Regulation of the pAD1 sex pheromone response of Enterococcus faecalis by direct interaction between the cAD1 peptide mating signal and the negatively regulating, DNA-binding TraA protein
- *Department of Biologic and Materials Sciences, School of Dentistry, and ‡Department of Microbiology/Immunology, School of Medicine, University of Michigan, Ann Arbor, MI 48109
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Communicated by Donald R. Helinski, University of California at San Diego, La Jolla, CA (received for review July 13, 1997)
Abstract
The Enterococcus faecalis conjugative plasmid pAD1 (60 kb) encodes a mating response to the recipient-produced peptide sex pheromone cAD1. The response involves two key plasmid-encoded regulatory proteins: TraE1, which positively regulates all or most structural genes relating to conjugation, and TraA, which binds DNA and negatively regulates expression of traE1. In vitro studies that included development of a DNA-associated protein-tag affinity chromatography technique showed that TraA (37.9 kDa) binds directly to cAD1 near its carboxyl-terminal end and, as a consequence, loses its affinity for DNA. Analyses of genetically modified TraA proteins indicated that truncations within the carboxyl-terminal 9 residues significantly affected the specificity of peptide-directed association/dissociation of DNA. The data support earlier observations that transposon insertions near the 3′ end of traA eliminated the ability of cells to respond to cAD1.
Footnotes
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↵ † Present address: Department of Microbiology, Gunma University School of Medicine, Maebashi, Gunma 371, Japan.
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↵ § To whom reprint requests should be addressed at the ∗ address. e-mail: dclewell{at}umich.edu.
- ABBREVIATIONS:
- DPAC,
- DNA-associated protein-tag affinity chromatography;
- TSAGE buffer,
- 100 mM Tris, pH 8.0/150 mM NaCl/0.02% sodium azide/10% glycerol/1 mM EDTA
- Copyright © 1998, The National Academy of Sciences
