Identification of a novel enhancin-like gene from Bacillus thuringiensis

Abstract

An enhancin-like gene was cloned from Bacillus thuringiensis (Bt) strain GS8 isolated from soil samples in china. The sequence analysis revealed that an open reading frame (ORF) of 2202 nucleotides encoding a protein containing 733 amino acids with a molecular mass of 84 kDa. The enhancin-like protein showed 100% identity to Bel protein (FJ644935) and 23%–41% identity to viral enhancin proteins; in the 252 to 261 amino-acid sequence of enhancin-like protein, a conserved metal binding motif (HEIAH) similar to that in the reported bacterial enhancin-like proteins was found (HEXXH in viral enhancin protein), which indicated that the enhancin-like protein belongs to metalloprotease. The purified enhancin-like protein was fed together with Cry9Ea to Spodopera exigua and Trichoplusia ni larvae, but no significant increase in toxicity was observed.

Keywords

Bacillus thuringiensis / enhancin-like gene / Cry9Ea protein / synergism

References

[1] Bischoff D S, Slavicek J M (1997). Molecular analysis of an enhancin gene in the Lymantria dispar nuclear polyhedrosis virus. J Virol, 71(11): 8133-8140

[2] Fang S L, Wang L, Guo W, Zhang X, Peng D H, Luo C, Yu Z, Sun M (2009). Bacillus thuringiensis bel protein enhances the toxicity of Cry1Ac protein to Helicoverpa armigera larvae by degrading insect intestinal mucin. Appl Environ Microbiol, 75(16): 5237-5243

[3] Galloway C S, Wang P, Winstanley D, Jones I M (2005). Comparison of the bacterial enhancin-like proteins from Yersinia and Bacillus spp. with a baculovirus Enhancin. J Invertebr Pathol, 90(2): 134-137

[4] Gill S S, Cowles E A, Pietrantonio P V (1992). The mode of action of Bacillus thuringiensis endotoxins. Annu Rev Entomol, 37(1): 615-634

[5] Granados R R, Fu Y, Corsaro B, Hughes P R (2001). Enhancement of Bacillus thuringiensis toxicity to lepidopterous species with the enhancin from Trichoplusia ni granulovirus. Biol Control, 20(2): 153-159

[6] Gunning R V, Dang H T, Kemp F C, Nicholson I C, Moores G D (2005). New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin. Appl Environ Microbiol, 71(5): 2558-2563

[7] Hajaij-Ellouze M, Fedhila S, Lereclus D, Nielsen-LeRoux C (2006). The enhancin-like metalloprotease from the Bacillus cereus group is regulated by the pleiotropic transcriptional activator PlcR but is not essential for larvicidal activity. FEMS Microbiol Lett, 260(1): 9-16

[8] Hayakawa T, Ko R, Okano K, Seong S I, Goto C, Maeda S (1999). Sequence analysis of the Xestia c-nigrum granulovirus genome. Virology, 262(2): 277-297

[9] Höfte H, Whiteley H R (1989). Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev, 53(2): 242-255

[10] Huang D F, Lin M (2001). Gene Bioengineering of Agricultural Microbe. Beijing: Science Press (in Chinese)

[11] Ivanova N, Sorokin A, Anderson I, Galleron N, Candelon B, Kapatral V, Bhattacharyya A, Reznik G, Mikhailova N, Lapidus A, Chu L, Mazur M, Goltsman E, Larsen N, D’Souza M, Walunas T, Grechkin Y, Pusch G, Haselkorn R, Fonstein M, Ehrlich SD, Overbeek R, Kyrpides N (2003). Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature, 423(6935): 87-91

[12] Lepore L S, Roelvink P R, Granados R R (1996). Enhancin, the granulosis virus protein that facilitates nucleopolyhedrovirus (NPV) infections, is a metalloprotease. J Invertebr Pathol, 68(2): 131-140

[13] Li Q, Li L, Moore K, Donly C, Theilmann DA, Erlandson M (2003). Characterization of Mamestra configurata nucleopolyhedrovirus enhancin and its functional analysis via expression in an Autographa californica M nucleopolyhedrovirus recombinant. J Gen Virol, 84(1): 123-132

[14] Liu T H, Guo W, Sun W M, Sun Y X (2009). Biological characteristics of Bacillus thuringiensis strain Bt11 and identification of its cry-type genes. Front Agric China, 3(2): 159-163

[15] Macaluso A, Mettus A M (1991). Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA. J Bacteriol, 173(3): 1353-1356

[16] Peng J X, Zhong J, Granados R R(1999). A baculovirus enhancin alters the permeability of a mucosal midgut peritrophic matrix from lepidopteran larvae. J Insect Physiol, 45(2): 159-166

[17] Roelvink P W, Corsaro B G, Granados R R (1995). Characterization of the Helicoverpa armigera and Pseudaletia unipuncta granulovirus enhancin genes. J Gen Virol, 76(11): 2693-2705

[18] Sambrook J, Fritsch E F, Manny Attice T (1992). Molecular Cloning Guide. (in Chinese, trans. Jin D Y, Li M F, Lin F). 2nd ed. Beijing: Science Press, 1999

[19] Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler D R, Dean D H (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev, 62(3): 775-806

[20] Slavicek J M, Popham H J R (2005). The Lymantria dispar nucleopolyhedrovirus enhancins are components of occlusion-derived virus. J Virol, 79(16): 10578-10588

[21] Song F P (2001). Studies on the specific cry genes from Bacillus thuringiensis strains. Dissertation for the Doctoral Degree. Harbin: Northeast Agricultural University (in Chinese)

[22] Tanada Y, Inoue H, Hess R T, Omi E M (1980). Site of action of a synergistic factor of agranulosis virus of the armyworm, Pseudaletia unipuncta. J Invertebr Pathol, 35: 249-255

[23] Wang P, Granados R R (1997). An intestinal mucin is the target substrate for a baculovirus enhancin. Proc Natl Acad Sci USA, 94(13): 6977-6982

[24] Wang P, Hammer D A, Granados R R (1994). Interaction of Trichoplusia ni granulosis virus-encoded enhancin with the midgut epithelium and peritrophic membrane of four lepidopteran insects. J Gen Virol, 75(8): 1961-1967

[25] Xu J, Yin X D, Zhu J L, Qi J H, Qin Q L (2003). Preliminary Study of the enhancement of Pseudaletia unipuncta granulovirus to Bacillus thuringiensis. Jiangsu Agricultural Sciences, 1: 30-31 (in Chinese)

[26] Yin J, Dan L, Song D X, Zhong J (2007). Site-directed mutagenesis of the zinc-binding domain of Trichoplusia ni granulovirus enhancin. Acta Entomologica Sinica, 50(11): 1111-1115 (in Chinese)

Acknowledgements

This work was supported in part by the National Basic Research Program of China (No. 2009CB118902), the National Science Foundation of China (No. 30771447), and the Earmarkal Fund for Modern Agriculture Technology Research System.

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg