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Applied and Environmental Microbiology, February 2005, p. 754-760, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.754-760.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Repeat-Length-Independent Broad-Spectrum Shuffling, a Novel Method of Generating a Random Chimera Library In Vivo

Koichi Mori ,1,{dagger},{ddagger} Takafumi Mukaihara,1,{dagger} Yoshiko Uesugi,1 Masaki Iwabuchi,1 and Tadashi Hatanaka1*

Research Institute for Biological Sciences, Okayama, Japan1

Received 11 May 2004/ Accepted 20 September 2004

We describe a novel method of random chimeragenesis based on highly frequent deletion formation in the Escherichia coli ssb-3 strain and a deletion-directed chimera selection system that uses the rpsL+ gene as a reporter. It enables the selection of chimeras without target gene expression and can therefore be applied to cytotoxic targets. When this system was applied to phospholipase D genes from Streptomyces septatus TH-2 and Streptomyces halstedii subsp. scabies K6 (examples of cytotoxic targets), chimeragenesis occurred between short identical sequences at the corresponding position of the parental genes with large variations. Chimeragenesis was >1,000 times more frequent in the ssb-3 background than in the ssb+ background. We called this system repeat-length-independent broad-spectrum shuffling. It enables the convenient chimeragenesis and functional study of chimeric proteins. In fact, we found two amino acid residues related to the thermostability of phospholipase D (Phe426 and Thr433) by comparing thermostability among the chimeric enzymes obtained.

* Corresponding author. Mailing address: Research Institute for Biological Sciences, Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan. Phone: 81-866-56-9452. Fax: 81-866-56-9454. E-mail: hatanaka{at}bio-ribs.com.

{dagger} K.M. and T.M. contributed equally to this work.

{ddagger} Present address: Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700-8530, Japan.

Applied and Environmental Microbiology, February 2005, p. 754-760, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.754-760.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Arima, J., Uesugi, Y., Uraji, M., Yatsushiro, S., Tsuboi, S., Iwabuchi, M., Hatanaka, T. (2006). Modulation of Streptomyces Leucine Aminopeptidase by Calcium: IDENTIFICATION AND FUNCTIONAL ANALYSIS OF KEY RESIDUES IN ACTIVATION AND STABILIZATION BY CALCIUM. J. Biol. Chem. 281: 5885-5894 [Abstract] [Full Text]  
  • Uesugi, Y., Mori, K., Arima, J., Iwabuchi, M., Hatanaka, T. (2005). Recognition of Phospholipids in Streptomyces Phospholipase D. J. Biol. Chem. 280: 26143-26151 [Abstract] [Full Text]  


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