Trichloroethylene (TCE) is a chlorinated solvent widely used in many industries
including textile, metal processing, electronic, printing, pulp and paper. It
is a common ground water pollutant throughout the United States (Amer
et al., 2008
). TCE also affects soil respiration and microbial biomass
(Kiyota et al., 2006
). Currently, there is concern
over the presence of this compound of drinking water supplies because they are
known to persist over time and many are toxic or carcinogenic or both for experimental
animals (Richmond et al., 2001
et al., 2002
). In anaerobic condition, Vinyl Chloride (VC) is produced
through TCE degradation which is more toxic than TCE. Bioremediation is one of
the environment friendly means of degrading toxic chemicals. Therefore, extensive
efforts have been made to study the biodegradation of TCE by bacteria. Although
no microbial growth on TCE as the sole carbon source
has been reported yet, we
were the pioneer in reporting microorganism capable of growing on TCE as the sole
(Dey and Roy, 2009
). However, TCE was found
to be cometabolize by some ammonia-oxidizing bacterial species and by some bacterial
species able to grow on hydrocarbons such as methane (Hazen
et al., 2009
), propane and isoprene (Ewers et
). The uses of Pseudomonas putida
are capable of degrading
TCE in presence of phenol (Ferhan, 2003
CEMB 10124 was best degrader of phenol as well as TCE (Ferhan
et al., 2002
). It was reported that the Toluene Dioxygenase (TDO)
enzyme catalyzes the degradation of TCE in presence of toluene of Pseudomonas
F1. This is a multicomponent enzyme in which terminal oxygenase component
is an iron-sulfur protein (ISPTOL
), has an α2
subunit composition. The cellular processes are either regulated or directly carried
out by proteins or protein complexes.
The aim of the study is to find out the proteins that are involved in TCE degradation in Bacillus cereus 2479. In the present work, we describe the changes in protein profiles of B. cereus 2479 which occur when cells grown in presence of TCE. A set of new genes can be express either some stress condition or altered condition.
MATERIALS AND METHODS
Bacterial strain: The strain 2479 was isolated from the soil of industrial
belt, situated at Rajbandh (West Bengal, India) in 2006 where the use of polychlorinated
hydrocarbons (including TCE) is abundant. The complete identification of the
isolated organism was done by the polyphasic approach and ribotyping was classified
as Bacillus cereus strain 2479 (Dey and Roy, 2009;
Mitra and Roy, 2010).
Growth conditions: The Bacillus cereus 2479 was grown in M9 medium (Na2HPO4-6 g; KH2PO4-3 g; NaCl-0.5 g; NH4Cl-1 g; MgSO4-0.002 M; CaCl2 -0.0001 M; Glucose-0.2%; H2O-1L. pH-7.0). In this experiment, peptone (0.2%) replaced glucose. Further, TCE (0.2%) (Merck, India pvt. Ltd.) was added with peptone for inducible protein analysis. Incubation was done for 2-3 days with shaking at 31°C. The bacterium was grown on TCE with peptone for analyzing the TCE inducible proteins.
Measurement of TCE degradation: B. cereus 2479 could degrade TCE efficiently was analyzed by Fujiwara test. To performed this test, TCE (0.2%) with Peptone (0.2%) containing M9 media are inoculated with B. cereus 2479 and E. coli. The incubation was done for 48 h at 31°C with shaking. Optical density (OD470) was measured at the beginning (just after inoculation) and repeated after 12 h, 24 h, 36 h and 48 h, respectively by UV-VIS Spectrophotometer (UV-1700 Pharma Spec, Shimadzu).
Preparation of cell extracts: The total proteome was extracted and profiled through one-dimensional SDS PAGE. The M9 media containing peptone (0.2%) or with both peptone and TCE (0.2%) grown cells were harvested at 10,000 g for 10 min at 4°C and then the cells denatured with 1% SDS and 1 mM phenylmethylsulfonyl fluoride, boiled it for 3 min.
Protein analysis: The extracted protein samples were boiled with Laemmli
buffer (Laemmli, 1970) [1% SDS, 5% Mercaptoethanol, 0.05%
Bromophenol blue in 25 mM Tris-HCl, 10% glycerol, pH 6.8] for 10 min and electrophoresed
on 12% SDS-PAGE. Protein electrophoretic profile in gel was stained with Coomassie
blue R-250 (0.2%) and the bands were scanned using a densitometer. It was calculated
the relative mobility of proteins from TCE with peptone grown cells, pattern
based on the migration rate of the standard molecular weight marker. Western-blot
analysis was done on identical SDS-PAGE (without staining) using antibodies
generated as follows:
Preparation of antibodies specific to TCE induced protein: The SDS extracted proteins from bacterial cells grown on TCE (as a sole carbon source) containing M9 medium were injected into rabbits to obtain polyclonal antibodies. The titre of the antiserum was first determined by Dot-blot in different dilution of anti sera (1:500; 1:1500; 1:2000) by using the spotted antigens. The polyclonal anti-serum was pre adsorbed by nitrocellulose membrane (Sigma-Aldrich) on which total cellular proteins from peptone grown cells was immobilized. Thus the antibodies reacting to the common antigens found in both peptone grown and TCE grown cells were removed. The preadsorbed antiserum was found to react specifically with TCE induced proteins in Western blot.
Dot blot assay: The SDS extracted proteins (cells were grown in peptone and peptone with TCE) and BSA were spotted (in different volumes; 1, 2 and 3 μL) onto three nitrocellulose strips (Sigma-Aldrich Chemical Co, USA) allowed to dry at room temperature. Each the strips were incubated overnight in 3% skimmed milk in buffer- A (10 mM Tris-HCl pH-8.0; 0.9% normal saline; 0.2% Tween 20) to block the residual binding sites on the paper. The strips were rinsed for 10 min in same buffer and then incubated with anti sera diluted 1:500; 1:1500; 1:2000 in buffer for 90 min at 37°C. After incubation, the strips were washed by the use of four changes of washing buffer-A and were further incubated for 90 min at 37°C with a goat anti-rabbit IgG (Sigma-Aldrich Chemical Co, USA) diluted 1:30,000 in buffer-A. The strips were washed four times and stained with BCIP/NBT in alkaline phosphatase buffer-B (100 mM Tris with 5 mM Mg2+ at pH 9.5) then allowed to dry.
Western blotting (Immunoblotting): Following electrophoresis, the proteins (without staining) were electrotransferred onto nitrocellulose membrane for 3 h at 20 volts. The membrane was blocked with 3% skimmed milk for 1 h at room temperature and incubated with pre adsorbed antiserum (raised against TCE inducible proteins) for 1 h. After this reaction, methods were same as above (Dot blot assay).
RESULTS AND DISCUSSION
TCE degradation by Strain 2479: Fujiwara test was done to examine that
the B. cereus 2479 (cells grown either in peptone or TCE with peptone
containing medium) could degrade TCE efficiently. Fujiwara test was performed
to estimate the concentration of the polychlorinated hydrocarbon in the medium
(Moss and Rylance, 1966). In the test, TCE was treated
with pyridine in an alkaline environment and the medium was heated. In presence
of TCE the red aqueous upper phase was developed and determined at 470 nm by
spectrophotometer. The absorbance of the red aqueous phase is proportional to
the concentration of free TCE. Figure 1 shows OD value of
red upper aqueous phase gradually decreases with time (h) in B. cereus
2479 grown in TCE with peptone medium during Fujiwara test. It indicates that
the medium contained no free TCE. The plot shows no declination of OD value,
in case of normal E.coli cells and uninoculated TCE containing medium.
TCE concentration was not changed. The mean value and standard deviation regarding
data of OD was calculated after triplicate observation. From the observations
in Fujiwara test, it can be concluded that the compound, TCE was metabolized
by the bacterium B. cereus strain 2479.
SDS-PAGE and densitometry: The protein profiles of B. cereus 2479 with and without exposure to TCE are shown in Fig. 2a.
Analysis of proteomes revealed that nine new proteins were produced responding
to TCE that was not expressed by the same strain in the absence of TCE. The
major polypeptides in the TCE with peptone grown cells had molecular masses
of 97.1, 67, 64, 57, 54, 40, 39, 36, 30 kDa.
||Optical density of the products of Fujiwara test with B.
cereus 2479 and E.coli cells grown in peptone with TCE and the
uninoculated M9 medium (Control) recorded at different time (h) intervals,
measured at OD470. The data are means and standard deviations
of triplicate reading
||(a) SDS-PAGE profile of B. cereus 2479 grown in peptone
or peptone with TCE. Lane 1, 2, 5, 6, 10, 11 shows proteins from cells grown
in peptone with TCE and Lane 3, 4, 7, 8, 9 are protein samples obtained
from bacterium grown in peptone as a sole carbon source. Lane M1 and M2
are protein molecular weight marker, high and low range, respectively and
(b) densitogram of stained proteins from B. cereus 2479 grown in
peptone and TCE with peptone after separation on a 12% polyacrylamide gel
These nine newly expressed proteins may be involved in TCE degradation. It
occurs when the bacterias medium composition varies even in the same strain
and same condition, causing different sets of protein expression (Neto
and Yamamoto, 1993). Densitometer tracing of the same gel showed the presence
of possible 9 high peaks in TCE containing medium that indicates these were
induced in presence of TCE. Such peaks were not found in protein sample extracted
from peptone grown cells (Fig. 2b). Hence, it may suggested
that alterations in cultural conditions may affected the proportion of the different
proteins in the cytoplasm.
Immunoblotting: In Dot blot, a clearly defined blue spot at the both lane (peptone and peptone with TCE grown cells proteins) where the antigens were spotted was considered a positive result and no such spots were found in BSA spotted site, considered a negative result (Fig. 3a). It is concluded that before preadsorbtion of antiserum was found to be reacted with both proteins from peptone and TCE grown cells. In Western Blot studies, three proteins (97.1, 40 and 30 kDa) from B. cereus 2479 specifically reacted with pre-adsorbed antiserum against TCE inducible protein (Fig. 3b). It suggested that the three proteins with molecular masses 97.1, 40 and 30 kDa are directly involved in TCE metabolism.
Proteomics or the systematic analysis of the proteins expressed by the genome
is not only a powerful tool for describing complete proteomes at the organelle,
cell, organ or tissue levels (Porubleva and Chitnis, 2000)
but also for comparing proteomes as affected by different physiological conditions,
such a those resulting from exposure to TCE or several other environmental factors.
||(a) The antigens were spotted onto each lane, strips were
developed by the technique described in Materials and Methods. Lane BSA
(negative control) shows no spot, lane peptone and TCE shows spots in different
volume (1, 2 and 3 μL). No reaction was found in lane BSA and (b) In
Western blot: lane 1 shows specific reaction of anti-serum against TCE inducible
proteins with proteins from TCE with peptone grown cells and lane 2 shows
no such reaction with proteins from peptone grown cells
It has been reported that when Escherichia coli (Jenkins
et al., 1988; Groat et al., 1986)
and Pseudomonas putida (Givsov et al., 1994)
Methylocystis sp. M. are starved, the cells acquire enhanced resistance
to a variety of stresses, thereby enhancing survival. Even enhancing the amounts
of stress proteins induced by the chemicals (especially TCE) may increase the
TCE degradation activity and therefore, facilitate enhanced degradation ability.
The change of culture media would induce some proteins which may be involved in TCE degradation. It may be suggested that the pattern of protein profile showed differences with exposure to the TCE in polyacrylamide gel electrophoretogram. In this study, the investigations reveal that 9 differentially expressed polypeptides are found in presence of TCE. Three of them are reacted immunologically to the antiserum raised against TCE inducible proteins. It may be concluded that some de novo proteins may have been synthesized in B. cereus 2479 which involved in TCE degradation.
The authors gratefully acknowledge Dr. Ashis Kumar Mondal for generously supporting the program. The authors would like to thank Sri Kaushik Dey of Durgapur College of Commerce and Science for providing the organism 2479 and Sri Sushil Kumar Sinha for providing technical assistance during computer work.