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Applied and Environmental Microbiology, April 2007, p. 2085-2092, Vol. 73, No. 7
0099-2240/07/$08.00+0     doi:10.1128/AEM.02755-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of the Sporulation Control Protein SsgA by Use of an Efficient Method To Create and Screen Random Mutant Libraries in Streptomycetes{triangledown} ,{dagger}

Bjørn A. Traag, Nicolas Seghezzi,{ddagger} Erik Vijgenboom, and Gilles P. van Wezel*

Microbial Development, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands

Received 24 November 2006/ Accepted 30 January 2007

Filamentous actinomycetes are commercially widely used as producers of natural products. However, the mycelial lifestyle of actinomycetes has been a major bottleneck in their commercialization, and screening is difficult due to their poor growth on microtiter plates. We previously demonstrated that the enhanced expression of the cell division activator protein SsgA results in the fragmented growth of streptomycetes, with enhanced growth rates and improved product formation. We here describe a novel and efficient method to create, maintain, and screen mutant libraries in streptomycetes and the application of this method for the functional analysis of Streptomyces coelicolor ssgA. The variants were amplified directly from deep-frozen biomass suspensions. Around 800 ssgA variants, including single-amino-acid-substitution mutants corresponding to more than half of all SsgA residues, were analyzed for their abilities to restore sporulation to an ssgA mutant. The essential residues were clustered in three main sections, and hardly any were in the carboxy-terminal third of the protein. The majority of the crucial residues were conserved among all SsgA-like proteins (SALPs). However, the essential residues L29, D58, and S89 were conserved only in SsgA orthologues and not in other SALPs, suggesting an SsgA-specific function.

* Corresponding author. Mailing address: Microbial Development, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Phone: 31 71 5274310. Fax: 31 71 5274340. E-mail: g.wezel{at}chem.leidenuniv.nl.

{triangledown} Published ahead of print on 9 February 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Institut de Genetique et Microbiologie, UMR CNRS 8621, Batiment 400 de l'Universite Paris 11, 91405 Orsay, France.

Applied and Environmental Microbiology, April 2007, p. 2085-2092, Vol. 73, No. 7
0099-2240/07/$08.00+0     doi:10.1128/AEM.02755-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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