SYNTHESIS AND ESTIMATION OF TOTAL EXTRACELLULAR PROTEIN CONTENT IN BACILLUS SUBTILIS UNDER MILD STRESS CONDITION OF CERTAIN ANTIMICROBIALS
Objective: The present study was investigated to determine the impact of certain antimicrobials on a novel bacterial isolate for the estimation of total
Methods: In this regard, isolation and molecular characterization of the isolate from poultry farm feces soil sample was done by serial dilution,
followed by morphological characteristics and biochemical tests of pure isolated culture. Further, the identification of bacterium as Bacillus subtilis
strain KPA was confirmed by subjecting its amplicon (483 bp) to 16S rRNA gene sequence analysis and pairwise alignment through basic local
alignment search tool. Different volumes of antimicrobial agents such as Allium sativum, ampicillin and mercuric chloride at their sub-minimal
inhibitory concentration (MIC) values were added to the lag phase culture of strain KPA. Total extracellular protein estimation was done through
Bradford test. Partially purified extracellular proteins were observed as spots through thin layer chromatography (TLC).
Results: The total extracellular protein content in strain KPA was found to be enhanced after 48 hrs of incubation in presence of antimicrobials tested
here. Mercuric chloride was able to enhance total protein in the bacteria even after 24 hrs of incubation. Separation of partially purified extracellular
proteins of treated samples by TLC was observed as different spots with different retention factor values, compared with non-treated or control
Conclusion: The stress response is a metabolic program activated due to unfavorable conditions. Hence, B. subtilis strain KPA in the presence of sub-
MIC of A. sativum, ampicillin and mercuric chloride could regulate bioactive proteins production.
Keywords: Antimicrobials, Bacillus subtilis, Sub-minimal inhibitory concentration, Total protein, Thin layer chromatography.
2. den Besten HM, Effraimidou S, Abee T. Catalase activity as a biomarker for mild-stress-induced robustness in Bacillus weihenstephanensis. Appl Environ Microbiol 2013;79(1):57-62.
3. Nagamitsu H, Murata M, Kosaka T, Kawaguchi J, Mori H, Yamada M. Crucial roles of MicA and RybB as vital factors for s-dependent cell lysis in Escherichia coli long-term stationary phase. J Mol Microbiol Biotechnol 2013;23(3):227-32.
4. Eun-Jin SK, An-Ping Z. Physiological response of Pseudomonas aeruginosa PA01 to oxidative stress in controlled microaerobic and aerobic cultures. Microbiol 2002;148:3195-202.
5. Tanaka M, Hasegawa T, Okamoto A, Torii K, Ohta M. Effect of antibiotics on group A Streptococcus exoprotein production analyzed by two-dimensional gel electrophoresis. Antimicrob Agents Chemother 2005;49(1):88-96.
6. Cowen LE, Steinbach WJ. Stress, drugs, and evolution: the role of cellular signaling in fungal drug resistance. Eukaryot Cell 2008;7(5):747-64.
7. Noor R, Islam Z, Munshi SH, Rahman F. Influence of temperature on Escherichia coli growth in different culture media. J Pure Appl Microb 2013;7:899-904.
8. den Besten HM, Mols M, Moezelaar R, Zwietering MH, Abee T. Phenotypic and transcriptomic analyses of mildly and severely salt-stressed Bacillus cereus ATCC 14579 cells. Appl Environ Microbiol 2009;75(12):4111-9.
9. Munna MS, Nur I, Rahman T, Noor R. Influence of exogenous oxidative stress on Escherichia coli cell growth, viability and morphology. Am J Biosci 2013;1:59-62.
10. Subramanian N, Jothimanivannan C, Moorthy K. Antimicrobial activity and preliminary phytochemical screening of Justicia gendarussa (Burm. F.) against human pathogens. Asian J Pharm Clin Res 2012;5(3):229-33.
11. Greenwood D, Eley A. Comparative antipseudomonal activity of some newer beta-lactam agents. Antimicrob Agents Chemother 1982;21(2):204-9.
12. Elliott TS, Greenwood D. The response of Pseudomonas aeruginosa to azlocillin, ticarcillin and cefsulodin. J Med Microbiol 1983;16(3):351- 62.
13. Roane TM, Pepper IL. Microbial responses to environmentally toxic cadmium. Microb Ecol 1999;38(4):358-64.
14. Eloff JN. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Plant Med 1998;64:711-3.
15. Lash BW, Mysliwiec TH, Gourama H. Detection and partial characterization of a broad-range bacteriocin produced by Lactobacillus plantarum (ATCC 8014). Food Microbiol 2005;22:199-204.
16. Goh EB, Yim G, Tsui W, McClure J, Surette MG, Davies J. Transcriptional modulation of bacterial gene expression by sub inhibitory concentrations of antibiotics. Proc Natl Acad Sci U S A 2002;99:17025-30.
17. Barrios-Gonzalez J, FernÃ¡ndez FJ, Tomasini A. Microbial secondary metabolites production and strain improvement. Indian J Biotechnol 2003;2:322-33.
18. Ismail IN, Noor HM, Muhamad HS, Radzi SM, Kadar AJ, Rehan MM, et al. Protein secreted by Bacillus subtilis ATCC 21332 in the presence of Allium sativum. Am J Biol Chem Pharm Sci 2013;1(6):35-41.
19. Davies J, Spiegelman GB, Yim G. The world of subinhibitory antibiotic concentrations. Curr Opin Microbiol 2006;9(5):445-53.
20. Ismail IN, Noor HM, Muhamad HS, Radzi SM, Kadar AJ, Rehan MM, et al. Bioactive protein produced by Lactobacillus plantarum ATCC 8014 in the presence of Allium sativum. Int J Med Sci Health Care 2013;1(10):1-8.
21. Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D. Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell 2000;5(5):897-904.
The publication is licensed under CC By and is open access. Copyright is with author and allowed to retain publishing rights without restrictions.