Chlorpyrifos Mediated Amendment in Protein Profiling of Bacillus spp.

Nistala Shweta¹, Jaswani Kamal¹, S. Keshavkant^1*

¹School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur 492 010, Chhattisgarh, India.

Abstract Chlorpyrifos 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

 1. Introduction

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 (CP), (O,O-diethyl-O-3,5,6-trichloro-2-pyridyl 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 security. 

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 stress.

 2. Materials and Methods

2.1 Maintenance and cultivation of Bacillus megaterium

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 -20^oC 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 27^oC and 37^oC for four consecutive days. Afterwards, growth pattern of the bacterium was monitored.

 2.3 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±2^oC) 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 27^oC in a shaker incubator maintained at 100 rpm, and in the dark.

 2.4 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±2^oC). 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 -20^oC 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 4^oC, and supernatant was collected and stored at -80^oC 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 ml^-1.

 2.5 Protein profiling of Bacillus megaterium by SDS-PAGE

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, the number of protein bands obtained, their intensities and molecular weights were analyzed and calculated using Image Lab software (BioRad, USA).

 2.6 Statistical analysis

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 replications.

 3. Results

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.

Fig. 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).

 3.2 Resistivity of Bacillus megaterium to different concentrations of chlorpyrifos

The Bacillus megaterium chosen in the present study was successfully grown at two different incubation temperatures i.e. 27 and 37^oC, 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 37^oC) 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 37^oC of incubation.

      ++ = Good growth; + = Average growth

 3.3 Total protein content

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.

 Fig. 2  Influence 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.   

Fig. 3 SDS-PAGE 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.

 4. Discussion

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 27^oC and 37^oC, 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 cereus 2D.

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. 2021).

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.

 5. Conclusions

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 interest.

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