Functional analysis of TraA, the sex pheromone receptor encoded by pPD1, in a promoter region essential for the mating response in Enterococcus faecalis

Abstract

Conjugative transfer of a bacteriocin plasmid, pPD1, of Enterococcus faecalis is induced in response to a peptide sex pheromone, cPD1, secreted from plasmid-free recipient cells. cPD1 is taken up by a pPD1 donor cell and binds to an intracellular receptor, TraA. Once a recipient cell acquires pPD1, it starts to produce an inhibitor of cPD1, termed iPD1, which functions as a TraA antagonist and blocks self-induction in donor cells. In this study, we discuss how TraA transduces the signal of cPD1 to the mating response. Gel mobility shift assays indicated that TraA is bound to a traA-ipd intergenic region, which is essential for cPD1 response. DNase I footprinting analysis suggested the presence of one strong (tab1) and two weak (tab2 and tab3) TraA-binding sites in the intergenic region. Primer extension analysis implied that the transcriptional initiation sites of traA and ipd were located in the intergenic region. Northern analysis showed that cPD1 upregulated and downregulated transcription of ipd and traA, respectively. The circular permutation assay showed that TraA bent a DNA fragment corresponding to the tab1 region, and its angle was changed in the presence of cPD1 or iPD1. From these data, we propose a model that TraA changes the conformation of the tab1 region in response to cPD1 and upregulates the transcription of ipd, which may lead to expression of genes required for the mating response.

FIG. 1.

Identification of TraA-binding site in pPD1. (A) Map of pPD1. ORFs relating to mating response are shown. The AluI sites of the 2.3-kb HincII-HincII fragment containing traA and ipd are indicated by arrowheads. The region between two speI sites is deleted in OG1X(pAM351AIM), exhibiting a pheromone-insensitive phenotype. (B) Agarose gel electrophoresis of AluI digests of the HincII-HincII fragment. The digests (0.3 μg) were incubated with (lane 1) or without (lane 2) TraA (50 ng) for 30 min at 37°C in 30 mM Tris-HCl buffer (pH 7.4) containing 0.6 mM EDTA, 30 mM KCl, 0.6 mM DTT, 0.2 mg of bovine serum albumin per ml, 0.2 mg of salmon sperm DNA (TAKARA) per ml, and 10% glycerol. Then, the incubated digests were separated by 4% agarose gel electrophoresis (NuSeive GTG; FMC bioproducts) and stained with ethidium bromide. The arrow indicates the 260-bp fragment which disappeared in lane 2.

FIG. 2.

The 260-bp traA-ipd intergenic region. Primers for the PCR amplification of the probes used for the gel shift assay (see Fig. 3A) are shown by arrows over the nucleotide sequence. A 68-bp fragment (used in Fig. 3B) is indicated with a line under the sequence. TraA binding sites determined by DNase I footprinting are boxed and named tab1, tab2, and tab3. Transcriptional initiation sites of traA and ipd determined by primer extension analysis (see Fig. 6) are indicated by bending arrows. S.D. indicates the putative Shine-Dalgarno sequence. −35 and −10 indicate the putative promoter sequence of ipd and traA.

FIG. 3.

Determination of the TraA binding site by using a gel shift assay. (A) Gel shift assay with TraA. Probes were prepared by PCR amplification with primers shown in Fig. 2: lanes 1 to 5, primers P1 and P3; lanes 6 to 10, primers P2 and P3; lanes 11 to 15, probe P2-P3 digested with DraI. Probes were incubated with the following: lanes 1, 6, and 11, no proteins and peptides; lane 2, 7, and 12, 0.05 μg of TraA; lane 3, 8, and 13, 0.5 μg of TraA; lane 4, 9, and 13, 1 μg of TraA; lane 5, 10, and 15, 3.5 μg of TraA. (B) Effects of cPD1 and iPD1 on mobility of the TraA-DNA complex. Each lane shows the shifted bands (TraA-DNA complexes) in the gel mobility shift assay. A 68-bp ³²P-labeled DNA probe containing tab1 sequence was incubated with the following: lane 1, no proteins and peptides; lane 2, TraA only; lane 3, TraA with cPD1; lane 4, TraA with cPD1 and iPD1; lane 5, TraA with iPD1.

FIG. 4.

DNase I footprinting. Forward chain (from traA to ipd) and reverse chain (from ipd to traA) analyses are shown. Lanes 1 to 3, double-stranded DNAs were incubated with 0, 1, and 7 μg of TraA, respectively. Protected regions (tab1, tab2, and tab3) are shown in Fig. 2.

FIG. 5.

Northern analysis. E. faecalis OG1X(pAM351) was grown in 10 ml of medium to an optical density at 660 nm of 0.5 at 37°C, and 50 nM of cPD1 was then added to the culture. Probes used are shown in panel A. Probe PA is double-stranded DNA incorporating [α-³²P]dCTP. Probe PI is an oligonucleotide labeled with ³²P at its 5′ ends. (B and C) Northern analysis of traA (B) and ipd (C). The probes PA and PI were used for traA and ipd, respectively. Arrows indicate sizes of observed transcripts. Lanes 1 to 6 are total RNAs extracted from induced cells in time course (0 [preinduced], 15, 30, 60, 90, and 120 min, respectively).

FIG. 6.

Primer extension analysis of traA and ipd. Reverse transcripts of traA (A) and ipd (B) by primer extension analysis are shown. Arrowheads indicate the nucleotide position +1. Sequences containing these initiation sites are regions indicated by lines. Lanes A, C, G, and T are positional markers using the same primers. Lanes 0 min and 15 min are reverse transcripts using RNAs from cells 0 and 15 min after induction,.

FIG. 7.

DNA bending in TraA-DNA complex. (A) Probes used in DNA bending assay. Each probe contains a 40-bp DNA fragment (position at 131 to 170) of pPD1 at different positions. Probes 1 to 10 were digested fragments of pFB6, a plasmid containing the tab1 region, using 10 restriction enzymes. The length of these probes was about 200 bp. (B to D) Electrophoretic pattern of TraA-DNA complexes in DNA bending assay. Lanes 1 to 10 are probes 1 to 10, respectively, incubated with TraA. Probes were incubated with TraA only (B), TraA plus cPD1 (C), and TraA plus iPD1 (D) are shown. Angles of DNA bending were calculated from mobilities of lanes 6 and 10.