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Author Notes:

Corresponding author. Mailing address: Department of Microbiology and Immunology, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322. Phone: (404) 727-0402. Fax: (404) 727-8999. E-mail: jrscott@emory.edu.

We dedicate this paper to the memory of Wolfgang Hillen, who graciously encouraged us in our work with the Ptet system and supplied us with useful hints and tips as we proceeded.

J.V.B. and B.J.F. contributed equally to this work.

We are grateful to V. Pancholi for his generous gift of anti-Hlp antiserum.

We are also grateful to C. Reynoso and J. Gallivan for the riboswitch.

Subject:

Research Funding:

This work was supported in part by NIH grant AI20723.

The Histone-Like Protein Hlp Is Essential for Growth of Streptococcus pyogenes: Comparison of Genetic Approaches To Study Essential Genes

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Journal Title:

Applied and Environmental Microbiology

Volume:

Volume 77, Number 13

Publisher:

, Pages 4422-4428

Type of Work:

Article | Final Publisher PDF

Abstract:

Selection of possible targets for vaccine and drug development requires an understanding of the physiology of bacterial pathogens, for which the ability to manipulate expression of essential genes is critical. For Streptococcus pyogenes (the group A streptococcus [GAS]), an important human pathogen, the lack of genetic tools for such studies has seriously hampered research. To address this problem, we characterized variants of the inducible Ptet cassette, in both sense and antisense contexts, as tools to regulate transcription from GAS genes. We found that although the three-operator Ptet construct [Ptet(O)3] had low uninduced expression, its induction level was low, while the two-operator construct [Ptet(O)2] was inducible to a high level but showed significant constitutive expression. Use of Ptet(O)3 in the chromosome allowed us to demonstrate previously that RNases J1 and J2 are required for growth of GAS. Here we report that the uninduced level from the chromosomally inserted Ptet(O)2 construct was too high for us to observe differential growth. For the highly expressed histone-like protein (Hlp) of GAS, neither chromosomal insertion of Ptet(O)2 or Ptet(O)3 nor their use on a high-copy-number plasmid to produce antisense RNA specific to hlp resulted in adequate differential expression. However, by replacing the ribosome binding site of hlp with an engineered riboswitch to control translation of Hlp, we demonstrated for the first time that this protein is essential for GAS growth.

Copyright information:

© 2011, American Society for Microbiology

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