Chlorpyrifos Mediated Amendment in Protein Profiling of Bacillus
Nistala Shweta1, Jaswani Kamal1,
1School of Studies in Biotechnology, Pt. Ravishankar Shukla
University, Raipur 492 010, Chhattisgarh, India.
is a popularly used organophosphate and has immense agricultural applications.
It is one of the major causes of soil contamination due to its higher
adsorption coefficient, hydrophobicity and relatively longer persistence. Conducted
study was attempted to monitor responses of Bacillus
megaterium, isolated from the paddy growing agricultural field towards
different doses of chlorpyrifos. The results highlighted the tolerance of this bacterium
to higher concentration (800 mg L-1) of chlorpyrifos. However, protein
is an important macromolecule in any living cells and is representative of all the
important functions going inside the cell. Thus, quantification and profiling of
protein of this bacterium in response to different doses of chlorpyrifos would probably
decipher expression, if any, of stress responsive gene(s). The overall
findings revealed that Bacillus
megaterium expressed a few enzymes/ or proteins in order to show adaptation
towards surrounding environment. It can be an efficient degrader of chlorpyrifos,
hence, could be exploited for remediation of chlorpyrifos contaminated sites.
Keywords: Bacillus megaterium; Chlorpyrifos; Protein Profiling; SDS-PAGE
Pesticides are a class of chemical formulations with
well known biocidal properties. They are known as plant protection agents, as are
used to prevent plants from adverse and damaging effects of weeds, insects,
pests, etc. (Latifi et al. 2011). In
India, scenario of pesticide market reveals that insecticides are highly
consumed (approximately 76%) among others due to climatic condition of the
country (Kanekar et al. 2003; Bhushan et al. 2013). Chlorpyrifos
phosphorothioate) is one of the widely used broad spectrum, Group II
organophosphorus insecticides applied against a wide range of insects-pests of
economically important crops (Nandhini et al. 2021; Bhende et al. 2022; Ore et
al. 2023). It is hydrophobic in nature and shows low water solubility which
accounts for its difficult biodegradability. In-spite of moderate toxicity, its
soil adsorption coefficient is very high (Singh and Walker 2006). Thus,
accumulation of CP in the soil could be a matter of serious concern. It can
affect soil fertility and thereby crop productivity/ yield and then food
However, the process of biodegradation has popularly
been suggested to be a reliable and cost effective technique to deal with the degradation
of CP (Latifi et al. 2011). The biodegradation of CP can be achieved by
microorganisms such as bacteria and fungi (Yu et al. 2006; Maria et al. 2017;
Shweta et al. 2021). Some of the bacterial strains can tolerate and utilize CP as
a source of energy (Singh 2008). Thus, CP compounds can be considered to be
biodegradable in nature. Prior to biodegradation microbial species also show other
symptomatic response to different environmental stresses. These stress mediated
preliminary responses are very well correlated with the proteomic analysis. The
proteomic analysis offers the crosslink between presences of actual stress
responsive genes, its product and reflects the protein profile of a cell under
the stress conditions (Babele et al. 2019). Further, various stress conditions can be
regulated by synthesis of few stress responsive proteins and/ or enzymes which
will assist in biodegradation and long term adaptation to prevailing
conditions. According to some of the earlier studies, overall protein content
of a cell may get increased indicating the stimulation in the expression
pattern of stress responsive gene thereby fostering bacteria to tolerate pesticide
toxicity (Asghar et al. 2006). The induction
of stress related protein and their profiling in response to different
pesticides such as cypermethrin, carbofuran, bifenithrin, etc., have
been previously analyzed in E. coli
(Asghar et al. 2006). Similar proteomics study was conducted in Cyanobacterial species under different
abiotic stresses (Babele et al. 2019). Hence, the present study was carried out
to study the responses and protein profiling of Bacillus megaterium against different doses of CP. Thereafter, it
would provide the necessary information about the response of Bacillus sp. to CP stress and would
advance our understanding to stress response signaling pathway activated under CP
Maintenance and cultivation of Bacillus
The bacterium exploited in the present study was
initially isolated from paddy growing chosen agricultural field of Raipur,
Chhattisgarh (21°14' N, 81° 38' E, 305 msl). The
bacterial isolate was then identified following standard procedures and was
confirmed as Bacillus megaterium
applying 16S rRNA test (Lane 1991; Mohanty and Jena 2017). Pure culture of Bacillus megaterium was maintained in
Nutrient Agar (NA) slants, stored at -20oC and was sub-cultured
fortnightly for further usage.
2.2 Resistivity of Bacillus megaterium to different concentrations of chlorpyrifos
In order to screen the resistivity of bacterium and to
scrutinize optimum concentration of CP in media for growth of Bacillus megaterium, toxicity tests were
performed following the procedure of Shafiani and Malick (2003). The sterilized
Minimal Salt (MS) Agar media was mixed separately with the filtered and sterilized
solutions (200, 400, 600 and 800 mg L-1 respectively) of CP. The
pure culture of Bacillus megaterium was then streaked on the MS Agar
plates and was incubated at both 27oC and 37oC for four
consecutive days. Afterwards, growth pattern of the bacterium was monitored.
Preparation of inoculum for total protein extraction and profiling
In view to
determine impacts of different concentrations (200, 400, 600 and 800 mg L-1)
of CP over total protein content of the bacterium, the pellets of pure bacterial
culture was obtained by centrifugation
at 6000 rpm at room temperature (25±2oC) for 10 min. Then, the pellets
thus obtained were washed with sterilized MSM broth and then re-suspended in 1
ml of similar MSM broth, and optical density (Lambda-25, Perkin Elmer, USA) of
it was adjusted to approximately 0.5-0.6 at 600 nm that correspond to
approximately 106-107 Colony Forming Unit (CFU) ml-1,
which was determined using dilution
plate count technique of Yang et al. (2006). Now, the obtained inoculum was further inoculated into sets of
treatment flasks containing MSM amended with 200, 400, 600, and 800 mg L-1
of CP, and control flask lacking CP. Now, all the flasks were incubated for
four consecutive days at 27oC in a shaker incubator maintained at
100 rpm, and in the dark.
Extraction and quantification of total protein from Bacillus megaterium
To extract total protein, bacterial pellets were
harvested from 5 ml of MSM broth cultures of both treated and control flasks by
centrifugation at 6000 rpm for 10 min at room temperature (25±2oC). These pellets were washed with sodium phosphate buffer
(pH 7.2, 100 mM), re-suspended in lysis buffer (pH 7.5) containing 50 mM
Tris-HCl, 100 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.2% (v/v) Triton-X100, 10%
(w/v) glycerol and 1 mg ml-1 lysozyme, and was then incubated at -20oC
for 30 min. Following incubation, the bacterial suspension was subjected to
cell lysis by sonication with an operating frequency of 20 KHz (SoniProbe,
Germany) (Deutscher 1990). Afterwards, sonicated
samples were centrifuged at 12000 rpm for 20 min at 4oC, and
supernatant was collected and stored at -80oC for future use. Now, the protein was precipitated following
the acetone precipitation method of Jiang et al. (2004) and quantified using the
method of Bradford (1976). Concentration of total protein
was calculated by comparing it with the standard curve and was expressed in
terms of µg
profiling of Bacillus megaterium by
Protein pellets obtained with respect to different
concentrations of CP and control was then dissolved in Laemmeli buffer and was
analyzed through SDS-PAGE (Laemmeli 1970). Finally,
of protein bands obtained, their intensities and molecular weights were
analyzed and calculated using Image Lab software (BioRad, USA).
The results obtained were evaluated following one-way
analysis of variance (ANOVA), followed by Duncan’s Multiple Range Tests at P
< 0.05 level. Data were expressed as means ± SE of three different
3.1 Maintenance and cultivation of Bacillus megaterium
Provided bacterial cells were successfully maintained
as pure culture in NA plates (Fig. 1a)
and were revived after every fortnight. The Gram’s staining and microscopic
observations revealed that the bacterial isolate, belonging to Bacillus group was Gram positive.
Surprisingly, this bacterium showed variable responses to Gram’s stain under
stressful conditions suggesting that it might experience a change in the cell
wall components (Fig. 1b).
Fig. 1a Pure
culture of Bacillus megaterium maintained
in nutrient agar plate.
1b Microscopic pictures of Bacillus
megaterium after Gram’s staining. A: Positive response to Gram’s stain i.e. Gram positive (young culture). B:
Negative response to Gram’s stain i.e.
Gram negative (old/ stressed culture).
of Bacillus megaterium to different
concentrations of chlorpyrifos
megaterium chosen in the present study was successfully grown at two
different incubation temperatures i.e.
27 and 37oC, which showed tolerance to a range of concentrations
such as 200, 400, 600, and 800 mg L-1 of CP added into MSM agar
plates. Observations regarding resistivity profiling revealed that the Bacillus megaterium can tolerate all the tested concentrations of CP.
However, a good growth of it was observed till 600 mg L-1 of CP (Table 1). Comparison of growth pattern
of Bacillus megaterium under two different incubation temperatures (27 and 37oC)
did not unveiled any significant difference, assuring that it would be
mesophilic in nature. The overall results regarding growth response and tolerance
analyses suggested that the Bacillus megaterium could tolerate relatively
higher concentration of CP hence, can be used for further investigation.
Table 1 Tolerance and growth responses of Bacillus megaterium to
different concentrations of chlorpyrifos, at 27 and 37oC of
Chlorpyrifos (mg L-1)
++ = Good growth; + = Average growth
Total protein extracted from both CP treated and
non-treated Bacillus megaterium was quantified following the
procedure of Bradford (1976). The 822 µg ml-1 of total protein
content was determined in the control sample, while its level was found to be
1174.14, 1250.12, 1838.18 and 1721.26 µg ml-1 in the bacterial cells
grown in 200, 400, 600 and 800 mg L-1 of CP respectively (Fig. 2). Accumulated data indicated a
significant impact (F=12.332; p<0.05) of applied concentrations of CP on content
of total protein.
of different concentrations of chlorpyrifos on total protein content of Bacillus megaterium. Data presented are
mean ± SE of 3 independent replications. Bars with similar alphabets do not
differ significantly at p < 0.05.
3.4 SDS-PAGE profiling of protein
Standard denaturing SDS-PAGE analysis was performed to
profile protein of Bacillus megaterium.
Significantly more numbers of bands were resolved out of the protein of CP
grown bacterial cells, compared to the control (Fig. 3). A total of 14 bands were obtained from control cultures,
while 13, 20, 18 and 20 bands were obtained from 200, 400, 600 and 800 mg L-1
of CP treated cultures respectively (Fig.
3). Moreover, intensities of a few of the bands such as 9 and 11 in lane 3
(400 mg L-1 CP), and 5 in lane 5 (800 mg L-1 CP), were
seen to be relatively high.
profiling of proteins extracted from Bacillus
megaterium. Lane 1 = Control, Lane 2 = 200 mg L-1 Cp, Lane 3 =
400 mg L-1 Cp, Lane 4 = 600 mg L-1 Cp, Lane 5 = 800 mg L-1
Cp, Lane 6 = Broad range molecular weight marker.
Bioremediation is an effective measure where metabolic
potential of microbial cells can be harnessed for the detoxification of
xenobiotics such as CP and its metabolites. The induction of responsive genes
and expression of proteins/ enzymes, required for degradation of contaminants
in the indigenous microbial communities can suggest the potential use of microbial
cells in detoxification of contaminated environments (Chishti et al. 2013;
Farhan et al. 2021). Resistivity testing of Bacillus
megaterium revealed strong ability of it, in terms of growth, over CP (200,
400, 600, and 800 mg L-1) supplemented MSM agar plates, and can
utilized CP as a source of carbon (Table
1). Moreover, it was also unveiled that although the Bacillus megaterium had maximum growth in 600 mg L-1 CP,
but was able to tolerate and grow in 800 mg L-1 concentration too (Table 1). This might be the resultant
of adaptation or adjustment of the organism with the prevailing environmental
conditions, as was also observed by Nair et al. (2014) and John et al. (2014). Similar kind of growth response was
also revealed by five different bacterial strains isolated by Latifi et al.
(2011) from effluents of pesticide manufacturing industries. These authors
inoculated the bacterial strains into the flasks containing MSM supplemented
with 50, 80, 110, 140, 170, 200, 300, 400, 600, 1000, 2000 and 3000 mg L-1
of CP, in which, one of the isolate showed maximum growth in 140 mg L-1
concentration but was also able to tolerate and grow in MSM supplemented with
200 mg L-1 CP. In another approach, CP responsive eleven different
bacterial isolates were successfully retrieved from OP contaminated soil, and
their growth efficiencies were monitored at 50, 75, 100 or 125 mg L-1
of CP. The Bacillus licheniformis ZHU-1 was seen to
utilize 100 mg ml-1 CP as sole source of carbon and energy in about
14 days, and better growth at both 27oC and 37oC, which revealed
mesophilic nature of it (Zhu et al. 2010). In addition to these
organisms, a number of bacterial and fungal strains have previously been
reported to be able to utilize CP, such as Agrobacterium
spp. (Horne et al. 2002), Aspergillus
spp. and Penicillium spp.
(Ningfeng et al. 2004), Providencia
stuartii MSO9 (Rani et al. 2008), Serratia
spp. and Pseudomonas spp. (Cycon
et al. 2009), and other bacterial and fungal consortium (Abraham and
Silambarasan 2018; Uniyal et al. 2021). Reports also showed that several
bacterial isolates utilize CP as the sole source of carbon, nitrogen or
phosphorus for their growth and development (Yang et al. 2005, 2006; Anwar et
al. 2007; Ghanem et al. 2007; Zhu et al. 2010; Duraisamy et al. 2018).
Determination of concentration of protein in an
aqueous sample is an important step in any of the enzymatic studies. Estimation
of precise quantity of protein available in a sample is essentially required
and for which a number of protocols are available these days (Walker 2002; De
Mey et al. 2008). Ganesh and Lin (2010) compared three different procedures of
protein quantification; 1) UV 280 assay: absorbance at 280 nm, 2) Bradford
assay: Bradford reagent and absorbance at 595 nm, and 3) Lowry assay:
Folin-Lowry reagent and absorbance at 720 nm. These authors suggested the UV
280 method to be the best procedure for protein quantification followed by the
Bradford and Lowry assays. The Bradford assay relies on the formation of a
complex between coommassie brilliant blue G-250 dye and proteins in the
solution. Hence, looking to the efficiency of the procedure to measure maximum
amount of proteins and its compatibility with reducing agents used in lysis
buffer (Johnson 2012), Bradford (1976) assay was followed in the present study.
In the present investigation, highest amount (1838.18 µg ml-1) of
total protein was quantified from cells of Bacillus
megaterium grown in the flask
containing 600 mg L-1 of CP as compared to the non-treated control flask
(Fig. 2). In congruent, Yang et al. (2005),
and Xu et al. (2008) extracted
comparatively more amount of protein from CP treated Alcaligenes faecalis strain DSP3 and Paracoccus spp.
respectively, than isolated from their respective controls. Similar proteomic
analysis was conducted by Gangola et al. (2021) to decipher the stress
responsive proteins/ enzymes. They found increased laccase enzyme production
and activity under pesticide stress in Bacillus
The SDS-PAGE profiling of Bacillus megaterium grown in MSM indicated the appearance of a
total of 14 protein bands. However, the application of CP also resulted in
appearance of 6 new and intense bands of proteins over the SDS-PAGE profile,
than that of control (Fig. 3). These
newly appeared bands may possibly be related with induction/ expression of new
enzymes/ proteins, responsible for growth of Bacillus megaterium in
such a toxic environment (600 mg L-1 of CP). Similar study was
carried out by Xu et al. (2008) in which a few novel protein bands were
recovered by resolving the whole cell proteins of Paracoccus spp. strain TRP cultivated in MSM supplemented with CP. Lu
et al. (2012) used both CP and TCP as the sole sources of carbon and reported
their efficient degradation by Cupriavidus
spp. DT-1. Further, these authors reported the amplified mpd (methyl
parathion hydrolase) gene, encoding CP hydrolyzing enzymes. The SDS-PAGE
profiling of the amplified enzyme showed a notable increase in intensities of
corresponding bands. Likewise, whole cell protein analysis of Alcaligenes faecalis strain DSP3 revealed
appearance of a few new protein bands when incubated in CP than in the control
(Yang et al. 2005). In one more study, proteomic remodelling in Pseudomonas spp. was studied in response
to CP and highlighted the up-regulation of proteins involved in electron
transport chain, carbohydrate metabolism, secondary metabolite biosynthesis, etc.,
suggesting adaptation of the bacteria to the adverse condition (Aswathi et al.
Furthermore, a comparative proteomic analysis in the
presence and absence of methyl parathion (organophosphorus pesticide) was
conducted in a methyl parathion degrading bacteria, Burkholderia zhejiangensis CEIB S4–3 (Castrejón-Godínez et al.
2022). The authors reported change
in protein expression pattern which was evaluated by 2D-PAGE and identified by
mass spectrometry. They finally concluded that few proteins were stress
responsive proteins and expressed only in presence of methyl parathion.
The results of the conducted study indicated that the Bacillus megaterium could be able to tolerate 800 mg L-1
concentration of CP, and could use it as a source of carbon for growth and
development. It was also reported that the bacterial species could degrade up
to 600 mg L-1 of CP efficiently. The accumulated data on the protein
quantification and profiling clearly demonstrated that CP-induced bacterial
cells expressed additional proteins and enzymes, which can be responsible for
degradation of this contaminant. Thus, it can be exemplified that the
supplementation of CP induced synthesis of hydrolyzing enzyme(s) in the Bacillus megaterium, hence can be exploited for bioremediation of CP
contaminated sites. However, more intensive proteomic studies are further
intended to precisely characterize and investigate the involvement(s) of CP
responsive proteins and/ or enzymes which can be utilized for their larger
scale production and decontamination of CP polluted sites.
Conflict of interest: The authors declare that they have no conflict of
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