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Parasporin 1Ac2, a Novel Cytotoxic Crystal Protein Isolated from Bacillus thuringiensis B0462 Strain

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Abstract

Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100 % identical even in nucleotide sequence level with that of parasporin-1Aa (PS1Aa1) from B. thuringiensis A1190 strain. The other (PS1Ac2) showed significant homology (99 % identity) to that of PS1Ac1 from B. thuringiensis 87-29 strain. The 15 kDa (S113–R250) and 60 kDa (I251–S777) fragments consisting of an active form of PS1Ac2 were expressed as His-tag fusion. Upon purification under denaturing condition and refolding, the recombinant polypeptides were applied to cancer cells to analyze their cytotoxicities. 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay revealed that either of 15 or 60 kDa polypeptide exhibited no cytotoxicity to HeLa cells, but they became cytotoxic upon mixed together. Our results suggested that PS1Ac2 was responsible for the cytotoxicity of B. thuringiensis B0462 strain, and that the formation of hetero-dimer of 15 and 60 kDa polypeptide was required for their cytotoxicity.

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Acknowledgments

This work was supported, in part, by Grant-in-Aid for Scientific Research (20380036, 23658048 and 24380034).

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Authors and Affiliations

  1. Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka 3-1-1 Kita-ku, Okayama, 700-8530, Japan

    Shouta Kuroda, Anowara Begum, Mizue Saga, Akina Hirao, Hiroshi Sakai & Tohru Hayakawa

  2. Department of Microbiology, University of Dhaka, Dhaka, 1000, Bangladesh

    Anowara Begum

  3. Biotechnology & Food Research Institute, Fukuoka Industrial Technology Center, 1465-5 Aikawa-machi, Kurume, Fukuoka, 839-0861, Japan

    Eiichi Mizuki

Authors
  1. Shouta Kuroda
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  2. Anowara Begum
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  3. Mizue Saga
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  4. Akina Hirao
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  5. Eiichi Mizuki
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  6. Hiroshi Sakai
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  7. Tohru Hayakawa
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Corresponding author

Correspondence to Tohru Hayakawa.

Electronic supplementary material

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284_2013_301_MOESM1_ESM.doc

Nucleotide sequence and the deduced amino acid sequence of the ps1Ac2 gene. The ps1Ac2 gene is 2,331 bp in length and codes for a polypeptide of 777 amino acids. Putative ribosome-binding site (RBS) is underlined. The amino acid sequence (XEPPST) detected by protein sequencing is double-underlined. Numerals on the left hand side are the numbers of nucleotide (upper) and amino acids (lower) Supplementary material 1 (DOC 40 kb)

284_2013_301_MOESM2_ESM.doc

Comparison of the deduced amino acid sequence of PS1Ac2 with PS1Aa1 and PS1Ac1. N- and C-termini of 15 and 56 kDa subunits are indicated. Amino acid sequences with underlines indicate five blocks that are amino acid sequences conserved among three-domain Cry toxins. Asterisks and dotted lines indicate identical amino acids and alignment gaps, respectively. Supplementary material 2 (DOC 37 kb)

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Kuroda, S., Begum, A., Saga, M. et al. Parasporin 1Ac2, a Novel Cytotoxic Crystal Protein Isolated from Bacillus thuringiensis B0462 Strain. Curr Microbiol 66, 475–480 (2013). https://doi.org/10.1007/s00284-013-0301-1

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  • DOI: https://doi.org/10.1007/s00284-013-0301-1

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