Biotransformation of Indole to 3-Methylindole by Lysinibacillus xylanilyticus Strain MA

1School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Republic of Korea 2Department of Microbiology, University of Chittagong, Chittagong 4331, Bangladesh 3División de Ciencias de la Salud, Departamento de Ingenieria Agroindustrial, Universidad de Guanajuato, Salvatierra, GTO,Mexico 4Escuela de Ingenieria en Alimentos, Biotecnologia y Agronomia, Instituto Tecnológico y de Estudios Superiores de Monterrey, Epigmenio Gonzalez 500, 76130 San Pablo, QRO, Mexico


Introduction
Indole is an industrially important heterocyclic aromatic compound that is an environmental pollutant due to its worldwide occurrence [1].Major contamination sources are industrial waste, coal tar waste, and wastewater from coking plants, coal gasification, and refineries and cigarette smoke [1].
In this study, we investigated a new mechanism of aerobic transformation of indole via a newly isolated bacterium, Lysinibacillus xylanilyticus strain MA.

Chemicals and Media.
Indole and its derivativeswere purchased from Sigma-Aldrich.All other chemicals, reagents, and solvents were purchased from Fisher Scientific.Minimal medium was prepared as described previously [13].

Bacterial Strain.
Twenty bacteria were isolated from the contaminated soil using minimal medium containing 10 mM sodium succinate and 0.5 mM indole.For isolation, 1 g soil was added to minimal medium containing 10 mM sodium succinate and 0.5 mM indole.After 48 h of incubation, the medium was serially diluted and plated on the minimal agar plates containing 10 mM sodium succinate and 0.5 mM indole.After incubation period, twenty different morphotypes were selected and streaked for purity.These bacteria were screened for their ability to mineralize or transform indole by growing on minimal media containing 0.5 mM indole in the presence or absence of 10 mM sodium succinate.Samples were collected at regular intervals to monitor indole depletion and extracted with ethyl acetate.The extracted samples were analyzed by high performance liquid chromatography by a previously described method [7], and the results showed that there was no indole depletion by any of the bacteria growing on minimal medium containing 0.5 mM indole.However, one bacteria-designated strain (MA) was able to deplete indole in the presence of additional carbon sources (i.e., sodium succinate).This bacterium was selected for further study.

16S rRNA Gene Sequencing and Phylogenetic Analysis.
The genomic DNA extraction for PCR amplification of the 16S rRNA gene of strain MA was carried out as described previously [14].Amplification and sequencing conditions for the 16S rRNA gene were done exactly the same as described previously [15].The 16S rRNA gene sequence of strain MA was aligned with other sequences obtained from the EzTaxon databases [16].The phylogenetic tree was constructed using neighbor-joining algorithms and evolutionary distance matrices were calculated with the Kimura twoparameter model [17].Bootstrap replications (1000) were performed with the MEGA6 program [17].

Growth and Indole Depletion.
The bacteria were grown on minimal medium containing 0.5 mM indole and 10 mM sodium succinate.Samples were collected at every 4 h interval up to 32 h.For growth measurement, the optical density of the culture was measured at 600 nm using a spectrophotometer.For indole depletion, samples were centrifuged and extracted with ethyl acetate.The extracted samples were dissolved in 20 L methanol and analyzed by HPLC (Waters 600 HPLC model) as described previously [7].

Identification of Metabolites.
The samples (0 h, 12 h, 24 h, and 32 h) were analyzed by gas-chromatography-mass spectrometry (GC-MS) to identify metabolites via an Agilent gas chromatography system model 7890A equipped with a high throughput time-of-flight mass spectrometer and HP-5 column (30 m × 0.320 mm × 0.25 m) [7].The column temperature was initially increased from 50 ∘ C to 280 ∘ C at the rate of 20 ∘ C/min and then held for 5 min [7].Helium was used as a carrier gas at 1.5 mL/min and the samples (1 L) were injected in splitless mode [7].The ion-source temperature and transfer line temperature were maintained at 250 ∘ C and 225 ∘ C, respectively [7].The electron energy was set at 70 eV [7].
2.6.Tryptophan 2-Monooxygenase Activity.Enzyme activity was determined in a 1 mL reaction mixture containing 100 mM Tris buffer (pH 7.8), 0.5 mM of L-tryptophan, and crude extracts.After the incubation at 10 min at room temperature, the reaction was stopped by adding 100 L of 5 N HCl.The reaction mixture without crude extracts served as the control.The reaction mixture was centrifuged, extracted with ethyl acetate, and dissolved in 20 L of methanol and analyzed with GC-MS to identify the product.

Results and Discussion
Strain MA was identified as a member of the genus Lysinibacillus xylanilyticus on the basis of the 16S rRNA gene sequencing.The 16S rRNA gene sequence of strain MA has been deposited in NCBI under the GenBank accession number KT030900.Phylogenetic analysis showed that strain MA fell within other members of Lysinibacillus with a cluster near Lysinibacillus xylanilyticus strain XDB9 (Figure 1).On the basis of the 16S rRNA gene sequencing and phylogenic analysis, strain MA was identified as Lysinibacillus xylanilyticus strain MA.
Figure 2 showed that strain MA grew well on minimal medium supplemented with 0.5 mM indole and 10 mM sodium succinate.During the initial 4 h, there was no bacterial growth due to lag phase whereas bacteria grow rapidly after 8 h due to the exponential phase.There was very slow growth after 24 h when the bacteria reached stationary phase.The maximum optical density of the culture was 1.7 after 32 h of incubation.No bacterial growth was observed on minimal medium supplemented with 0.5 mM indole because it is the sole source of carbon and energy.These data indicate that strain MA did not utilize indole as its sole source of carbon and energy.Strain MA transforms indole in the presence of additional carbon source (i.e., sodium succinate).Indole transformation was measured by HPLC, and the results showed complete indole depletion within 32 h.
The GC-MS studies showed transformation of indole into three metabolites.These metabolites were identified on the basis of their mass spectra comparisons with those of authentic standards.The mass spectrum of metabolite I had a molecular ion at / 174 and quinolinium ion at / 130.This metabolite was identified as indole-3acetamide (Figure 3(a)).The mass spectrum of metabolite II contains a parent ion at / 175 and quinolinium ion at / 130.This metabolite was identified as indole-3-acetic acid (Figure 3(b)).The mass spectrum of metabolite III had ions at / 131, 130, 103, 102, 77, and 78.This metabolite was identified as 3-methylindole (Figure 3(c)).
The GC-MS analysis of the enzyme reaction mixture indicated the formation of a product with a mass spectrum corresponding to indole-3-acetamide.However, this product was not detected in the control.On the basis of transformation products, we proposed a new pathway of indole transformation.Indole is initially converted to indole-3-acetamide via tryptophan.Indole-3acetamide was then transformed into indole-3-acetic acid, which was decarboxylated to 3-methylindole (Figure 4).This is the first report of formation of 3-methylindole from indole.
Previous studies showed that indole-3-acetic acid formation occurs via either tryptophan-dependent pathway [18,19] or tryptophan-independent pathway [7,20,21].The Arthrobacter sp.SPG converted indole to indole-3-acetic acid without forming tryptophan.This suggested the involvement of a tryptophan-independent pathway [7].However, in this study, we observed tryptophan 2-monooxygenase activity in the crude extracts of indole-induced cells of strain MA suggesting the involvement of a tryptophan-dependent pathway.Two mechanisms are known for formation of indole-3-acetic acid from tryptophan [18,19].The first mechanism involves a tryptophan aminotransferase-catalyzed conversion of tryptophan to indole-3-pyruvic acid, which is decarboxylated to indole-3-acetaldehyde by an indole-3pyruvic acid decarboxylase.This is then further oxidized to indole-3-acetic acid [18].We have not detected indole-3-pyruvic acid and indole-3-acetaldehyde as metabolites of indole degradation, suggesting that this mechanism is not involved in the transformation.In the second mechanism, the initial step is catalyzed by tryptophan 2-monooxygenase and involves conversion of tryptophan to indole-3-acetamide that is then transformed to indole-3-acetic acid by indole-3acetamide hydrolase [19].In this study, indole-3-acetamide was detected as a metabolite, indicating involvement of the indole-3-acetamide pathway.Furthermore, the tryptophan 2monooxygenase activity confirmed the formation of indole-3-acetamide from L-tryptophan.
This study differs from all previous studies of indole biotransformation due to involvement of new transformation mechanism.In the previous study, Arthrobacter sp.SPG also transformed indole to indole-3-acetic acid that was further converted to indole-3-glyoxylic acid and indole-3aldehyde [7].In this case, indole-3-acetic acid is transformed to 3-methylindole.Several bacteria transformed indole to indigo via indoxyl [1]; however, strain MA did not produce indoxyl or indole.Recently, Fukuoka et al. [26] reported biotransformation of indole in Cupriavidus sp.strain KK10 via an N-heterocyclic ring cleavage or carbocyclic aromatic ring cleavage of indole; however, in this study, neither Nheterocyclic ring cleavage nor carbocyclic aromatic ring cleavage occurred.