Isolation and cloning of the Pseudomonas fluorescens chitinase gene – An ecofriendly approach for its use as a specific biopesticide

Suganthi M; Abirami G; Jayanthi M; Ashok Kumar K; Deepan S; Senthilkumar P

doi:10.7324/JABB.2023.144437

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Abstract

Insect pests are one of the major biotic factors limiting the yield of agricultural food crops. In routine agricultural practice, chemical pesticides are used to control insect pests; however, repeated spraying leads to numerous environmental and health concerns. The search for eco-friendly and sustainable alternatives to chemical pesticides has led to the exploitation of biological control agents. Among these, chitinase plays a target-specific insecticidal activity by degrading the chitin in the insect exoskeleton and gut regions, making it a desirable alternative to chemical pesticides. Therefore, the objective of the study was to isolate and clone the Pseudomonas fluorescens chitinase gene for its use as a specific biopesticide. Full-length chitinase gene coming under family 18 Group D glycosyl hydrolases was isolated from P. fluorescens genomic DNA using polymerase chain reaction amplification and cloned into an expression vector pET32C+ and transformed into Escherichia coli. The sequencing results showed that the chi gene contained an 1800 bp long open reading frame encoding 353 amino acids. The deduced amino acid sequence showed that the protein consisted of the chitin-binding domain, a catalytic domain, and a fibronectin Type III domain also an amino terminus signal peptide. This study allows the identification of new, target-specific bacterial metabolite as a biopesticide for safe, environment-friendly pest management strategies.

Keyword: Agricultural practice Biopesticide Chitin Cloning Domains Expression vector Pest management

Citation:

Suganthi M, Jayanthi M, Ashok Kumar K, Abirami G, Deepan S, Senthilkumar P. Isolation and cloning of the Pseudomonas fluorescens gene – An ecofriendly approach for its use as a specific biopesticide. J App Biol Biotech. 2023;11(Suppl 1):27-33. http://doi.org/10.7324/JABB.2023.144437

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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Reference

1. Bernardes MF, Pazin M, Pereira LC, Dorta DJ. Impact of pesticides on environmental and human health. In: Toxicology Studies- Cells, Drugs and Environment. London, UK: IntechOpen; 2015. p. 195-233. https://doi.org/10.5772/59710
2. Bankar R, Ray AK, Kumar A, Adeppa K. Organochlorine pesticide residues in vegetables of three major markets in Uttar Pradesh, India. Acta Biol 2012;1:77-80.
3. Tudi M, Daniel Ruan H, Wang L, Lyu J, Sadler R, Connell D, et al. Agriculture development, pesticide application and its impact on the environment. Int J Environ Res Public Health 2021;18:1112. https://doi.org/10.3390/ijerph18031112
4. Mishra P, Sharma A, Sharma DA. Study on harmful effects of pesticide residue in vegetables. Int J Recent Res Rev 2014;7:45-8.
5. Shternshis MV, Ovchinnikova LA, Duzhak AB, Tomilova OG. The efficiency of the viral and bacterial entomopathogens formulated with chitinase for biocontrol of lepidopteran cabbage pests. Arch Phytopathol Plant Prot 2002;35:161-9. https://doi.org/10.1080/03235400214209
6. Suganthi M, Arvinth S, Senthilkumar P. Comparative bioefficacy of Bacillus and Pseudomonas chitinase against Helopeltis theivora in tea (Camellia sinensis (L.) O. Kuntze. Physiol Mol Biol Plants 2020;26:2053-60. https://doi.org/10.1007/s12298-020-00875-2
7. Gangwar M, Singh V, Pandey AK, Tripathi CK, Mishra BN. Purification and characterization of chitinase from Streptomyces violascens NRRL B2700. Indian J Exp Biol 2016;54:64-71.
8. Bhattacharya D, Nagpure A, Gupta RK. Bacterial chitinases: Properties and potential. Crit Rev Biotechnol 2007;27:21-8. https://doi.org/10.1080/07388550601168223
9. Mendonsa ES, Vartak PH, Rao JU, Deshpande MV. An enzyme from Myrothecium verrucaria that degrades insect cuticles for biocontrol of Aedes aegypti mosquito. Biotechnol Lett 1996;18:373-6. https://doi.org/10.1007/BF00143454
10. Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. J Biochem 1991;280:309-16. https://doi.org/10.1042/bj2800309
11. Morimoto K, Karita S, Kimura T, Sakka K, Ohmiya K. Cloning, sequencing, and expression of the gene encoding Clostridium paraputrificum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain. J Bacteriol 1997;179:7306-14. https://doi.org/10.1128/jb.179.23.7306-7314.1997
12. Suganthi M, Arvinth S, Chandrashekara KN, Raj Kumar R, Senthilkumar P. Molecular characterization of bacterial biocontrol agents and their chitinase genes from tea soil. Indian J Exp Biol 2018;56:395-401.
13. Suganthi M, Senthilkumar P, Arvinth S, Chandrashekara KN. Chitinase from Pseudomonas fluorescens and its insecticidal activity against Helopeltis theivora. J Gen Appl Microbiol 2017;63:222-7. https://doi.org/10.2323/jgam.2016.11.001
14. Chandrashekara KN, Prasannakumar MK, Deepa M, Vani A. Coleus, a new host for Ralstonia solanacearum race biovar in India. J Plant Pathol 2011;93:233-5.
15. Wanwimolruk S, Phopin K, Boonpangrak S, Prachayasittikul V. Food safety in Thailand 4: Comparison of pesticide residues found in three commonly consumed vegetables purchased from local markets and supermarkets in Thailand. PeerJ 2016;4:e2432. https://doi.org/10.7717/peerj.2432
16. Grewal AS, Singla A, Kamboj P, Dua JS. Pesticide residues in food grains, vegetables and fruits: A hazard to human health. J Med Chem Toxicol 2017;2:1-7. https://doi.org/10.15436/2575-808X.17.1355
17. Cook RJ. Assuring the safe use of microbial biocontrol agents: A need for policy based on real rather than perceived risks. Can J Plant Pathol 1996;18:439-45. https://doi.org/10.1080/07060669609500602
18. Jabeen F, Qazi JI. Isolation of chitinase yielding Bacillus cereus JF68 from soil employing an edible crab shell chitin. J Sci Ind Res 2014;73:771-6.
19. Suganthi M, Senthilkumar P, Arvinth S, Raj Kumar R, Chandrashekara KN. RAPD and SCAR marker linked to tea-mosquito resistance in tea. J Crop Improv 2014;28:795-803. https://doi.org/10.1080/15427528.2014.943355
20. Gomaa EZ. Chitinase production by Bacillus thuringiensis and Bacillus licheniformis: Their potential in antifungal biocontrol. J Microbiol 2012;50:103-11. https://doi.org/10.1007/s12275-012-1343-y
21. Karthik N, Akanksha K, Pandey A. Production, purification and properties of fungal chitinases--a review. Indian J Exp Biol 2014;52:1025-35.
22. Liu C, Shen N, Wu J, Jiang M, Shi S, Wang J, et al. Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002. PeerJ 2020;5:8:e8964. https://doi.org/10.7717/peerj.8964
23. Zhong WF, Fang JC, Cai PZ, Yan WZ, Wu J, Guo HF. Cloning of the Bacillus thuringiensis serovar sotto chitinase (Schi) gene and characterization of its protein. Genet Mol Biol 2005;28:821-6. https://doi.org/10.1590/S1415-47572005000500026
24. Songjang K, Donchai T, Chaiyawat P, Christopher R, Meyer. Cloning and expression of chitinase gene isolated from insect pathogenic fungi, Beauveria bassiana in Escherichia coli. Sci 2006;33:347-55.
25. Stefanidi E, Vorgias CE. Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium Moritella marina and biochemical characterization of the recombinant enzyme. Extremophiles 2008;12:541-52. https://doi.org/10.1007/s00792-008-0155-9
26. Watanabe T, Ito Y, Yamada T, Hashimoto M, Sekine S, Tanaka H. The roles of the C-terminal domain and Type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J Bacteriol 1994;176:4465-72. https://doi.org/10.1128/jb.176.15.4465-4472.1994
27. Chernin LS, Winson MK, Thompson JM, Haran S, Bycroft BW, Chet I, et al. Chitinolytic activity in Chromobacterium violaceum: Substrate analysis and regulation by quorum sensing. J Bacteriol 1998;180:4435-41. https://doi.org/10.1128/JB.180.17.4435-4441.1998
28. Little E, Bork P, Doolittle RF. Tracing the spread of fibronectin Type III domains in bacterial glycohydrolases. J Mol Evol 1994;39:631-43. https://doi.org/10.1007/BF00160409
29. Doolittle RF, Bork P. Evolutionarily mobile modules in proteins. Sci Am 1993;269:50-6. https://doi.org/10.1038/scientificamerican1093-50
30. Henrissat B, Bairoch A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 1993;293:781-8. https://doi.org/10.1042/bj2930781
31. Fahmy AH, Hassanein RA, Hashem HA, Ibrahim AS, El Shihy OM, Qaid EA. Developing of transgenic wheat cultivars for improved disease resistance. J Appl Biol Biotechnol 2018;6:31-40.
32. Wang SL, Liang TW, Lin BS, Wang CL, Wu PC, Liu JR. Purification and characterization of chitinase from a new species strain Pseudomonas sp. TKU008. J Microbiol Biotechnol 2010;20:1001-5. https://doi.org/10.4014/jmb.0911.11017
33. Brammacharry U, Paily K. Chitinase like activity of metabolites of Pseudomonas fluorescens Migula on immature stages of the mosquito, Culex quinquefasciatus (Diptera: Culicidae). African J Microbiol Res 2012;6:2718-26. https://doi.org/10.5897/AJMR11.1270

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1. Bernardes MF, Pazin M, Pereira LC, Dorta DJ. Impact of pesticides on environmental and human health. In: Toxicology Studies- Cells, Drugs and Environment. London, UK: IntechOpen; 2015. p. 195-233. https://doi.org/10.5772/59710

2. Bankar R, Ray AK, Kumar A, Adeppa K. Organochlorine pesticide residues in vegetables of three major markets in Uttar Pradesh, India. Acta Biol 2012;1:77-80.

3. Tudi M, Daniel Ruan H, Wang L, Lyu J, Sadler R, Connell D, et al. Agriculture development, pesticide application and its impact on the environment. Int J Environ Res Public Health 2021;18:1112. https://doi.org/10.3390/ijerph18031112

4. Mishra P, Sharma A, Sharma DA. Study on harmful effects of pesticide residue in vegetables. Int J Recent Res Rev 2014;7:45-8.

5. Shternshis MV, Ovchinnikova LA, Duzhak AB, Tomilova OG. The efficiency of the viral and bacterial entomopathogens formulated with chitinase for biocontrol of lepidopteran cabbage pests. Arch Phytopathol Plant Prot 2002;35:161-9. https://doi.org/10.1080/03235400214209

6. Suganthi M, Arvinth S, Senthilkumar P. Comparative bioefficacy of Bacillus and Pseudomonas chitinase against Helopeltis theivora in tea (Camellia sinensis (L.) O. Kuntze. Physiol Mol Biol Plants 2020;26:2053-60. https://doi.org/10.1007/s12298-020-00875-2

7. Gangwar M, Singh V, Pandey AK, Tripathi CK, Mishra BN. Purification and characterization of chitinase from Streptomyces violascens NRRL B2700. Indian J Exp Biol 2016;54:64-71.

8. Bhattacharya D, Nagpure A, Gupta RK. Bacterial chitinases: Properties and potential. Crit Rev Biotechnol 2007;27:21-8. https://doi.org/10.1080/07388550601168223

9. Mendonsa ES, Vartak PH, Rao JU, Deshpande MV. An enzyme from Myrothecium verrucaria that degrades insect cuticles for biocontrol of Aedes aegypti mosquito. Biotechnol Lett 1996;18:373-6. https://doi.org/10.1007/BF00143454

10. Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. J Biochem 1991;280:309-16. https://doi.org/10.1042/bj2800309

11. Morimoto K, Karita S, Kimura T, Sakka K, Ohmiya K. Cloning, sequencing, and expression of the gene encoding Clostridium paraputrificum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain. J Bacteriol 1997;179:7306-14. https://doi.org/10.1128/jb.179.23.7306-7314.1997

12. Suganthi M, Arvinth S, Chandrashekara KN, Raj Kumar R, Senthilkumar P. Molecular characterization of bacterial biocontrol agents and their chitinase genes from tea soil. Indian J Exp Biol 2018;56:395-401.

13. Suganthi M, Senthilkumar P, Arvinth S, Chandrashekara KN. Chitinase from Pseudomonas fluorescens and its insecticidal activity against Helopeltis theivora. J Gen Appl Microbiol 2017;63:222-7. https://doi.org/10.2323/jgam.2016.11.001

14. Chandrashekara KN, Prasannakumar MK, Deepa M, Vani A. Coleus, a new host for Ralstonia solanacearum race biovar in India. J Plant Pathol 2011;93:233-5.

15. Wanwimolruk S, Phopin K, Boonpangrak S, Prachayasittikul V. Food safety in Thailand 4: Comparison of pesticide residues found in three commonly consumed vegetables purchased from local markets and supermarkets in Thailand. PeerJ 2016;4:e2432. https://doi.org/10.7717/peerj.2432

16. Grewal AS, Singla A, Kamboj P, Dua JS. Pesticide residues in food grains, vegetables and fruits: A hazard to human health. J Med Chem Toxicol 2017;2:1-7. https://doi.org/10.15436/2575-808X.17.1355

17. Cook RJ. Assuring the safe use of microbial biocontrol agents: A need for policy based on real rather than perceived risks. Can J Plant Pathol 1996;18:439-45. https://doi.org/10.1080/07060669609500602

18. Jabeen F, Qazi JI. Isolation of chitinase yielding Bacillus cereus JF68 from soil employing an edible crab shell chitin. J Sci Ind Res 2014;73:771-6.

19. Suganthi M, Senthilkumar P, Arvinth S, Raj Kumar R, Chandrashekara KN. RAPD and SCAR marker linked to tea-mosquito resistance in tea. J Crop Improv 2014;28:795-803. https://doi.org/10.1080/15427528.2014.943355

20. Gomaa EZ. Chitinase production by Bacillus thuringiensis and Bacillus licheniformis: Their potential in antifungal biocontrol. J Microbiol 2012;50:103-11. https://doi.org/10.1007/s12275-012-1343-y

21. Karthik N, Akanksha K, Pandey A. Production, purification and properties of fungal chitinases--a review. Indian J Exp Biol 2014;52:1025-35.

22. Liu C, Shen N, Wu J, Jiang M, Shi S, Wang J, et al. Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002. PeerJ 2020;5:8:e8964. https://doi.org/10.7717/peerj.8964

23. Zhong WF, Fang JC, Cai PZ, Yan WZ, Wu J, Guo HF. Cloning of the Bacillus thuringiensis serovar sotto chitinase (Schi) gene and characterization of its protein. Genet Mol Biol 2005;28:821-6. https://doi.org/10.1590/S1415-47572005000500026

24. Songjang K, Donchai T, Chaiyawat P, Christopher R, Meyer. Cloning and expression of chitinase gene isolated from insect pathogenic fungi, Beauveria bassiana in Escherichia coli. Sci 2006;33:347-55.

25. Stefanidi E, Vorgias CE. Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium Moritella marina and biochemical characterization of the recombinant enzyme. Extremophiles 2008;12:541-52. https://doi.org/10.1007/s00792-008-0155-9

26. Watanabe T, Ito Y, Yamada T, Hashimoto M, Sekine S, Tanaka H. The roles of the C-terminal domain and Type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J Bacteriol 1994;176:4465-72. https://doi.org/10.1128/jb.176.15.4465-4472.1994

27. Chernin LS, Winson MK, Thompson JM, Haran S, Bycroft BW, Chet I, et al. Chitinolytic activity in Chromobacterium violaceum: Substrate analysis and regulation by quorum sensing. J Bacteriol 1998;180:4435-41. https://doi.org/10.1128/JB.180.17.4435-4441.1998

28. Little E, Bork P, Doolittle RF. Tracing the spread of fibronectin Type III domains in bacterial glycohydrolases. J Mol Evol 1994;39:631-43. https://doi.org/10.1007/BF00160409

29. Doolittle RF, Bork P. Evolutionarily mobile modules in proteins. Sci Am 1993;269:50-6. https://doi.org/10.1038/scientificamerican1093-50

30. Henrissat B, Bairoch A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 1993;293:781-8. https://doi.org/10.1042/bj2930781

31. Fahmy AH, Hassanein RA, Hashem HA, Ibrahim AS, El Shihy OM, Qaid EA. Developing of transgenic wheat cultivars for improved disease resistance. J Appl Biol Biotechnol 2018;6:31-40.

32. Wang SL, Liang TW, Lin BS, Wang CL, Wu PC, Liu JR. Purification and characterization of chitinase from a new species strain Pseudomonas sp. TKU008. J Microbiol Biotechnol 2010;20:1001-5. https://doi.org/10.4014/jmb.0911.11017

33. Brammacharry U, Paily K. Chitinase like activity of metabolites of Pseudomonas fluorescens Migula on immature stages of the mosquito, Culex quinquefasciatus (Diptera: Culicidae). African J Microbiol Res 2012;6:2718-26. https://doi.org/10.5897/AJMR11.1270