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Applied and Environmental Microbiology, March 2008, p. 1324-1331, Vol. 74, No. 5
0099-2240/08/$08.00+0     doi:10.1128/AEM.01901-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Bombyx mori Midgut Membrane Protein P252, Which Binds to Bacillus thuringiensis Cry1A, Is a Chlorophyllide-Binding Protein, and the Resulting Complex Has Antimicrobial Activity{triangledown}

Ganesh N. Pandian, Toshiki Ishikawa, Makoto Togashi, Yasuyuki Shitomi, Kohsuke Haginoya, Shuhei Yamamoto, Tadayuki Nishiumi, and Hidetaka Hori*

Laboratories of Applied Biosciences, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan

Received 13 June 2007/ Accepted 28 December 2007

The epithelial cell membrane 252-kDa protein (P252) isolated in our laboratory from Bombyx mori midgut was shown to bind strongly with Cry1Aa, Cry1Ab, and Cry1Ac toxins of Bacillus thuringiensis (15). In the current paper, P252 was shown to bind with chlorophyllide (Chlide) to form red fluorescent protein (RFP) complex, termed Bm252RFP, with absorbance and fluorescence emission peaks at 600 nm and 620 nm, respectively. P252 at a concentration of 1 µM is shown to bind with about 50 µM Chlide in a positively cooperative reaction to form Bm252RFP under aerobic conditions and in the presence of light at 37°C. Various parameters influencing this reaction have been optimized for efficient in vitro chemical synthesis of Bm252RFP. Circular dichroism spectra revealed that P252 is composed of a β-structure (39.8% ± 2.2%, based on 5 samples) with negligible contribution of {alpha}-helix structure. When bound to Chlide, the β-structure content in the complex is reduced to 21.6% ± 3.1% (n = 5). Since Chlide had no secondary structure, the observed reduction suggests significant conformational changes of P252 during the formation of Bm252RFP complex. Bm252RFP had antimicrobial activity against Escherichia coli, Serratia marcescens, B. thuringiensis, and Saccharomyces cerevisiae with 50% effective concentrations of 2.82, 2.94, 5.88 µM, and 21.6 µM, respectively. This is the first report ever to show clear, concrete binding characteristics of the midgut protein to form an RFP having significant antimicrobial activity.

* Corresponding author. Mailing address: Laboratories of Applied Biosciences, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan. Phone and fax: 81 25 262 7637. E-mail: hide-hri{at}gs.niigata-u.ac.jp

{triangledown} Published ahead of print on 11 January 2008.

Applied and Environmental Microbiology, March 2008, p. 1324-1331, Vol. 74, No. 5
0099-2240/08/$08.00+0     doi:10.1128/AEM.01901-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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