Abstract
Bacillus thuringiensis is the most successful microbial insecticide against different pests in agriculture and vectors of diseases. Its activity is mostly attributed to the Cry proteins expressed during its sporulation phase. However, these proteins are not exclusive to B. thuringiensis. Some cry genes have been found in other Bacillus species, or even in other genera. In this work, cry genes were searched in 223 acrystalliferous bacillaceous strains. From these strains 13 amplicons were obtained, cloned, and sequenced; however, only 6 amplicons tested positive for cry-like genes, and the 6 isolates showed to be the same strain. We report the characterization of an unusual strain of B. cereus (LBIC-004) which is unable to form protein inclusions during the sporulation phase. LBIC-004 showed a high identity to B. cereus using the sequences of 16S rRNA, gyrB and hag genes; in addition, a unique plasmid pattern of the strain was obtained. A 1953-bp cry gene was identified, coding for a 651 amino acid protein with a molecular weight of 74.9 kDa. This protein showed a predicted three-domain structure, similar to all Cry proteins. However, the amino acid sequence of the protein showed only 41% identity its highest hit: the Cry8Ca1 protein, indicating the uniqueness of this cry-like gene. It was cloned and transferred into a mutant acrystalliferous B. thuringiensis strain which was used in bioassays against Caenorhabditis elegans, Aedes aegypti, Manduca sexta and Phyllophaga sp. The recombinant strain showed no crystal formation and no toxicity to the tested species.
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References
Adalat R, Saleem F, Crickmore N, Naz S, Shakoori AR (2017) In vivo crystallization of three-domain Cry toxins. Toxins (basel) 9(3):80
Barboza-Corona JE, López-Meza JE, Ibarra JE (1998) Cloning and expression of the cry1Ea4 gene of Bacillus thuringiensis and the comparative toxicity of its gene product. World J Microbiol Biotechnol 14(3):437–441
Barboza-Corona JE, Park HW, Bideshi DK, Federici BA (2012) The 60-kilodalton protein encoded by orf2 in the cry19A operon of Bacillus thuringiensis ssp. jegathesan functions like a C-terminal crystallization domain. Appl Environ Microbiol 78:2005–2012. https://doi.org/10.1128/AEM.06750-11
Barloy F, Delécluse A, Nicolas L, Lecadet MM (1996) Cloning and expression of the first anaerobic toxin gene from Clostridium bifermentans subsp. malaysia, encoding a new mosquitocidal protein with homologies to Bacillus thuringiensis delta-endotoxins. J Bacteriol 178:3099–3105. https://doi.org/10.1128/jb.178.11.3099-3105.1996
Barloy F, Lecadet MM, Delécluse A (1998) Cloning and sequencing of three new putative toxin genes from Clostridium bifermentans CH18. Gene 211:293–299. https://doi.org/10.1016/S0378-1119(98)00122-X
Bravo A, Gómez I, Porta H, García-Gómez BI, Rodriguez-Almazan C, Pardo L, Soberón M (2013) Evolution of Bacillus thuringiensis Cry toxins insecticidal activity. Microb Biotechnol 6:17–26. https://doi.org/10.1111/j.1751-7915.2012.00342.x
Castillo-Esparza JF, Hernández-González I, Ibarra JE (2019) Search for Cry proteins expressed by Bacillus spp. genomes, using hidden Markov model profiles. 3 Biotech. https://doi.org/10.1007/s13205-018-1533-3
Crickmore N, Berry C, Panneerselvam S, Mishra R, Connor TR, Bonning BC (2020) A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins. J Invertebr Pathol 107438
Evdokimov AG, Moshiri F, Sturman EJ, Rydel TJ, Zheng M, Seale JW, Franklin S (2014) Structure of the full-length insecticidal protein Cry1 A c reveals intriguing details of toxin packaging into in vivo formed crystals. Protein Sci 23(11):1491–1497
Ge AZ, Shivarova NI, Dean DH (1989) Location of the Bombyx mori specificity domain on a Bacillus thuringiensis delta-endotoxin protein. Proc Natl Acad Sci USA 86(11):4037–4041
Gómez-Lunar Z, Hernández-González I, Rodríguez-Torres MD, Souza V, Olmedo-Álvarez G (2016) Microevolution analysis of Bacillus coahuilensis UNVEILS differences in phosphorus acquisition strategies and their regulation. Front Microbiol 7:58. https://doi.org/10.3389/fmicb.2016.00058
Hernández Flores JL, Salinas Landaverde D, Pacheco Huerta Y, Guerra Castillo VL, Barrios Sánchez M, Arvizu Hernández I, Ramos López MÁ, Álvarez Hidalgo E, Jones H, Campos Guillén J (2020) Phylogenetic analysis of Bacillus cereus sensu lato Isolates from commercial bee pollen using tRNACys-PCR. Microorganisms 8(4):524. https://doi.org/10.3390/microorganisms8040524
Hernández-Soto A, Del Rincón-Castro MC, Espinoza AM, Ibarra JE (2009) Parasporal body formation via overexpression of the Cry10Aa toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa synergism. Appl Environ Microbiol 75:4661–4667. https://doi.org/10.1128/AEM.00409-09
Ibarra JE, del Rincón MC, Ordúz S, Noriega D, Benintende G, Monnerat R, Regis L, de Oliveira CM, Lanz H, Rodriguez MH, Sánchez J, Peña G, Bravo A (2003) Diversity of Bacillus thuringiensis strains from Latin America with insecticidal activity against different mosquito species. Appl Environ Microbiol 69(9):5269–5274. https://doi.org/10.1128/aem.69.9.5269-5274.2003
Jones GW, Nielsen-Leroux C, Yang Y, Yuan Z, Dumas VF, Gomes Monnerat R, Berry C (2007) A new Cry toxin with a unique two-component dependency from Bacillus sphaericus. FASEB J 21:4112–4120. https://doi.org/10.1096/fj.07-8913com
Jouzani GS, Valijanian E, Sharafi R (2017) Bacillus thuringiensis: a successful insecticide with new environmental features and tidings. Appl Microbiol Biotechnol 101:2691–2711. https://doi.org/10.1007/s00253-017-8175-y
Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10(6):845–858. https://doi.org/10.1038/nprot.2015.053
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685. https://doi.org/10.1038/227680a0
Lambert B, Buysse L, Decock C, Jansens S, Piens C, Saey B, Seurinck J, Van Audenhove K, Van Rie J, Van Vliet A, Peferoen M (1996) A Bacillus thuringiensis insecticidal crystal protein with a high activity against members of the family Noctuidae. Appl Environ Microbiol 62(1):80–86. https://doi.org/10.1128/AEM.62.1.80-86.1996
Manasherob R, Zaritsky A, Ben-Dov E, Saxena D, Barak Z, Einav M (2001) Effect of accessory proteins P19 and P20 on cytolytic activity of Cyt1Aa from Bacillus thuringiensis subsp. israelensis in Escherichia coli. Curr Microbiol 43(5):355–364. https://doi.org/10.1007/s002840010316
Noguera PA, Ibarra JE (2010) Detection of new cry genes of Bacillus thuringiensis by use of a Novel PCR primer system. Appl Environ Microbiol 76:6150–6155. https://doi.org/10.1128/AEM.00797-10
Ohgushi A, Saitoh H, Wasano N, Uemori A, Ohba M (2005) Cloning and characterization of two novel genes, cry24B and s1orf2, from a mosquitocidal strain of Bacillus thuringiensis serovar sotto. Curr Microbiol 51:131–136. https://doi.org/10.1007/s00284-005-7529-3
Palma L, Muñoz D, Berry C, Murillo J, Caballero P (2014) Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins 6:3296–3325. https://doi.org/10.3390/toxins6123296
Park SH, Kim HJ, Kim JH, Kim TW, Kim HY (2007) Simultaneous detection and identification of Bacillus cereus group bacteria using multiplex PCR. J Microbiol Biotechnol 17(7):1177–1182
Peng DH, Pang CY, Wu H, Huang Q, Zheng JS, Sun M (2015) The expression and crystallization of Cry65Aa require two C-termini, revealing a novel evolutionary strategy of Bacillus thuringiensis Cry proteins. Sci Rep 5:19–21. https://doi.org/10.1038/srep08291
Reinoso-Pozo Y, Del Rincón-Castro MC, Ibarra JE (2016) Characterization of a highly toxic strain of Bacillus thuringiensis serovar kurstaki very similar to the HD-73 strain. FEMS Microbiol Lett 363:1–6. https://doi.org/10.1093/femsle/fnw188
Reyes-Ramírez A, Ibarra JE (2008) Plasmid patterns of Bacillus thuringiensis type strains. Appl Environ Microbiol 74(1):125–129. https://doi.org/10.1128/AEM.02133-07
Ruiu L, Falchi G, Floris I, Marche MG, Mura ME, Satta A (2015) Pathogenicity and characterization of a novel Bacillus cereus sensu lato isolate toxic to the Mediterranean fruit fly Ceratitis capitata Wied. J Invertebr Pathol 126:71–77. https://doi.org/10.1016/j.jip.2015.01.010
Soufiane B, Côté JC (2009) Discrimination among Bacillus thuringiensis H serotypes, serovars and strains based on 16S rRNA, gyrB and aroE gene sequence analyses. Antonie Van Leeuwenhoek 95(1):33–45. https://doi.org/10.1007/s10482-008-9285-4
Sun Y, Zhao Q, Xia L, Ding X, Hu Q, Federici BA, Park HW (2013) Identification and characterization of three previously undescribed crystal proteins from Bacillus thuringiensis subsp. jegathesan. Appl Environ Microbiol 79:3364–3370. https://doi.org/10.1128/AEM.00078-13
Tabassum R (2019) Molecular cloning and 3D model of first cytochrome P450 from CYP3A subfamily in saltwater crocodile (Crocodylus porosus). Biochem Biophys Res Commun 516(3):1046–1052. https://doi.org/10.1016/j.bbrc.2017.10.079
Verduzco-Rosas LA, García-Suárez R, López-Tlacomulco JJ, Ibarra JE (2021) Selection and characterization of two Bacillus thuringiensis strains showing nematicidal activity against Caenorhabditis elegans and Meloidogyne incognita. FEMS Microbiol Lett. https://doi.org/10.1093/femle/fnaa186
Wu D, Federici BA (1993) A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis. J Bacteriol 175(16):5276–5280. https://doi.org/10.1128/jb.175.16.5276-5280.1993
Xu D, Côté JC (2006) Sequence diversity of the Bacillus thuringiensis and B. cereus sensu lato flagellin (H antigen) protein: comparison with H serotype diversity. Appl Environ Microbiol 72(7):4653–4662. https://doi.org/10.1128/AEM.00328-06
Yokoyama T, Tanaka M, Hasegawa M (2004) Novel cry gene from Paenibacillus lentimorbus strain Semadara inhibits ingestion and promotes insecticidal activity in Anomala cuprea larvae. J Invertebr Pathol 85(1):25–32. https://doi.org/10.1016/j.jip.2003.12.009
Yu Z, Bai P, Bai P, Ye W, Zhang F, Ruan L, Yu Z, Sun M (2008) A novel negative regulatory factor for nematicidal Cry protein gene expression in Bacillus thuringiensis. J Microbiol Biotechnol 18(6):1033–1039
Zhang J, Hodgman TC, Krieger L, Schnetter W, Schairer HU (1997) Cloning and analysis of the first cry gene from Bacillus popilliae. J Bacteriol 179:4336–4341. https://doi.org/10.1128/jb.179.13.4336-4341.1997
Zhang J, Schairer HU, Schnetter W, Lereclus D, Agaisse H (1998) Bacillus popilliae cry18Aa operon is transcribed by sigmaE and sigmaK forms of RNA polymerase from a single initiation site. Nucleic Acids Res 26(5):1288–1293. https://doi.org/10.1093/nar/26.5.1288
Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7(1–2):203–214. https://doi.org/10.1089/10665270050081478
Acknowledgements
JFCE received a PhD fellowship from Consejo Nacional de Ciencia y Tecnología (CONACYT, México).
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JFCE made practically all the experiments of this work, analyzed the data, and wrote the first version of this manuscript; JLB: made part of the experimental work, provided biological material and contributed to the writing of the manuscript; JEI: designed and coordinated the work, analyzed the data and wrote the last version of this manuscript.
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Francisco Castillo-Esparza, J., Luévano-Borroel, J. & Ibarra, J.E. Identification and characterization of a new cry-like gene found in a Bacillus cereus strain. Antonie van Leeuwenhoek 114, 1759–1770 (2021). https://doi.org/10.1007/s10482-021-01635-2
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DOI: https://doi.org/10.1007/s10482-021-01635-2