Structure of cCF10, a Peptide Sex Pheromone Which Induces Conjugative Transfer of the Streptococcus fueculis Tetracycline Resistance Plasmid, pCF 10"

The peptide pheromone, cCF10, which induces ag- gregation and high frequency plasmid transfer in Streptococcus faecalis cells carrying the tetracycline resistance plasmid, pCF10, was isolated and its struc- ture determined. The molecular weight of cCFlO is 789, and its amino acid sequence is H-Leu-Val-Thr- Leu-Val-Phe-Val-OH. Pheromone activity, as determined by a clumping induction assay, was detectable at a concentration of 2.5 X lo-" M. A peptide of the same sequence as that of the cCFlO produced by S. cells was The

same sequence as that of the cCFlO produced by S. faecalis cells was synthesized by the liquid-phase method. The synthetic pheromone showed biological activity and chromatographic behavior that was identical to that of the cCFlO of bacterial origin. When the response of S. faecalis cells to various concentrations of synthetic cCFlO was monitored by measuring both the frequency of plasmid transfer and the synthesis of pheromone-inducible antigens, an excellent correlation was observed between donor ability and the appearance of a 150-kilodalton protein that appears to be involved in formation of mating aggregates. The dose-response data in the range of concentrations where the amount of pheromone became limiting (10-"-10-12 M) were consistent with the notion that as few as one or two molecules per donor cell may be sufficient to induce a mating response.
In Streptococcus fuecalis, conjugal transfer of certain plasmids can be enhanced by low molecular weight, heat-stable, protease-sensitive sex pheromones (1)(2)(3). The pheromones are excreted by the recipient cells, and their interaction with plasmid-containing donor cells induces expression of several surface antigens that are involved in processes such as formation of mating aggregates (clumps) (4, 5) and entry exclusion of incoming plasmids in donor cells (6). In addition, one or more plasmid transfer functions, distinct from aggregation, are also induced (7). If a pheromone-inducible plasmid is transferred into a pheromone-producing strain, culture filtrates of the new donor strain do not contain detectable pheromone activity, when assayed against responder strains * This work was supported by United States Public Health Service Grant A119310 (to G. D.) and by grants from the Ministry of Education, Science, and culture of Japan (to M. M. and A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
11 To whom correspondence should be addressed. carrying the same plasmid. However, the strain continues to excrete pheromones active against strains carrying unrelated conjugative plasmids (2,8). Thus, there seems to be a plasmidencoded inhibitor function that specifically interferes with production of the pheromone to which the plasmid determines a response. Recent evidence indicates that the donor cell produces a plasmid-encoded peptide that competes with active pheromone for binding to responder cells, but fails to induce a response (9)(10)(11). The pheromones, also termed clumpinginducing agents, or CIAs' are named after the plasmid whose transfer functions they induce, e.g. pheromone CAD1 induces clumping and transfer in cells carrying the plasmid, pAD1, etc. (12). Similarly, the inhibitcr peptide for PAD1 is termed iADl (9). Several pheromones and two inhibitors have been purified, their structures determined, and they have been synthesized chemically (9,(13)(14)(15). All of these compounds seem to be short, very hydrophobic peptides which exhibit biological activity at extremely low concentrations. In this paper we describe the isolation, amino acid sequence, and synthesis of cCF10, the pheromone that induces transfer of the tetracycline resistance plasmid, pCF10. This plasmid was the first R-factor described that encoded pheromone response functions (3), and its conjugal transfer system has been the subject of a considerable amount of genetic, molecular, and immunological analysis (16). We also compare the sequence of cCFlO to those of previously analyzed pheromones and inhibitors, we demonstrate directly that the synthetic pheromone induces clumping, mating, and synthesis of surface antigens, and (using synthetic cCFlO in dose-response experiments) we provide evidence that a single pheromone molecule may be sufficient to induce a mating response in a S. faecalis cell carrying pCF10.
CIA activity was assayed using the microtiter assay described previously (2). One unit of activity was defined as the smallest amount of material that induced clumping of responder cells in the 100 pl volume used in the microtiter assay. For mating experiments, overnight cultures of donor (OGlSSp (pCF10)) and recipient (OGlRF) cells were diluted (separately) 10-fold into fresh medium. In the case of the donor cells, this medium contained the pure or crude cCFlO preparation to he tested. The cultures were incubated for 60 min, and 1 volume of the donor culture was added to 9 volumes of the recipient culture. After 15 min incubation, the mixtures were plated on media selective for donors (1000 pg/ml streptomycin + 20 pg/ml spectinomycin) or transconjugants (100 pg/ml rifampicin + 20 pg/ml fusidic acid + 10 pg/ml tetracycline). For any specific experiment, the times of pheromone induction and mating were kept constant.
Analysis of Pheromone-induced Cell Surface Antigens-Synthesis of cell surface antigens by responder cells exposed to synthetic cCFlO was monitored by Western blot analysis using a slight modification of a technique described previously (4). A fresh overnight culture of responder cells was diluted 10-fold into fresh BYGT containing synthetic cCF10, and the culture was incubated 4 h at 37 "C. The cells were pelleted, resuspended in 200 pl of a solution of lysozyme (5 mg/ml in distilled H,O), and incubated for 45 min at room temperature. This mixture was placed in a 1.5-ml microcentrifuge tube and centrifuged for 5 min in a Fisher Microfuge. We added 10 p1 of Trisbuffered protease inhibitors (4) to the supernatant material, added an equal volume of Laemmli's loading buffer (17), and loaded 25 pl on a 7.5% sodium dodecyl sulfate-polyacrylamide gel. Electrophoresis, electrophoretic transfer to nitrocellulose, and immunodevelopment of blots with rabbit antiserum raised against pheromone-induced cells carrying a derivative of pCFlO were exactly as described (4), except that we used 0.22 pm nitrocellulose filter paper (Shleicher & Schuell) in the present studies. Although the lysozyme in the extract interfered with accurate determination of protein content, silver staining of these gels indicated that the use of the pheromone induction and antigen extraction protocols described above resulted in very uniform loading of lanes representing different extracts. The antiserum used to develop the blots was raised by three to four intravenous injections of rabbits with about 10' formalin-treated, pheromone-induced OGlSSp (pCF11) cells, as described previously (4). Since pCFll is a derivative of pCFlO that overproduces pheromone-inducible products inducible antigens Tral3O and Tral50 (16).
(4), the antibodies in the antiserum are directed primarily against the Zsolution of cCFlO-The pheromone-producing strain, FA2-2 (pAM351) was grown anaerobically in 20-liter batches with gentle stirring for 20 h. The cells were removed by centrifugation, and the supernatant was passed through an Amberlite XAD-2 column (Rohm and Haas, 5 X 20 cm). The active material adsorbed was recovered by elution with 50% pyridine (800 ml) after washing with 3% butanol and 5% pyridine (600 ml each). The 300-ml eluate containing the cCFlO activity was diluted with H20 to 10% pyridine, and applied to a DEAE-Sephadex A-25 column (Pharmacia LKB Biotechnology Inc., 3.4 X 12.7 cm, AcO-form). The column was washed with 200 ml of 25% ethanol and eluted with a gradient of 0.05 M (200 ml) to 0.5 M (200 ml) of ammonium acetate (pH 6.8, in 25% ethanol). The active fractions from this column (200 ml) were combined and subjected to reverse-phase HPLC (RP HPLC) on an LRP-2 column (2 X 30 cm, Whatman). The column was washed with 20% acetonitrile in 0.1% trifluoroacetic acid (200 ml) and eluted with a gradient of 20-50% acetonitrile in 0.1% trifluoroacetic acid for 60 min at 10 ml/ min. The 60 ml of active fractions from this column were diluted to 200 ml to bring the acetonitrile concentration to 20% and divided into two equal aliquots. Each aliquot was loaded on an SSC-ODS-742 RP HPLC column (10 X 250 mm, Senshukagaku), and the column was washed with 20% acetonitrile in 10 mM triethylammonium phosphate buffer (pH 5.0). This was followed with a gradient elution with 20-35% acetonitrile in the same buffer for 30 min at 4 ml/min. The active fractions were combined and diluted to 100 ml, bringing the acetonitrile concentration to 5%. This material was then loaded onto another RP HPLC column, Senshupak CN-4251-N (10 X 250 mm, Senshukagaku), washed with 5% acetonitrile in 0.1% trifluoroacetic acid, and eluted for 40 min at 4 ml/min with a 5-25% acetonitrile (in 0.1% trifluoroacetic acid) gradient. The active fractions eluted at approximately 19% acetonitrile and were diluted to 30 ml to bring the acetonitrile concentration to 10%. This material was loaded onto an SSC-ODs-252 (6 X 100 mm, Senshukagaku) RP HPLC column, washed with 20 ml of 10% acetonitrile in 10 mM ammonium acetate, and eluted with a 10-25% gradient of acetonitrile in 10 mM ammonium acetate for 5 min, followed by a 25-29% acetonitrile (in 10 mM ammonium acetate) gradient for 20 min at a flow rate of 1 ml/min. The active fractions eluted at approximately 28% acetonitrile, and material from the two aliquots described above, each representing 10liters of culture supernatant, was recombined and diluted to 10 ml and 10% acetonitrile. This sample was reapplied to the same column, washed with 10% acetonitrile in 0.1% trifluoroacetic acid, and eluted with 10-26% acetonitrile for 5 min, and 26-31% acetonitrile for 25 min at 1 ml/min. For the final purification step, the active fractions from three 20-liter batches were combined in a volume of 2.8 ml and divided into 400-pl aliquots. Each aliquot was then purified on a Senshupak SC4-1251 (4.6 X 250 mm, Senshukagaku) RP HPLC column with an isocratic elution of 33% acetonitrile in 0.1% trifluoroacetic acid at 1 ml/min. The purification steps are outlined in Table  I. During this procedure, the active fractions were never evaporated to dryness, since this compound is very hydrophobic, and it was impossible to recover complete activity if fractions were dried.
FAB Mass Spectrum-One pl of a 50% acetonitrile solution containing approximately 0.4 pg of cCFlO was added to a matrix of diethanolamine on a stainless steel probe tip, which was introduced with a JEOL JMS DX-303 mass spectrometer using xenon as the into the ion source of the mass spectrometer. Analysis was performed fast atom.
Amino Acid Sequence Analysis-About 4.5 nmol of cCFlO were degraded by the manually operated Edman method (18), and the resulting anilinothiazolinone was converted to a phenylthiohydantoin with 25% trifluoroacetic acid at 58 "C for 10 min. The phenylthiohydantoin amino acid derivatives were identified by HPLC on an Ultrasphere ODS column (4.6 X 250, Altex) with multistep gradient of 15-20% acetonitrile in 10 mM sodium acetate (pH 4.5).

RESULTS
Purification, Structural Analysis, and Synthesis of cCF10-The S. faecalis sex pheromone cCFlO was purified from 60 liters of culture filtrate by the eight steps shown in Table I and described under "Materials and Methods." We achieved a 1.45 X 108-fold purification and obtained 4.1 pg of pure compound. The biological activity after each purification step was monitored by a clumping induction assay, and the total weight estimated, as shown in Table I. The specific activity of pure cCF10, 2 pg/unit, is similar to the values determined for other S. faecalis sex pheromones (13)(14)(15).
Because treatment of partially purified cCFlO with either  Pronase or chymotrypsin abolished its biological activity (data not shown), we assumed that, like the other S. faecalis pheromones, cCFlO would be a peptide. As shown in Fig. 1, the FAB mass spectrum revealed the presence of three quasimolecular ion peaks at m/z 790 (M + H)+, 812 (M + Na)+, and 895 (M + DEA + H)+. Therefore the molecular mass of cCFlO was deduced to be 789. The amino acid sequence of cCFlO was determined by a direct Edman method, with HPLC identification of the phenylthiohydantoin amino acid derivative from each degradation step. The sequence determined by this method for cCF10, H-Leu-Val-Thr-Leu-Val-Phe-Val-OH, gives a predicted molecular mass identical to that obtained by FAB mass spectroscopy. Using the liquid-phase method, we derived a synthetic peptide with the same amino acid sequence as that of the purified cCF10. The synthetic cCFlO showed HPLC retention times and biological activities that were identical to those of the cCFlO of bacterial origin.

60-
Purified cCFlO showed no CIA activity against responder strains for the other pheromones. Although cCFlO actually shows some amino acid sequence homology with the inhibitor peptides iADl and iPDl (see Fig. 2), it displayed no inhibitor activity in the appropriate assays. However, iADl did show a very weak (100 ng/unit) CIA activity against a responder strain carrying pCF10. Two cCFlO analogs, H-Val-Ala-Thr-Leu-Val-Phe-Val-OH and H-Ala-Leu-Gly-Leu-Val-Phe-Val-OH, were synthesized, and only the former showed any CIA activity (30 ng/unit).

Dose-Response Analysis using
Synthetic cCF10"Exposure of S. fuecalis cells to culture filtrates containing CIA has a number of effects on the responder cells, as noted in the Introduction. Most of the genetic and biochemical analyses of pheromone-inducible plasmid transfer systems to date support the notion that cell clumping, synthesis of unique surface antigens, and enhanced plasmid transfer are all components of a single, coordinated response to a particular peptide pheromone. The availability of large quantities of synthetic cCFlO in pure form made it possible to test this notion more thoroughly for the pCFlO system and to compare quite precisely the pheromone induction process as measured by different criteria.
We exposed identical cultures of S. fuecalis responder cells to various concentrations (based on the results of clumping inducing assays) of synthetic cCFlO and measured both the donor potential of the cells and the production of surface antigens previously associated with the pheromone response.
A typical result of the mating assay is depicted in Fig. 3. At concentrations of pheromone above 2 X lo-" M, the responder cells are essentially saturated and the induction is maximal. As the pheromone is further diluted and becomes limiting, there is a decline in mating induction, presumably due to the lack of a sufficient number of pheromone molecules to interact with all of the cells. The linear portion of the dose-response curve was in the range of -10-"-10-'* M. Interestingly, we observed a >40-fold increase in donor potential over uninduced cells at a cCFlO concentration of 3.8 X lo-'* M (or about 2 X lo9 molecules/ml), and nearly a 10-fold increase at 1.9 X M. Since the concentration of donor cells in our induction mixtures is -1-5 X 10' colony-forming units/ml, these results provide further evidence for the extreme sensitivity of the responder cells to the pheromone. As can be seen in Fig. 3, the slope of portion of the dose-response curve below the saturation point is about 2.
We also looked at the response of S. fuecalis cells to synthetic cCFlO by examining the effects of pheromone induction on the bacterial cell surface. Previous results (4-6) have indicated that the S. fuecalis pheromone response is associated with the appearance of novel proteinaceous antigens on the cell surface. In the case of cells carrying pCF10, these include Tral30, which mediates pheromone-inducible surface exclusion (6), and Tral50 which, based on recent cloning and immunological studies: is directly involved in formation of mating aggregates. Neither of these antigens is produced by plasmid-free cells. Cells carrying pCFlO produce several lower molecular wzight forms (120-130 kilodaltons) of Tral30 (pre-Tral30) constitutively. Upon exposure to CIA, the amount of this protein increases, and the 130-kilodalton mature Tral30 form becomes predominant. Our previously published data (6) indicate that the change in molecular weight, which seems to be required for biological activity, may be the result of a pheromone-inducible post-translational modification. In contrast, Tral5O has only been detected on the surface of pheromone-induced donor cells. We used Western blot analysis of cell surface antigen extracts to examine the antigenic changes in the donor cell surface resulting from exposure to various amounts of synthetic cCF10. The results of this analysis are shown in Fig. 4. While the Tral30 antigen shows a slight increase in amount and size at the higher cCFlO concentrations, the most striking result of this study was the direct relationship between the concentration of cCFlO used in the induction and the amount of Tral50 that could be extracted from the cells. There was an excellent correlation between the mating response of cells and the synthesis of Tral50 resulting from exposure to cCFlO at concentrations of 10"'lo-" M. However, the sensitivity of the Western blot assay for Tral50 as a measure of pheromone response was somewhat less than the mating assay in that we detected virtually no Tral50 on cells exposed to 3.8 x M cCFlO even though a significant mating response was observed at this concentration (Fig. 3). Both of these assays were more sensitive than the clumping assay.

DISCUSSION
The structure of cCFlO is similar to that of other S. faecal6 sex pheromones and inhibitors (Fig. 2) in that it is a small P. Christie, J. Adsit hydrophobic peptide with no acidic or basic amino acid residues. Whereas the carboxyl-terminal amino acid residue of the other peptides in this group are hydrophilic (Gly or Ser), that of cCFlO is Val. The three sex pheromones isolated previously contain a Leu in the 3rd residue from the carboxyl terminus, while cCFlO contains a Val. Comparison of the sequences of the various pheromone and inhibitor peptides, along with the analysis of the specificity of the CIA activity of the various peptides shows that, in spite of the extreme hydrophobicity of all of these compounds, the precise amino acid sequence is a critical determinant of biological activity.
The dose-response analyses presented in Figs. 3 and 4 provide illustrations of the extreme sensitivity of S. faecalis to pheromone induction. We previously published data on the response of S. faecalis donor cells to crude pheromone preparations utilizing an enzyme-linked immunosorbent assay for the Tral30 antigen as a measure of the response (19). These data indicated that the pheromone response might follow single-hit kinetics, which would give a slope of 1, in contrast to the slope of 2 that was observed in the linear portion of the curve shown in Fig. 3. Although it is possible that the differences between the previous and current results could be due to experimental error, both the previously published data (19) and the experiment shown in Fig. 3 were repeated several times with very similar results. An alternative explanation for the differences is that, in measuring mating frequency, there may be chain length effects. Under the growth conditions used in the induction and mating experiments, the donor cells are predominantly in pairs, with a few chains of three to six cells. Induction of a single cell in a chain of two or more cells would give the same result as induction of the entire chain, whereas measurements of the amount of a cell surface antigen per unit mass of donor cells would not be subject to chain length effects. In any case Fig. 3 shows clearly that there is a significant donor response at 1-10 cCFlO molecules/ donor cell and that the system becomes saturated at 25-40 molecules/donor cell.
There was a very good correlation between induction of mating and expression of the Tral50 antigen on the surface of donor cells (Fig. 4). Interestingly, the appearance of Tral50 and the increase in the amount of Tral30 also correlated with the disappearance of a 80-kilodalton antigen. We have not previously detected a reproducible pheromone-inducible antigenic change in this molecular mass range. It is likely that the use of the pure synthetic cCFlO in the inductions reduced the antigenic background in these immunoblots, since they are of better quality than any similar blots we have prepared from cells induced with crude CIA from culture filtrates.
The isolation and synthesis of S. faecalis sex pheromones, and the demonstration of their biological activities as reported here and previously (13)(14)(15)(16), has served to support the relationship of clumping, synthesis of novel surface antigens, and plasmid transfer as originally postulated (2). We feel that further investigation of the mechanism of pheromone binding and signal transduction would be of considerable interest, in light of the extreme sensitivity and specificity of the recognition system that has been demonstrated in these studies. The results reported here should provide a good foundation for such an investigation.