Abstract
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.
Highlights
Bacillus thuringiensis is a Gram-positive, spore-forming bacterium with one of its most important features being the formation of parasporal crystals
In a few strains, such as in B. thuringiensis subsp. finitimus strains [2,3] and B. thuringiensis subsp. oyamensis strain LBIT-113 [4], the parasporal crystals are located between the exosporium and the spore coat and continue to adhere to the spore after mother cell lysis
We found that two native, large plasmids, pBMB26 and pBMB28, were essential for the formation of spore-crystal association (SCA) phenotype, and two 35 kb fragments located on plasmids pBMB26 and pBMB28 were able to recover the SCA phenotype in an acrystalliferous mutant strain BMB171
Summary
Bacillus thuringiensis is a Gram-positive, spore-forming bacterium with one of its most important features being the formation of parasporal crystals. B. thuringiensis strains carry one or more different crystal genes which are usually located on large, transmissible plasmids [1]. Parasporal crystals are separated from spores after lysis of the mother cell. Oyamensis strain LBIT-113 [4], the parasporal crystals are located between the exosporium and the spore coat and continue to adhere to the spore after mother cell lysis. In a few strains, such as in B. thuringiensis subsp. This phenotype has been previously described as spore-crystal association (SCA) [5]
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