Chemotaxis and biodegradation of 3-methyl- 4-nitrophenol by ralstonia sp. sj98
Biochemical and Biophysical Research Communications 275, 129 –133 (2000)
doi:10.1006/bbrc.2000.3216, available online at http://www.idealibrary.com on
Chemotaxis and Biodegradation of 3-Methyl-4-Nitrophenol by Ralstonia sp. SJ98
Bharat Bhushan,*,1 Sudip K. Samanta,*,2 Ashvini Chauhan,*Asit K. Chakraborti,† and Rakesh K. Jain*,3*Institute of Microbial Technology, Sector-39A, Chandigarh 160036, India; and †Department of Medicinal Chemistry,National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar 160062, India
Nitroaromatic compounds (NACs) are widely spread
3-Methyl-4-nitrophenol is one of the major break-
in the environment because of their extensive use
down products of fenitrothion [O,O-dimethyl O-(3-
in the manufacturing of pharmaceuticals, pesticides,
methyl-4-nitrophenyl) thiophosphate], a recalcitrant
plasticizers, azo dyes, and explosives (1–3). The NACs
organophosphate insecticide used in agriculture. Be-
and their incomplete degradative products have a high
ing the non-polar methylated aromatic compound,
level of toxicity and some of them are potential car-
3-methyl-4-nitrophenol is highly toxic and, therefore,
cinogens (1, 2, 4). Once released into the environment,
a complete degradation of this compound is important
NACs undergo complex physical, chemical, and biolog-
for environmental decontamination/bioremediation
ical changes. Nitrophenolic compounds can also accu-
purposes. A gram negative, motile Ralstonia sp. SJ98
mulate in the soil as a result of hydrolysis of several
was isolated by selective screening from a soil sample contaminated with pesticides. The microorganism was
methyl parathion, and fenitrothion (3, 5–7), and may
capable of utilizing 3-methyl-4-nitrophenol as the sole
enter the ground water resources where they cause
source of carbon and energy. Thin layer chromatogra-
adverse effects to the biological systems. phy (TLC), gas chromatography (GC), gas chroma-
Although, there are several reports on biodegrada-
tography-mass spectrometry (GC-MS), and high per-
tion of different NACs (1– 4), little is known on the
formance liquid chromatography (HPLC) were per-
biodegradation of 3-methyl-4-nitrophenol which is one
formed to determine the possible intermediates in the
of the major breakdown products of fenitrothion, a
degradative pathway of this compound. Taken to-
recalcitrant organophosphate insecticide, and is highly
gether, catechol was found to be one of the major
toxic. Only recently, the involvement of a plasmid in
intermediate of the pathway. Furthermore, the che-
the degradation of fenitrothion has been reported in
motactic behavior of Ralstonia sp. SJ98 towards
which a Burkholderia sp. strain NF100 was shown to
3-methyl-4-nitrophenol was tested using three differ-
first hydrolyze the organophosphate bond of fenitro-
ent methods i.e., drop assay, swarm plate assay and
thion forming 3-methyl-4-nitrophenol which was fur-
capillary assay, which were found to be positive to-
ther converted to methylhydroquinone, the substrate
wards this compound. This is the first report clearly
for oxygenase-catalyzed ring fission (8). We have re-
indicating the involvement of a microorganism in the
cently reported the degradation and chemotactic activity
chemotaxis and biodegradation of methyl-4-nitro-
towards four NACs viz- p-nitrophenol, 4-nitrocatechol,
phenol and formation of catechol as an intermediate o-nitrobenzoate and p-nitrobenzoate by a Ralstonia sp. in the degradative pathway. 2000 Academic Press
SJ98 (9). In the present study the involvement of this
Key Words: Ralstonia sp. SJ98; 3-methyl-4-nitro- Ralstonia sp. SJ98 in chemotaxis and biodegradation
phenol degradation; catechol formation; chemotaxis.
of 3-methyl-4-nitrophenol has been elucidated.
1 Current address: Department of Biochemistry and Molecular
Biology, Wayne State University, School of Medicine, 540, East Can-
Microorganism and culture conditions.
isolated in our laboratory by “chemotactic enrichment technique” from
2 Current address: Department of Microbiology, University of
pesticide contaminated soil sample (9). The composition of the minimal
Iowa, 3-401 Bowen Science Building, Iowa City, IA 52242.
medium (MM) used in the present study was same as described earlier
3 To whom correspondence should be addressed. Fax: ϩ91-172-
(10). 3-Methyl-4-nitrophenol was added as the filter sterilized solution
690585/690632. E-mail: [email protected]
into MM at a final concentration of 0.5 mM. The medium was inocu-
0006-291X/00 $35.00Copyright 2000 by Academic PressAll rights of reproduction in any form reserved.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A NACs degrading, gram negative, motile bacterium
Ralstonia sp. SJ98 was isolated in our laboratory (9). Its degradation capacity and chemotactic ability weretested for a methylated nitroaromatic compound,3-methyl-4-nitrophenol, which was utilized as the solesource of carbon and energy. The complete degradationof this compound occurred via oxidative route withcorresponding release of nitrite molecules (Fig. 1). Inorder to identify the intermediates of the degradativepathway of 3-methyl-4-nitrophenol, TLC, GC, andGC-MS studies were performed on the extractedsamples following growth of Ralstonia sp. SJ98 on3-methyl-4-nitrophenol. These studies showed the
Degradation of 3-methyl-4-nitrophenol by Ralstonia sp.
presence of two major compounds in the degradative
SJ98. 3-methyl-4-nitrophenol is metabolized with concomitant re-
pathway along with 3-methyl-4-nitrophenol. Com-
lease of nitrite. The inoculum used was obtained from washed cells of
pound I with an R value of 0.46 in TLC studies and
Ralstonia sp. SJ98 grown overnight on 3-methyl-4-nitrophenol. F,The optical density (OD) of culture; Œ, 3-methyl-4-nitrophenol con-
retention time of 2.23 min in GC studies was apparent
centration; ‚, nitrite concentration.
in this study which corresponded well with the authen-tic catechol indicating that this may be an intermedi-
lated with overnight grown seed culture and incubated at 30°C under
ate in the degradation pathway. The GC-MS studies
shaking conditions (200 rpm). Growth was monitored by measuring
also revealed the presence of catechol with a retention
absorbance (OD) at 600 nm. The depletion of 3-methyl-4-nitrophenol
time of 4.22 min and molecular ion at m/z 110 corre-
was monitored by measuring absorbance at 320 nm which is the absor-
sponding to the molecular mass of catechol and frag-
bance maximum of 3-methyl-4-nitrophenol at pH 7.0, and the residual
mentation ion at m/z 82 and 81 corresponding to the
compound was calculated from a standard curve prepared using au-thentic 3-methyl-4-nitrophenol as shown earlier in case of o-nitroben-
losses of Mϩ Ϫ CO and Mϩ Ϫ CHO were identical to
zoate (11). Nitrite concentrations in the samples were determined by
that produced by authentic catechol. These results
comparison of values with those of a standard calibration curve pre-
therefore clearly showed the presence of catechol in the
pared using sodium nitrite as described earlier (12).
degradative pathway of 3-methyl-4-nitrophenol. Com-
Extraction of intermediates, analytical methods, and chemotaxis
pound II having an R value of 0.43 in TLC studies and
The extraction of intermediates of 3-methyl-4-nitrophenol
retention time of 2.25 min in GC studies was also
degradative pathway, analytical methods (TLC, GC, and GC-MS)and the chemotaxis methods used in the present study were exactly
evident. However, attempts to identify this compound
same as used in our previous studies (9, 11, 13).
were unsuccessful as it could not be correlated to any of
In order to test whether the energy source of flagellar motors in
the likely intermediates before the formation of cate-
Ralstonia sp. SJ98 was Naϩ or Hϩ driven motive force, amiloride, a
chol in the biodegradation of 3-methyl-4-nitrophenol as
specific inhibitor of Naϩ driven flagellar motors (14, 15), was mixed
checked by TLC, GC, and GC-MS studies (data not
in the drop assay and swarm plate assay media of chemotaxis. Thestock solution of amiloride was prepared in dimethyl sulfoxide
shown). A recent report by Hayatsu et al. (8) has shown
(DMSO) and then added to chemotaxis medium at a final concentra-
the degradation of 3-methyl-4-nitrophenol via the for-
tion in the range of 1 to 5 mM. In motility restoration experiment,
mation of methylhydroquinone. However, we were un-
sodium chloride was supplemented in the chemotaxis medium at
able to detect this compound as an intermediate in the
different levels between 50 to 400 mM and the threshold inhibitory
degradative pathway indicating that methylhydroqui-
concentration (2 mM) of amiloride was kept constant in the medium.
none is not involved in the degradation of 3-methyl-4-
High performance liquid chromatography (HPLC) analysis.
nitrophenol by Ralstonia sp. SJ98.
Quantitative analysis of intermediates in 3-methyl-4-nitrophenoldegradation was performed by HPLC as shown earlier (13, 16). Theculture was harvested during late log-phase following growth ofRalstonia sp. SJ98 on MM supplemented with 3-methyl-4-nitro-
phenol (0.5 mM) and succinate (10 mM), and the washed concen-
Capillary Assay for Chemotaxis of Ralstonia sp. SJ98
nitrophenol (0.5 mM). 2,2-Dipyridyl was also added in order to detectthe accumulating intermediates from 3-methyl-4-nitrophenol (13).
Acetonitrile-water containing 13.5 mM trifluoroacetic acid (20:80)
was the mobile phase at a flow rate of 1 ml/min. Compounds wereidentified and quantified by comparison of HPLC retention times and
UV-visible spectra to those of standards.
3-Methyl-4-nitrophenol was purchased from Aldrich
Chemical Co. whereas catechol, amiloride, and 2,2Ј-dipyridyl werepurchased from Sigma Chemical Co. (USA). All other chemicals used
Note. S.D., Standard deviation. a Aspartic acid was used as positive control.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HPLC chromatograms of the conversion of 3-methyl-4-nitrophenol by Ralstonia sp. SJ98. Concentrated cell suspensions grown
on 3-methyl-4-nitrophenol and succinate were incubated with 3-methyl-4-nitrophenol and intermediates were detected when the ringcleavage was blocked using 2,2Ј-dipyridyl; (A) sample analyzed at 4 h growth interval; (B) sample analyzed at 6 h growth interval; and (C)sample analyzed at 10 h growth interval. The intermediate at retention time of 2.66 min is an unidentified metabolite.
Attempts were then made to determine the stoichi-
presence and absence of 2,2Ј-dipyridyl, a ring cleavage
ometry and rate of conversion of 3-methyl-4-nitro-
inhibitor (13, 16). HPLC studies revealed the formation
phenol into catechol by HPLC studies. The concen-
of catechol from 3-methyl-4-nitrophenol during its deg-
trated cell suspension of Ralstonia sp. SJ98 was incu-
radation. In presence of 2,2Ј-dipyridyl, catechol (reten-
bated with 3-methyl-4-nitrophenol (0.5 mM) in the
tion time of 3.49 min; Fig. 2A) started appearing after
Chemotactic response of Ralstonia sp. SJ98 in drop assay towards: (A) 3-methyl-4-nitrophenol; (B) 3-methyl-4-nitrophenol along
with amiloride at a concentration of 2 mM in the medium; (C) aspartic acid used as positive control; (D) negative control.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Chemotactic response of Ralstonia sp. SJ98 in swarm plate assay towards: (A) 3-methyl-4-nitrophenol; (B) 3-methyl-4-
nitrophenol along with amiloride at a concentration of 2 mM in the medium; (C) aspartic acid used as positive control; (D) negative control.
4 h of incubation (0.05 mM); after 6 h of incubation 0.10
known selective inhibitor of Naϩ driven flagellar motor
mM of catechol was detected (Fig. 2B) and after 10 h its
(14, 15). When amiloride was mixed in the chemotaxis
concentration in the medium was 0.20 mM (Fig. 2C).
medium at a threshold inhibitory concentration of 2
Catechol increased up to a concentration of 0.28 mM
mM, it inhibited the chemotactic activity of the micro-
after 14 h with corresponding depletion of 3-methyl-4-
organism towards 3-methyl-4-nitrophenol which indi-
nitrophenol (0.32 mM; retention time of 4.07 min).
cated that motility in Ralstonia sp. SJ98 is driven by
However, in absence of 2,2Ј-dipyridyl, there was a com-
Naϩ motive force (Figs. 3 and 4). Although restoration
plete degradation of 3-methyl-4-nitrophenol within
of motility of microorganisms by increasing the concen-
10 h and no intermediates were detected; the maxi-
tration of Naϩ ions in the medium has been reported
mum catechol released was 0.40 mM after 8 h of
earlier in some cases (14, 15), in the present study, the
incubation which corresponded to the depletion of
restoration of motility and chemotaxis could not be
3-methyl-4-nitrophenol (0.42 mM). Unidentified com-
achieved even up to a concentration of 400 mM of
pound II as indicated above could not be identified and
sodium chloride indicating that the motility inhibition
future investigations are necessary to identify the
phenomenon may be irreversible in Ralstonia sp. SJ98.
same. On the basis of studies carried out by TLC, GC,
This is the first report in which 3-methyl-4-
GC-MS, and HPLC, it could be established that cate-
nitrophenol is shown to be degraded via the formation
chol is one of the intermediates in the degradative
of catechol (Fig. 5). This indicates that Ralstonia sp.
SJ98 converts the non-polar methylated nitroaromatic
Since our group recently reported that Ralstonia sp.
compound into highly polar catechol which is then de-
SJ98 is chemotactic towards several NACs (9), the
graded further by oxygenase(s) enzyme. Furthermore,
chemotactic behavior of Ralstonia sp. SJ98 towards
chemotaxis of any microorganism towards 3-methyl-4-
3-methyl-4-nitrophenol was also tested by three differ-
nitrophenol, an immediate byproduct of fenitrothion,
ent methods, i.e., drop assay, swarm plate assay, and
has also been shown for the first time suggesting the
capillary assay. All these methods demonstrated the
role of Ralstonia sp. SJ98 in efficient degradation of
chemotaxis of Ralstonia sp. SJ98 towards 3-methyl-4-
nitrophenol. The results of drop and swarm plate assayin the form of migrating rings of the microorganism
have been shown in Figs. 3 and 4, respectively. Incapillary assay, it was observed that Ralstonia sp.
We are thankful to Mr. Dhan Prakash and Mr. R. Sureshkumar for
technical help. We are also grateful to Mr. Vikas Grower and Mrs.
SJ98 was chemotactic towards 3-methyl-4-nitrophenol
Kamaljeet Kaur, NIPER, Mohali for help in recording the HPLC and
at an optimum concentration of 200 M with a chemo-
GC-MS spectra. This work was supported by CSIR and DBT, India.
taxis index (C.I.) of 9.0 (Table 1).
S.K.S. and A.C. were supported by Senior Research Fellowships
In order to test whether chemotactic activity in Ral-
awarded by CSIR, Government of India. This is IMTECH communi-
stonia sp. SJ98 is driven by Naϩ motive force, experi-
ments were performed with amiloride which is a well
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