Crystals in Bacillus thuringiensis are usually formed in the mother cell compartment during sporulation and are separated from the spores after mother cell lysis. In a few strains, crystals are produced inside the exosporium and are associated with the spores after sporulation. This special phenotype, named ‘spore crystal association’ (SCA), typically occurs in B. thuringiensis subsp. finitimus. Our aim was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020. Plasmid conjugation experiments indicated that the SCA phenotype in this strain was tightly linked with two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed. Six fragments from BAC clones were screened from this library and discovered to cover the full length of pBMB26; four others were found to cover pBMB28. Using fragment complementation testing, two fragments, each of approximately 35 kb and located on pBMB26 and pBMB28, were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable to the SCA phenotype. Gene disruption and frame-shift mutation analyses revealed that two of the five genes from pBMB28, which showed low similarity to crystal proteins, determined the location of crystals inside the exosporium. Gene disruption revealed that the three remaining genes, similar to spore germination genes, contributed to the stability of the SCA phenotype in strain YBT-020. Our results thus identified the genes determining the SCA phenotype in B. thuringiensis subsp. finitimus.
References
[1]
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, et al. (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62: 775–806.
[2]
Debro L, Fitz-James PC, Aronson A (1986) Two different parasporal inclusions are produced by Bacillus thuringiensis subsp. finitimus. J Bacteriol 165: 258–268.
[3]
Wojciechowska JA, Lewitin E, Revina LP, Zalunin IA, Chestukhina GG (1999) Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimus. FEBS Lett 453: 46–48.
[4]
Lopez-Meza JE, Ibarra JE (1996) Characterization of a Novel Strain of Bacillus thuringiensis. Appl Environ Microbiol 62: 1306–1310.
[5]
Ji F, Zhu Y, Ju S, Zhang R, Yu Z, et al. (2009) Promoters of crystal protein genes do not control crystal formation inside exosporium of Bacillus thuringiensis ssp. finitimus strain YBT-020. FEMS Microbiol Lett 300: 11–17.
[6]
Hannay CL (1961) Fowler's bacillus and its parasporal body. J Biophys Biochem Cytol 9: 285–298.
[7]
Short JA, Walker PD, Thomson RO, Somerville HJ (1974) The fine structure of Bacillus finitimus and Bacillus thuringiensis spores with special reference to the location of crystal antigen. J Gen Microbiol 84: 261–276.
[8]
Vidal-Quist JC, Castanera P, Gonzalez-Cabrera J (2009) Diversity of Bacillus thuringiensis strains isolated from citrus orchards in Spain and evaluation of their insecticidal activity against Ceratitis capitata. J Microbiol Biotechnol 19: 749–759.
[9]
Jv SY, Ji F, Zhu ZM, Yu ZN, Sun M, et al. (2007) The plasmid harboring cry26Aa may contribute to the phenomenon of spore-crystal connection in Bacillus thuringiensis subsp. finitimus. Wei Sheng Wu Xue Bao 47: 88–91.
[10]
Zhu Y, Shang H, Zhu Q, Ji F, Wang P, et al. (2011) Complete Genome Sequence of Bacillus thuringiensis Serovar finitimus Strain YBT-020. J Bacteriol 193: 2379–2380.
[11]
Kim HS, Saitoh H, Yamashita S, Akao T, Park YS, et al. (2003) Cloning and characterization of two novel crystal protein genes from a Bacillus thuringiensis serovar dakota strain. Curr Microbiol 46: 33–38.
[12]
Berry C, O'Neil S, Ben-Dov E, Jones AF, Murphy L, et al. (2002) Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 68: 5082–5095.
[13]
Abdoarrahem MM, Gammon K, Dancer BN, Berry C (2009) Genetic basis for alkaline activation of germination in Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 75: 6410–6413.
[14]
Stein C, Jones GW, Chalmers T, Berry C (2006) Transcriptional analysis of the toxin-coding plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 72: 1771–1776.
[15]
Aronson A (2002) Sporulation and delta-endotoxin synthesis by Bacillus thuringiensis. Cell Mol Life Sci 59: 417–425.
[16]
Guo S, Liu M, Peng D, Ji S, Wang P, et al. (2008) New strategy for isolating novel nematicidal crystal protein genes from Bacillus thuringiensis strain YBT-1518. Appl Environ Microbiol 74: 6997–7001.
[17]
Liu XY, Ruan LF, Hu ZF, Peng DH, Cao SY, et al. (2010) Genome-wide Screening Reveals the Genetic Determinants of an Antibiotic Insecticide in Bacillus thuringiensis. J Biol Chem 285: 39191–39200.
[18]
Gerhardt P, Ribi E (1964) Ultrastructure of the Exosporium Enveloping Spores of Bacillus Cereus. J Bacteriol 88: 1774–1789.
[19]
Henriques AO, Moran CP Jr (2007) Structure, assembly, and function of the spore surface layers. Annu Rev Microbiol 61: 555–588.
[20]
Steichen CT, Kearney JF, Turnbough CL Jr (2007) Non-uniform assembly of the Bacillus anthracis exosporium and a bottle cap model for spore germination and outgrowth. Mol Microbiol 64: 359–367.
[21]
Andrup L, Damgaard J, Wassermann K (1993) Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation. J Bacteriol 175: 6530–6536.
[22]
Sambrook JRD (2001) Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
[23]
Zhong C, Peng D, Ye W, Chai L, Qi J, et al. (2011) Determination of plasmid copy number reveals the total plasmid DNA amount is greater than the chromosomal DNA amount in Bacillus thuringiensis YBT-1520. PLoS One 6: e16025.
[24]
Baum JA, Gilbert MP (1991) Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids. J Bacteriol 173: 5280–5289.
[25]
Peng D, Luo Y, Guo S, Zeng H, Ju S, et al. (2009) Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis. J Appl Microbiol 106: 1849–1858.
[26]
Liu X, Peng D, Luo Y, Ruan L, Yu Z, et al. (2009) Construction of an Escherichia coli to Bacillus thuringiensis shuttle vector for large DNA fragments. Appl Microbiol Biotechnol 82: 765–772.
[27]
Baum JA (1994) Tn5401, a new class II transposable element from Bacillus thuringiensis. J Bacteriol 176: 2835–2845.
[28]
Fang S, Wang L, Guo W, Zhang X, Peng D, et al. (2009) Bacillus thuringiensis bel protein enhances the toxicity of Cry1Ac protein to Helicoverpa armigera larvae by degrading insect intestinal mucin. Appl Environ Microbiol 75: 5237–5243.
[29]
Andrup L, Smidt L, Andersen K, Boe L (1998) Kinetics of conjugative transfer: a study of the plasmid pXO16 from Bacillus thuringiensis subsp. israelensis. Plasmid 40: 30–43.
[30]
Shao Z, Liu Z, Yu Z (2001) Effects of the 20-kilodalton helper protein on Cry1Ac production and spore formation in Bacillus thuringiensis. Appl Environ Microbiol 67: 5362–5369.
[31]
Hernandez-Soto A, Del Rincon-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.
[32]
Bailey-Smith K, Todd SJ, Southworth TW, Proctor J, Moir A (2005) The ExsA protein of Bacillus cereus is required for assembly of coat and exosporium onto the spore surface. J Bacteriol 187: 3800–3806.
[33]
He J, Shao X, Zheng H, Li M, Wang J, et al. (2010) Complete genome sequence of Bacillus thuringiensis mutant strain BMB171. J Bacteriol 192: 4074–4075.
[34]
Arantes O, Lereclus D (1991) Construction of cloning vectors for Bacillus thuringiensis. Gene 108: 115–119.
[35]
Guerout-Fleury AM, Shazand K, Frandsen N, Stragier P (1995) Antibiotic-resistance cassettes for Bacillus subtilis. Gene 167: 335–336.
