpleasant smell [1,[12][13][14].Indole-3-acetic acid (auxin) is a naturally occurring plant hormone that has a significant role in plant growth and development.
Indole and its derivatives are discharged into the environment through industrial waste, coal tar waste, and wastewater from coking plants, coal gasification [5,15,16] and refineries [6], and cigarette smoke.
Human beings can be exposed to indole via (i) ambient air, (ii) tobacco smoke, (iii) food, and (iv) skin contact with vapors and other products, such as perfumes that contain indole.
Indole and its derivatives are considered environmental pollutants due to their toxicity and worldwide occurrence in soils, coastal areas, groundwater, surface waters, and even indoor environments [15,32].Several reviews are available for applications and microbial production of indole; however, there are very few reviews on microbial degradation of indole [4,11].Recently, rapid progress was made in the study of microbial degradation of indole and its derivatives and a few new pathways were proposed for microbial degradation of indole and its derivatives [28,33,34].The aim of this review is to summarize the microbial degradation of indole, indole-3-acetate, 4-chloroindole, and methylindole and highlight recent developments in the field.

Bacterial Mineralization of Indole.
A few indolemineralizing bacteria have been isolated and characterized for aerobic biodegradation of indole [35][36][37]46].Three major pathways for indole mineralization have been proposed and these pathways are the catechol pathway, the gentisate pathway, and the anthranilate pathway.The catechol pathway was studied in an indole-mineralizing Gram-negative bacterium isolated from tap water [35].The first step of the catechol pathway was hydroxylation of indole to indoxyl, which was further hydroxylated to 2,3-dihydroxyindole (Figure 1(a)).Further degradation proceeded via isatin, N-formylanthranilic acid, anthranilic acid, salicylic acid, and catechol [35].The anthranilate pathway of indole degradation was studied in a Gram-positive coccus that utilized indole as its sole source of carbon and energy and degraded it via 2,3-dihydroxyindole, N-carboxyanthranilic acid, and anthranilic acid (Figure 1(b)) [36].Claus and Kutzner [37] reported the gentisate pathway of indole degradation in an indole-mineralizing bacterium, Alcaligenes sp.In 3 isolates from activated sludge.In this pathway, indole degradation occurred via indoxyl, isatin, anthranilic acid, and gentisic acid (Figure 1(c)); the formation of gentisic acid was a key feature of this pathway, formed due to hydroxylation of anthranilic acid.The possibility of new indole degradation pathways, aside from these 3 isolates, has been suggested.Doukyu and Aono [46] reported the mineralization of indole via isatin and isatic acid in Pseudomonas sp.strain ST-200.Yin et al. [47] studied indole degradation in an indole-mineralizing bacterium, Pseudomonas aeruginosa Gs isolated from mangrove sediments, and detected two major metabolites; however, they could not identify either metabolite.
Several enzymes, such as monooxygenases, dioxygenases, and cytochrome P450, were characterized for indigo production [50].Many genes encoding these enzymes were cloned and used to construct engineering bacteria for efficient indigo production [50].Ensley et al. [51] cloned and expressed a DNA fragment of a Pseudomonas plasmid, containing naphthalene oxidation genes, in E. coli and observed that the recombinant E. coli synthesized indigo in nutrient-rich medium; indigo production increased in the presence of tryptophan or indole.Wu et al. [58] transferred a plasmid containing naphthalene degrading genes from Pseudomonas sp.S13 to E. coli.The recombinant E. coli was able to synthesize indigo [58].Qu et al. [59] showed that E. coli that expressed biphenyl dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase efficiently transformed indole to indigo.E. coli that expressed cytochrome P450 also oxidized indole to indigo.The immobilization of E. coli BL21 expressing P450 BM-3 showed better rates of indigo production than nonimmobilized cells [60].The xylA gene that encodes xylene oxygenase was cloned from the TOL plasmid pWW53 of P. putida MT53 and is responsible for indigo production [61].Nagayama et al. [62] constructed a cosmid library of metagenomic DNA in E. coli and introduced it into P. putida-derived strains that produced little indigo on indolecontaining agar plates.Screening results showed that 29 cosmid clones generated indigo on the indole-containing agar plates [62].Six representative cosmids were selected for sequencing and in vitro transposon mutagenesis, leading to the identification of genes encoding putative classes B and D flavo protein monooxygenases, a multicomponent hydroxylase, and a reductase that were responsible for indigo formation [62].
Another fungal metabolic pathway of indole was studied in an endophytic fungus, Phomopsis liquidambari, which utilized indole as its sole source of carbon and nitrogen [38].In this fungus, indole was initially oxidized to oxindole and isatin.In the next step, isatin was transformed to 2dioxindole.The 2-dioxindole was further converted to 2aminobenzoic acid via pyridine ring cleavage (Figure 4(b)) [38].Katapodis et al. [63] reported indole degradation by a thermophilic fungus, Sporotrichum thermophile, using a persolvent fermentation system containing a large amount of indole (the medium contained 20% soybean oil by volume and up to 2 g/L indole).They reported that most of the indole was partitioned in the organic solvent layer and complete indole degradation was observed after 6 days when the fungus was grown on media containing indole at 1 g/L [63].
To date, only one pure culture of bacteria capable of utilizing indole as its sole source of carbon and energy, that is, the sulfate reducer Desulfobacterium indolicum, has been isolated and characterized.This bacterium was initially isolated from enriched marine sediments by Bak and Widdel [64].Several studies investigated indole degradation in Desulfobacterium indolicum, which degrades indole via oxindole [39,73], including Johansen et al. [39], who proposed the biodegradation pathway of indole for D. indolicum.Initially, indole was hydroxylated at the C-2 position to form oxindole that was further hydroxylated at C-3 to form isatin. Isatin underwent ring cleavage between the C-2 and C-3 atoms on the pyrrole ring of indole to produce isatoic acid, which was decarboxylated to anthranilic acid (Figure 5).The further degradation of anthranilic acid achieved complete mineralization.Similar results were reported for indole degradation by a denitrifying microbial community [68].Hong et al. [74] studied two anaerobic, indole-decomposing microbial communities under both denitrifying and sulfate-reducing conditions.In the denitrifying bioreactor, most of the dominant bacteria were -proteobacteria, predominantly Alicycliphilus, Alcaligenes, and Thauera genera.In the sulfate-reducing bioreactor, Clostridia and Actinobacteria were the dominating indole-degrading species [74].
Some bacteria promote plant growth by degrading exogenous indole-3-acetic acid in plant roots [86]; for example, Zúñiga et al. [86] reported that bacterial degradation of indole-3-acetic acid plays a key role in plant growthpromoting traits and is necessary for efficient rhizosphere colonization.They reported that wild-type Burkholderia phytofirmans promotes the growth of Arabidopsis plant roots in the presence of exogenously added indole-3-acetic acid; however, a mutant strain with destructed iacC was unable to promote the growth of the plant root [86].
further degradation of indole proceeded via isatin, anthranilic acid, and salicylic acid (Figure 7(a)).The enzyme activities for 4-chloroindole dehalogenase and anthranilic acid deaminase were detected in the crude extract of the 4-chloroindoles-induced cells of Exiguobacterium sp.PMA, confirming indole and salicylic acid formation in the degradation pathway of 4-chloroindole.Exiguobacterium sp.PMA also degraded 4-chloroindole in sterile and nonsterile soil [28].The degradation rate was faster in sterile soil than in nonsterile soil [28].
sediment obtained from the Mai Po Nature Reserve of Hong Kong; a pure culture of Pseudomonas aeruginosa Gs isolated from this enrichment utilized 1-methylindole and 3methylindole as its sole source of carbon and energy and completely degraded 1-methylindole and 3-methylindole after more than 40 days and 24 days, respectively, when the concentration of 3-methylindole or 1-methylindole was 2.0 mM in the culture [87].Indoline-3-carboxylic acid and indoline-3-ol were identified as metabolites of 3-methylindole in P. aeruginosa Gs (Figure 7(b)) [44].Gu and Berry [32] reported the degradation of 3-methylindole via 3-methyloxindole using a methanogenic consortium derived from enrichment of wetland soil.The removal of 3-methylindole was monitored by the four strains of lactic acid bacteria (Lactobacillus brevis 1.12 (L.brevis 1.12), L. plantarum 102, L. casei 6103, and L. plantarum ATCC8014); L. brevis 1.12 was the best at removing 3-methylindole [88].Gu et al. [45] reported that a methanogenic bacterial consortia derived from marine sediment from Victoria Harbour transformed 3-methylindole to 3methyloxindole, whereas a sulfate-reducing consortium mineralized 3-methylindole completely via 3-methyloxindole and -methyl-2-aminobenzeneacetic acid (Figure 7(c)).Sharma et al. [89] isolated a new 3-methylindole-degrading purple nonsulfur bacterium, Rhodopseudomonas palustris WKU-KDNS3, from a swine waste lagoon using an enrichment technique.This bacterium could remove >93% of the total 3-methylindole in the medium by 21 days.

Conclusions and Future Perspectives
(i) Microbes degrade indole either by mineralization or cometabolism (biotransformation).In mineralization, microbes utilized indole as the sole source of carbon and energy and degraded it completely via a series of chemical reactions; however, in the process of biotransformation, indole was transformed to other compounds in the presence of an additional carbon source.These biotransformed products may be more or less toxic than indole and sometimes used as useful products; for example, several bacteria convert indole to indigo, a compound of industrial value.Similarly, Arthrobacter sp.SPG biotransformed indole to indole-3-acetic acid (a plant growth-promoting hormone), indole-3-glyoxylic acid, and indole-3aldehyde.A few microbes adopt detoxification mechanisms via biotransformation and convert indole to less toxic or nontoxic compounds; for example, Cupriavidus sp.strain KK10 transformed indole to less toxic or nontoxic products via N-heterocyclic ring cleavage or carbocyclic aromatic ring cleavage.
(ii) Three major pathways for aerobic bacterial mineralization of indole have been proposed.However, the genes and the enzymes involved in these pathways could not yet be characterized.
(iii) Anaerobic degradation of indole has been studied under methanogenic, sulfate-reducing and denitrifying conditions.However, a few indole-mineralizing bacteria are known for anaerobic degradation of indole.More indole degrading anaerobic bacteria should be isolated to understand the mechanism of anaerobic degradation of indole.
(iv) More biochemical studies should be carried out to elucidate the metabolic pathways of degradation of 4chloroindole and methylindole.
(v) Four major pathways of aerobic bacterial degradation of indole-3-acetic acid have been elucidated.However, the genetics of bacterial degradation pathway of indole-3-acetic acid was studied in Pseudomonas putida 1290 that contains iac gene cluster for indole-3-acetic acid degradation.Furthermore, complete characterization of iac genes would be very helpful to understand the mechanism of biodegradation of indole-3-acetic acid.
-Chloroindole was initially dehalogenated and