Characterization of the Sporulation Control Protein SsgA by Use of an Efficient Method To Create and Screen Random Mutant Libraries in Streptomycetesv t

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...

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Bibliographic Details
Published in:Applied and environmental microbiology 2007-04, Vol.73 (7), p.2085
Main Authors: Traag, Bjørn A, Seghezzi, Nicolas, Vijgenboom, Erik, van Wezel, Gilles P
Format: Article
Language:English
Subjects:
Bacteria
Biochemistry
Cell division
Genetics
Mutation
Proteins
Online Access:Get full text
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Summary: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 Streptomvces coelicolor ssgA. The variants were amplified directly from deep- frozen biomass suspensions. Around 800 ssgA variants, including single-amino-acid-substitution mutants colTesponding 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. [PUBLICATION ABSTRACT]
ISSN:0099-2240
1098-5336