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Journal of Chemistry
and solvents were purchased from Fisher Scientific. Minimal
medium was prepared as described previously [13].
injected in splitless mode [7]. e ion-source temperature
and transfer line temperature were maintained at 250∘C and
225∘C, respectively [7]. e electron energy was set at 70 eV
[7].
2.2. 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. Afer 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. Afer incubation period, twenty different morpho-
types were selected and streaked for purity. ese 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. e extracted
samples were analyzed by high performance liquid chro-
matography 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). is bacterium was selected
for further study.
2.6. Tryptophan 2-Monooxygenase Activity. Enzyme activ-
ity 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. Afer the incubation at 10 min at room tem-
perature, the reaction was stopped by adding 100 ꢀL of 5 N
HCl. e reaction mixture without crude extracts served as
the control. e reaction mixture was centrifuged, extracted
with ethyl acetate, and dissolved in 20 ꢀL of methanol and
analyzed with GC-MS to identify the product.
3. Results and Discussion
Strain MA was identified as a member of the genus Lysini-
bacillus xylanilyticus on the basis of the 16S rRNA gene
sequencing. e 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 xylanilyti-
cus 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 bac-
terial growth due to lag phase whereas bacteria grow rapidly
afer 8 h due to the exponential phase. ere was very slow
growth afer 24 h when the bacteria reached stationary phase.
e maximum optical density of the culture was 1.7 afer 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. ese 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.
2.3. 16S rRNA Gene Sequencing and Phylogenetic Analysis.
e 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]. e 16S rRNA gene sequence of
strain MA was aligned with other sequences obtained from
the EzTaxon databases [16]. e phylogenetic tree was con-
structed using neighbor-joining algorithms and evolutionary
distance matrices were calculated with the Kimura two-
parameter model [17]. Bootstrap replications (1000) were
performed with the MEGA6 program [17].
2.4. Growth and Indole Depletion. e 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. e extracted samples were dissolved in
20 ꢀL methanol and analyzed by HPLC (Waters 600 HPLC
model) as described previously [7].
e GC-MS studies showed transformation of indole
into three metabolites. ese metabolites were identified
on the basis of their mass spectra comparisons with those
of authentic standards. e mass spectrum of metabolite
I had a molecular ion at ꢁꢂ/ 174 and quinolinium ion
at ꢁꢂ/ 130. is metabolite was identified as indole-3-
acetamide (Figure 3(a)). e mass spectrum of metabolite
II contains a parent ion at ꢁꢂ/ 175 and quinolinium ion at
ꢁꢂ/ 130. is metabolite was identified as indole-3-acetic
acid (Figure 3(b)). e mass spectrum of metabolite III had
ions at ꢁꢂ/ 131, 130, 103, 102, 77, and 78. is metabolite was
identified as 3-methylindole (Figure 3(c)).
2.5. Identification of Metabolites. e samples (0 h, 12 h, 24 h,
and 32 h) were analyzed by gas-chromatography-mass spec-
trometry (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]. e 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
e 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.
Arora, Pankaj Kumar
