Production of Lysinibacillus sphaericus Mosquitocidal Protein Mtx2 from Bacillus subtilis as a Secretory Protein

Author(s): Chutchanun Trakulnaleamsai, Boonhiang Promdonkoy, Sumarin Soonsanga*

Journal Name: Protein & Peptide Letters

Volume 28 , Issue 9 , 2021

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Background: Mtx2 is a mosquitocidal toxin produced during the vegetative growth of Lysinibacillus sphaericus. The protein shows synergism with other toxins against mosquito larvae; hence it could be used in mosquito control formulations. The protein expression system is needed for Mtx2 development as a biocontrol agent.

Objective: This study aimed to set up a Bacillus subtilis system to produce Mtx2 as a secreted protein since the protein contains a putative signal peptide.

Methods: Initially, four different promoters (P43, Pspac, PxylA, and PyxiE) were compared for their strength using GFP as a reporter in B. subtilis. Subsequently, six different signal peptides (SacB, Epr, AmyE, AprE, LipA, and Vip3A) were tested in conjunction with the selected promoter and mtx2 to evaluate levels of Mtx2 secreted by B. subtilis WB800, an extracellular protease-deficient strain.

Results: The promoter PyxiE showed the highest GFP intensity and was selected for further study. Mtx2 was successfully produced as a secreted protein from signal peptides LipA and AmyE, and it exhibited larvicidal activity against Aedes aegypti.

Conclusion: B. subtilis was successfully developed as a host for the production of secreted Mtx2, and the protein retained its larvicidal activity. Although the Mtx2 production level still needs improvement, the constructed plasmids could be used to produce other soluble proteins.

Keywords: Lysinibacillus sphaericus, Bacillus subtilis, Mtx2, protein production, signal peptide, secretory protein.

Silva Filha, M.H.N.L.; Berry, C.; Regis, L. Lysinibacillus sphaericus: Toxins and mode of action, applications for mosquito control and resistance management. In: Advances in Insect Physiology; Dhadialla, T.S.; Gill, S.S., Eds.; Elsevier Ltd., 2014; 47, pp. 89-176.
Park, H.W.; Bideshi, D.K.; Federici, B.A. Properties and applied use of the mosquitocidal bacterium, Bacillus sphaericus. J. Asia Pac. Entomol., 2010, 13(3), 159-168.
[] [PMID: 28883761]
Kalfon, A.; Charles, J.F.; Bourgouin, C.; de Barjac, H. Sporulation of Bacillus sphaericus 2297: an electron microscope study of crystal-like inclusion biogenesis and toxicity to mosquito larvae. J. Gen. Microbiol., 1984, 130(4), 893-900.
[PMID: 6736921]
Narasu, M.L.; Gopinathan, K.P. Purification of larvicidal protein from Bacillus sphaericus 1593. Biochem. Biophys. Res. Commun., 1986, 141(2), 756-761.
[] [PMID: 3801026]
Broadwell, A.H.; Baumann, P. Sporulation-associated activation of Bacillus sphaericus larvicide. Appl. Environ. Microbiol., 1986, 52(4), 758-764.
[] [PMID: 3777925]
Baumann, P.; Unterman, B.M.; Baumann, L.; Broadwell, A.H.; Abbene, S.J.; Bowditch, R.D. Purification of the larvicidal toxin of Bacillus sphaericus and evidence for high-molecular-weight precursors. J. Bacteriol., 1985, 163(2), 738-747.
[] [PMID: 3926751]
Payne, J.M.; Davidson, E.W. Insecticidal activity of the crystalline parasporal inclusions and other components of the Bacillus sphaericus 1593 spore complex. J. Invertebr. Pathol., 1984, 43(3), 383-388.
[] [PMID: 6725980]
Yousten, A.A.; Davidson, E.W. Ultrastructural analysis of spores and parasporal crystals formed by Bacillus sphaericus 2297. Appl. Environ. Microbiol., 1982, 44(6), 1449-1455.
[] [PMID: 16346157]
Aly, C.; Mulla, M.S.; Federici, B.A. Ingestion, dissolution, and proteolysis of the Bacillus sphaericus toxin by mosquito larvae. J. Invertebr. Pathol., 1989, 53(1), 12-20.
[] [PMID: 2915145]
Broadwell, A.H.; Baumann, P. Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin. Appl. Environ. Microbiol., 1987, 53(6), 1333-1337.
[] [PMID: 2886104]
Silva-Filha, M.H.; Nielsen-LeRoux, C.; Charles, J.F. Identification of the receptor for Bacillus sphaericus crystal toxin in the brush border membrane of the mosquito Culex pipiens (Diptera: Culicidae). Insect Biochem. Mol. Biol., 1999, 29(8), 711-721.
[] [PMID: 10451923]
Nielsen-Leroux, C.; Pasteur, N.; Prètre, J.; Charles, J.F.; Sheikh, H.B.; Chevillon, C. High resistance to Bacillus sphaericus binary toxin in culex pipiens (Diptera: Culicidae): The complex situation of west Mediterranean countries. J. Med. Entomol., 2002, 39(5), 729-735.
[] [PMID: 12349855]
Mulla, M.S.; Thavara, U.; Tawatsin, A.; Chomposri, J.; Su, T. Emergence of resistance and resistance management in field populations of tropical Culex quinquefasciatus to the microbial control agent Bacillus sphaericus. J. Am. Mosq. Control Assoc., 2003, 19(1), 39-46.
[PMID: 12674533]
Darboux, I.; Charles, J.F.; Pauchet, Y.; Warot, S.; Pauron, D. Transposon-mediated resistance to Bacillus sphaericus in a field-evolved population of Culex pipiens (Diptera: Culicidae). Cell. Microbiol., 2007, 9(8), 2022-2029.
[] [PMID: 17394558]
Nielsen-Leroux, C.; Pasquier, F.; Charles, J.F.; Sinègre, G.; Gaven, B.; Pasteur, N. Resistance to Bacillus sphaericus involves different mechanisms in Culex pipiens (Diptera:Culicidae) larvae. J. Med. Entomol., 1997, 34(3), 321-327.
[] [PMID: 9151498]
Berry, C. The bacterium, Lysinibacillus sphaericus, as an insect pathogen. J. Invertebr. Pathol., 2012, 109(1), 1-10.
[] [PMID: 22137877]
Liu, J.W.; Porter, A.G.; Wee, B.Y.; Thanabalu, T. New gene from nine Bacillus sphaericus strains encoding highly conserved 35.8-kilodalton mosquitocidal toxins. Appl. Environ. Microbiol., 1996, 62(6), 2174-2176.
[] [PMID: 8787415]
Thanabalu, T.; Hindley, J.; Jackson-Yap, J.; Berry, C. Cloning, sequencing, and expression of a gene encoding a 100-kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1. J. Bacteriol., 1991, 173(9), 2776-2785.
[] [PMID: 1840581]
de Maagd, R.A.; Bravo, A.; Berry, C.; Crickmore, N.; Schnepf, H.E. Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu. Rev. Genet., 2003, 37, 409-433.
[] [PMID: 14616068]
Thanabalu, T.; Porter, A.G. A Bacillus sphaericus gene encoding a novel type of mosquitocidal toxin of 31.8 kDa. Gene, 1996, 170(1), 85-89.
[] [PMID: 8621095]
Chan, S.W.; Thanabalu, T.; Wee, B.Y.; Porter, A.G. Unusual amino acid determinants of host range in the Mtx2 family of mosquitocidal toxins. J. Biol. Chem., 1996, 271(24), 14183-14187.
[] [PMID: 8662969]
Phannachet, K.; Raksat, P.; Limvuttegrijeerat, T.; Promdonkoy, B. Production and characterization of N- and C-terminally truncated Mtx2: a mosquitocidal toxin from Bacillus sphaericus. Curr. Microbiol., 2010, 61(6), 549-553.
[] [PMID: 20411263]
Rungrod, A.; Tjahaja, N.K.; Soonsanga, S.; Audtho, M.; Promdonkoy, B. Bacillus sphaericus Mtx1 and Mtx2 toxins co-expressed in Escherichia coli are synergistic against Aedes aegypti larvae. Biotechnol. Lett., 2009, 31(4), 551-555.
[] [PMID: 19082531]
Wirth, M.C.; Berry, C.; Walton, W.E.; Federici, B.A. Mtx toxins from Lysinibacillus sphaericus enhance mosquitocidal cry-toxin activity and suppress cry-resistance in Culex quinquefasciatus. J. Invertebr. Pathol., 2014, 115, 62-67.
[] [PMID: 24144574]
Wirth, M.C.; Yang, Y.; Walton, W.E.; Federici, B.A.; Berry, C. Mtx toxins synergize Bacillus sphaericus and Cry11Aa against susceptible and insecticide-resistant Culex quinquefasciatus larvae. Appl. Environ. Microbiol., 2007, 73(19), 6066-6071.
[] [PMID: 17704274]
Tjalsma, H.; Bolhuis, A.; Jongbloed, J.D.; Bron, S.; van Dijl, J.M. Signal peptide-dependent protein transport in Bacillus subtilis: A genome-based survey of the secretome. Microbiol. Mol. Biol. Rev., 2000, 64(3), 515-547.
[] [PMID: 10974125]
Ferrari, E.; Jarnagin, A.S.; Schmidt, B.F. Commercial production of extracellular enzymes. In: Bacillus subtilis and other gram-positive bacteria: Biochemistry, physiology, and molecular genetics; Sonenshein, A.L.; Hoch, J.A.; Losick, R., Eds.; American Society for Microbiology: Washington, DC, 1993.
Wu, S.C.; Yeung, J.C.; Duan, Y.; Ye, R.; Szarka, S.J.; Habibi, H.R.; Wong, S.L. Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production. Appl. Environ. Microbiol., 2002, 68(7), 3261-3269.
[] [PMID: 12089002]
Murashima, K.; Chen, C.L.; Kosugi, A.; Tamaru, Y.; Doi, R.H.; Wong, S.L. Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes. J. Bacteriol., 2002, 184(1), 76-81.
[] [PMID: 11741846]
Zhang, X.Z.; Cui, Z.L.; Hong, Q.; Li, S.P. High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800. Appl. Environ. Microbiol., 2005, 71(7), 4101-4103.
[] [PMID: 16000826]
Ping Lu, ; Zi, R.X.; Wei, F.L.; Jiang, B.S.; Lu, P.; Chun, X.H. Protein secretion pathways in Bacillus subtilis: Implication for optimization of heterologous protein secretion. Biotechnol. Adv., 2007, 25(1), 1-12.
[] [PMID: 16997527]
Zhang, A.L.; Liu, H.; Yang, M.M.; Gong, Y.S.; Chen, H. Assay and characterization of a strong promoter element from B. subtilis. Biochem. Biophys. Res. Commun., 2007, 354(1), 90-95.
[] [PMID: 17210127]
Nguyen, H.D.; Nguyen, Q.A.; Ferreira, R.C.; Ferreira, L.C.; Tran, L.T.; Schumann, W. Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid, 2005, 54(3), 241-248.
[] [PMID: 16005967]
Dunn, A.K.; Handelsman, J. A vector for promoter trapping in Bacillus cereus. Gene, 1999, 226(2), 297-305.
[] [PMID: 9931504]
Wang, P.Z.; Doi, R.H. Overlapping promoters transcribed by Bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases. J. Biol. Chem., 1984, 259(13), 8619-8625.
[] [PMID: 6330116]
Brockmeier, U.; Caspers, M.; Freudl, R.; Jockwer, A.; Noll, T.; Eggert, T. Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in gram-positive bacteria. J. Mol. Biol., 2006, 362(3), 393-402.
[] [PMID: 16930615]
Heng, C.; Chen, Z.; Du, L.; Lu, F. Expression and secretion of an acid-stable alpha-amylase gene in Bacillus subtilis by SacB promoter and signal peptide. Biotechnol. Lett., 2005, 27(21), 1731-1737.
[] [PMID: 16247683]
Brockmeier, U.; Wendorff, M.; Eggert, T. Versatile expression and secretion vectors for Bacillus subtilis. Curr. Microbiol., 2006, 52(2), 143-148.
[] [PMID: 16450069]
Soonsanga, S.; Rungrod, A.; Audtho, M.; Promdonkoy, B. Tyrosine-776 of Vip3Aa64 from Bacillus thuringiensis is important for retained larvicidal activity during high-temperature storage. Curr. Microbiol., 2019, 76(1), 15-21.
[] [PMID: 30302555]
Yang, Y.; Wang, L.; Gaviria, A.; Yuan, Z.; Berry, C. Proteolytic stability of insecticidal toxins expressed in recombinant bacilli. Appl. Environ. Microbiol., 2007, 73(1), 218-225.
[] [PMID: 17098916]
Liu, Y.; Shi, C.; Li, D.; Chen, X.; Li, J.; Zhang, Y.; Yuan, H.; Li, Y.; Lu, F. Engineering a highly efficient expression system to produce BcaPRO protease in Bacillus subtilis by an optimized promoter and signal peptide. Int. J. Biol. Macromol., 2019, 138, 903-911.
[] [PMID: 31356949]
Yao, D.; Su, L.; Li, N.; Wu, J. Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection. Microb. Cell Fact., 2019, 18(1), 69.
[] [PMID: 30971250]
Heinrich, J.; Drewniok, C.; Neugebauer, E.; Kellner, H.; Wiegert, T. The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilis. Microb. Cell Fact., 2019, 18(1), 31.
[] [PMID: 30732606]

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2021
Published on: 25 August, 2021
Page: [1054 - 1060]
Pages: 7
DOI: 10.2174/0929866528666210616103337
Price: $65

Article Metrics

PDF: 254