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

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.

Figure 1

Map of pAD1 pheromone response regulatory region. (A) Relative locations and transcriptional orientations (arrows) of specific determinants. TTS1 and TTS2 are two transcription terminators located just upstream of traE1. (B) Structure of TraA-related fusion derivatives with modifications at the carboxyl terminus.

Figure 2

Purification from E. coli of TraA-tag and cAD1 affinity chromatography. The proteins were examined by SDS/PAGE and stained with silver stain. (A) Purified TraA-tag (lane A) with molecular size markers (lane M). (B) Amino terminus-fixed cAD1 affinity chromatography of TraA-tag. Crude extract with overexpressed TraA-Tag (lane C) or purified TraA-tag (lane P) was mixed with amino terminus-fixed cAD1 affinity matrix. After incubation and washing, protein was eluted with 60 μM cAD1 (30 μl). Lane eC represents eluate from the matrix that had been mixed with crude extract. Lane eP is eluate from matrix mixed with purified TraA-Tag. Molecular size markers are in lane M.

Figure 3

Pheromone specificity of TraA and TraA-related proteins. The proteins were examined by SDS/PAGE and stained with silver stain. (A) Elution of amino terminus-fixed cAD1 affinity matrix-bound TraA-tag or TraA(1–310)-tag with various peptides. Protein extracts were mixed with the cAD1 matrix and, after washing, were eluted with 60 μM cAD1, iAD1, cPD1, iPD1, or cCF10. Each fraction (e1, e2, e3, and e4) represents 30 μl of eluate. (B) Amino terminus-fixed cAD1 and iAD1 affinity chromatography of TraA-tag, TraA(1–310)-tag, and TraA(1–300)-tag. Protein was eluted with 30 μl of 60 μM cAD1 or iAD1 in TSAGE buffer. In the case where protein was bound to the streptavidin matrix, elution was with 5 mM diaminobiotin.

Figure 4

DNA binding of TraA-tag using the DPAC method. Extracts containing overexpressed TraA-tag and RsaI- or DraI-digested pAM7500 were used in the DPAC procedure as described in the Materials and Methods. Eluted material was extracted with phenol-chloroform, submitted to agarose gel electrophoresis, and stained with ethidium bromide. The restriction fragment “held” to the matrix in the case of RsaI is seen in lane A; the fragment held in the case of DraI is seen in lane B. The lane labeled 1 kb represents marker DNA (1-kb ladder).

Figure 5

Modulation of DNA-binding affinity of TraA and TraA-related proteins by pheromone and other peptides. (A) Effect of exposure to cAD1 on the DNA binding of TraA using the DPAC method. After binding the protein to the streptavidin matrix and washing, the bound material was exposed to either 6 μM cAD1 or solvent (0.5% DMSO). RsaI-digested pAM7500 then was added to the matrix complex. After elution with diaminobiotin 25-μl fractions were obtained from which 20 μl was extracted with phenol-chloroform and analyzed by agarose gel electrophoresis (A-1). A 5-μl portion of the eluate was run directly on SDS/PAGE and silver-stained (A-2). (B) Effect of pheromone and other peptides on the DNA binding of TraA and TraA-related proteins. In the case of 6His-TraA, a Ni-NTA matrix was used (see Materials and Methods); all others made use of the streptavidin matrix. Peptide concentrations were all at 6 μM. The bands shown all correspond to the single RsaI band shown above to bind to TraA.