A Method for Structure–Activity Analysis of Quorum-Sensing Signaling Peptides from Naturally Transformable Streptococci

Many species of streptococci secrete and use a competence-stimulating peptide (CSP) to initiate quorum sensing for induction of genetic competence, bacteriocin production, and other activities. These signaling molecules are small, unmodified peptides that induce powerful strain-specific activity at nano-molar concentrations. This feature has provided an excellent opportunity to explore their structure–function relationships. However, CSP variants have also been identified in many species, and each specifically activates its cognate receptor. How such minor changes dramatically affect the specificity of these peptides remains unclear. Structure–activity analysis of these peptides may provide clues for understanding the specificity of signaling peptide–receptor interactions. Here, we use the Streptococcus mutans CSP as an example to describe methods of analyzing its structure–activity relationship. The methods described here may provide a platform for studying quorum-sensing signaling peptides of other naturally transformable streptococci.


Introduction
Natural genetic transformation is a process by which bacteria are able to take up and integrate exogenous free DNA from their environment (1,2). This process enables the recipient organisms to acquire novel genes, thereby promoting the emergence of antibiotic resistance, genetic variation, and rapid evolution of virulence factors (1)(2)(3)(4). Many members of the genus Streptococcus are naturally transformable and each depends on a signaling peptide-mediated quorum-sensing system for induction of genetic competence (3)(4)(5). Activation of quorum sensing for genetic competence and other coordinated activities in these species requires at least six gene products encoded by comCDE, comAB, and comX (Fig. 1). The comC gene encodes a competence-stimulating peptide (CSP) precursor, which is cleaved and exported through a peptide-specific ABC transporter encoded by comAB, releasing CSP into a extracellular environment (2)(3)(4)(5)(6). The comDE encodes a two-component system consisting of a histidine kinase sensor protein (ComD) and its cognate response regulator (ComE) that specifically senses and responds to CSP. At a critical concentration, the CSP activates autophosphorylation of the ComD of neighboring cells. The phosphate group is then transferred to the ComE, which in turn activates its target genes, including comX that encodes a competence-specific sigma factor recognizing a consensus sequence (com-box) at the promoter regions of late competence genes, triggering the signaling cascade for genetic competence (2)(3)(4)(5). The quorum-sensing system in Streptococcus mutans represents a unique regulatory mechanism, since this system regulates both bacteriocin production and genetic competence (2,6,7). In contrast to S. mutans, Streptococcus pneumoniae requires two separate signaling systems, the ComCDE and BlpRH, to regulate genetic competence and bacteriocin production (8).
Many CSPs from naturally transformable streptococci have been identified and their activity for genetic competence has been validated (2)(3)(4). These signaling molecules are small, unmodified peptides, ranging from 14 to 25 residues in length, and induce powerful strain-specific activity at nano-molar concentrations. Some CSPs have been chemically synthesized and used as a tool to induce genetic competence during molecular cloning. Recently, the structure-activity relationships of CSPs from both S. pneumoniae and S. mutans have been analyzed (9,10). The CSPs from these organisms have been found to adopt an amphipathic, α-helical conformation with a defined hydrophobic face that contributes to the CSP specificity. Furthermore, the CSP from S. mutans reveals two functional domains (10). The C-terminal structural motif consisting of a sequence of polar hydrophobic charged residues is crucial for activating the signal transduction pathway, while the core α-helical structure extending from residue 5 through to the end of the peptide is required for receptor binding. Sequence alignment of CSPs from various streptococci shows that almost all CSPs have such a motif at the C-termini (10), suggesting that these CSPs likely have similar functional domains. However, CSP variants have been identified in many species and each induces competence in a highly strain-specific manner. Thus, the CSP variants may provide an excellent model to study peptide-receptor interactions in these bacteria. Here, we extend our technical information and describe methods and protocols for structure-activity analysis of CSP from S. mutans Fig. 1. A schematic diagram describing the quorum-sensing system and its controlled cooperative activities in Streptococcus mutans. The signaling peptide or CSP induces quorum-sensing cascade when reaching to a critical concentration. This in turn activates the production of numerous bacteriocins and genetic competence, resulting in killing of other species, DNA release, and gene exchange.
by combining activity assays with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies (see Appendix). The methods described herein may provide a platform and rationale for analyzing CSPs from other naturally transformable streptococci.

Chemical Synthesis of Signaling Peptides
Efficient structural characterization of signaling peptides by CD and NMR requires purified (≥95% purity) and soluble peptides. In our study, all the peptides were commercially synthesized and purified by reversed-phase high-pressure liquid chromatography. Their identity was confirmed by a mass spectrometry (10). The peptides were lyophilized and stored at -20°C until use. For CD and NMR studies, each peptide was dissolved in an appropriate solvent. For activity assays, each peptide was freshly dissolved in sterile distilled water at a concentration of 1.0 mM (stock solution), which was further diluted as required.

Sample Preparation
The first step was to optimize sample conditions by adjusting pH, ionic strength, and temperature to mimic physiological conditions with the constraint in which the conditions were compatible with data acquisition (11). For CD spectroscopy, peptide concentrations were typically on the order of 50µM with 200µL of solution (12). Buffers, co-solvents, and additives (e.g., detergent molecules) were non-chiral, sodium-free, and non-absorbing within the wavelength range (typically 250-180 nm). For NMR studies, buffers, co-solvents, and additives were hydrogen-free or deuterated. Samples could be dissolved in as little as 10µL, but typically, spectrometers were designed for sample volumes of at least 500µL. Due to the inherently low sensitivity, concentrations of peptides ranged from 1.0 to 10 mM (13).
In our study, synthetic peptides CSP (UA159sp) and TPC3 for CD and NMR were dissolved in 95/5/0%, 70/ 0/30%, 30/0/70%, or 0/0/100% aqueous buffer/D 2 O/ TFE-d 2 , or 95/5 aqueous buffer/D 2 O with 300 mM DPCd 38 (98 atom %D), where TFE refers to trifluoroethanol and DPC-d 38 refers to perdeuterated dodecylphosphocholine (10,13). The aqueous buffer used for sample preparation contained 50 mM K 2 HPO 4 /KH 2 PO 4 at pH7.0. The concentration of peptides for solutions containing 100% TFE-d 2 or DPC-d 38 was 2 and 5 mM. For diffusion studies, we analyzed the solutions containing peptides and DPC-d 38 by lyophilizing and dissolving them in an equivalent volume of D 2 O (99.9 atom %D) followed by transferring into a 5 mm OD, D 2 O magnetic susceptibility-matched NMR tube (BMS-3; Shigemi, Tokyo). The sample height of 1.2 cm ensured that the entire sample was within the radio frequency coils, which was essential for artifact-free and accurate diffusion measurements.

CD Spectroscopy
CD spectra for peptides and reference solutions were recorded at 298 K on a Jasco J-920 CD spectrometer with a 1-mm quartz cuvette. Spectra were collected and averaged over 16 scans from wavelengths 190 to 250 nm with a 0.1-nm step resolution. CD measurements were performed and analyzed to confirm the effects of TFE on the secondary structure of the peptides (14,15). In our study, we analyzed the peptides by using CD analysis package DICHROWEB (www.cryst.bbk.ac.uk/cdweb/html) (16).

NMR Spectroscopy
Initial experiments in our study included 1 H-1 H COSY (correlation spectroscopy) and 1 H-1 H TOCSY (total correlation spectroscopy). The COSY spectra were used to correlate 1 H atoms that were ≤3 bonds apart, and to determine the 3 J H N H α coupling values between H N and H α , whereas the TOCSY spectra were used to correlate all 1 H atoms within a spin system that were ≤3 bonds apart (17). In addition, 1D and 2D 1 H NMR data sets of the peptides were collected on a Bruker AVANCE 500 spectrometer. Unless otherwise stated, 1 H detected spectra were acquired at a repetition rate of 1.5 s −1 at 293 K with water frequency centered on carrier frequency. For structural determination, phase-sensitive 2D 1 H-1 H NOESY, 1 H-1 H COSY, and 1 H-1 H TOCSY data sets (MLEV17) were recorded at 293 K with a presaturation or with a 3-9-19 pulse sequence for water suppression. 1 H detected 2D DOSY data sets were recorded using a stimulated-echo sequence with bipolar-gradient pulses and 32 t 1 blocks of 64 transients each. NMR data sets were processed using Bruker XwinNMR 3.5.

Structural Determination
Structural determination of the peptides followed the procedures as described previously (10). Briefly, spin systems (i.e., amino acid residues) were identified through chemical shifts and TOCSY cross-peak patterns. Distance constraints determined from integration of the 250-ms NOESY spectra were classified into four groups: strong, medium, weak, and very weak corresponding to interproton distance ranges of G2.3, 2.0-3.5, 3.3-5.0, and 4.8-6.0Å, respectively. The 3  From the NMR-derived structural constraints, two series of structural calculations were performed with or without added hydrogen bonds to assess the effect of adding suspected hydrogen bonds into the structure calculation. All structural calculations were based on previous studies using the XPLOR 3.1 software package (10). Final calculated structures were energy minimized with 2,000 conjugate gradient steps before processing to the restrained annealing molecular dynamics calculation. A total of 33 and 21 UA159sp and 43 and 36 TPC3 (with and without hydrogen bonds included within the simulation) lowest energy structures were retained that had no violations of NOESY constraints >0.5Å (18). The overall quality of these refined structures was examined with the program PROCHECK (19). Except for random coil sections, all backbone dihedral angles resided in the well-defined, acceptable regions of the Ramachandran plot. Three-dimensional structures of UA159sp and TCP3 determined from NMR were constructed by computer simulation using the VMD software (http://www. ks.uiuc.edu/Development).

Construction of comC Mutant That Was Defective in Producing CSP
To assay quorum sensing (QS) activation in response to addition of a peptide without interference by endogenous CSP from a wild-type strain, a comC mutant that was unable to produce, but still responded to CSP was constructed in S. mutans GS5 using the strategy as described previously (20). Following genetic recombination via allelic exchange, the comC deletion mutant was created by insertion of a spectinomycin (Spec r ) resistance cassette (21) into the comC gene of strain GS5. The mutant was then confirmed by polymerase chain reaction (PCR) and sequencing of the junction site of the Spec cassette as described previously (20). The resulting mutant was named SMdC and was used as a negative background strain of signaling peptide (CSP) for CSP-dependent assays of genetic competence, lacZ reporter, and bacteriocin production.

Construction of lacZ Transcriptional Reporter Strains
To assay QS activation in response to addition of a peptide, we constructed two types of lacZ transcriptional reporter strains that represented two levels of QS-activated gene expression. The first was a lacZ reporter gene fused to the comDE promoter, which allowed monitoring of the activity of the comDE promoter in response to a test peptide. The second strain was a lacZ reporter gene fused to the promoter of nlmAB, the gene encoding a nonlantibiotic bacteriocin and its expression directly controlled by the ComE (6,22). This strain allowed assay of comDE-controlled bacteriocin production.
For the first reporter strain, we simply transformed previously constructed vector pYH2 (10) into the comC mutant (SMdC) to generate SMdC-pYH2 (PcomDE::lacZ, comC − , Spec r , Kan r ). pYH2 was constructed in such a way that the comDE promoter was fused to a promoterless lacZ gene on the backbone of Streptococcus-Escherichia coli shuttle vector pSL. The vector pSL was also transformed into the same mutant SMdC and used as a background control (SMdC-pSL). These strains were then assessed for lacZ reporter activity in response to addition of peptides.
For the second reporter strain, we generated a chromosomal nlmAB-lacZ fusion strain by transforming a PnlmAB::lacZ fusion construct pOMZ47 (6) into both comC mutant SMdC and wildtype GS5. The resulting reporter strains were confirmed by PCR and sequencing of the fusion sites as described previously (6). The confirmed reporter strains were respectively named SMdC-PnlmAB (PnlmAB::lacZ, comC − , Kan r , Spec r ) and SMGS5-PnlmAB (PnlmAB::lacZ, Kan r ). The strain SMGS5-PnlmAB was used a positive control.

CSP-Dependent Transformation Assay
To determine if synthetic peptides activated quorum sensing for induction of genetic competence, we used the comC deletion mutant that was unable to produce, but still responded to CSP, to assay peptide-dependent genetic transformation using the method as described previously (2,10). A streptococcal suicide vector pVA-gtfA, which harbored a 2.4-kb fragment of the S. mutans gtfA gene and an erythromycin resistance marker, was used as transforming DNA. The wild-type GS5 was used as a positive control, while both ComC (SMdC) and ComE mutants (SMdE) were used as negative controls. The wild-type strain was grown on Todd-Hewitt yeast extract (THYE) medium, whereas the mutants were maintained on THYE plus spectinomycin. When culture reached to early mid-log phase (OD 600 ≈0.25), an aliquot of CSP was added into the culture at a final concentration of 50 nM (AC 50 ≈0.25 nM). After 20 min of incubation at 37°C, an aliquot of transforming DNA of plasmid pVA-gtfA conferring erythromycin resistance was added at a final concentration of 1µg mL −1 . The culture was incubated for additional 2 h before plated on THYE agar plates plus 10µg mL −1 erythromycin. Prior to addition of transforming DNA, an aliquot of the cell suspension was plated on THYE plates containing the same antibiotic to monitor spontaneous mutation. Transformation frequency was expressed as percentage of transformants against total viable recipient cells per milliliter cell suspension.

β-Galactosidase Activity Assay
A lacZ reporter assay was performed to monitor quorum-sensing activation using the same method as described previously (10). The newly constructed lacZ reporter strains were used to assay and quantify β-galactosidase activity in response to test peptides. Aliquots of samples were taken to prepare cell lysates at different time points T 0 , T 15 , T 30 , T 60 , and T 120 after addition of a test peptide. Protein concentrations of the supernatants were determined by Bio-Rad protein assay (Bio-Rad). Specific β-gal activities (A 420 min −1 mL −1 mg −1 protein) were calculated from triplicate samples from two independent experiments. The data from the β-gal activity experiments were then normalized against negative or background controls and plotted as percentages of maximal activation versus log peptide concentrations. The half maximal activation concentration (AC 50 ) was determined from the sigmoidal dose-response curve using Prism 4 (Graphpad, San Diego, CA, USA).

Assay for QS-Controlled Bacteriocin Production
The S. mutans comC mutant SMdC as well as its parent GS5 and wild-type strain UA159 (positive controls) were used to assay for bacteriocin production using a modified agar plate method (7,(23)(24)(25). Briefly, an aliquot of mid-log phase cells of the S. mutans wild-type strains and mutant SMdC grown under a condition with or without use of CSP or a test peptide were stabbed onto THYE agar plates. The plates were incubated anaerobically at 37°C for 6 h before carefully overlaid with 100µL of cell suspension (about 10 6 CFU/mL −1 ) of an indicator strain Streptococcus sanguinis SK108. The plates were further incubated anaerobically at 37°C for 24 h before inspection of bacteriocin production. The S. mutans strains showing an inhibitory zone around the stabs were scored as positive for bacteriocin production. The plates were then photographed for records.

CD and NMR Structural Analyses of the Signaling Peptides
CD and NMR spectroscopy are non-destructive and non-invasive techniques that have been widely used to determine secondary folding features of proteins or peptides (14)(15)(16)(17). In addition, NMR spectroscopy records data from "spin-active" nuclei, such as 1 H, 13 C, 15 N, or 31 P. These nuclei are sensitive to their chemical environment, which is determined by the type and number of bonds and the position and proximity of other chemical entities and the solvent. Thus, NMR is a powerful tool that can be used to determine atomic level structural information, dynamics (chemical exchange, motional fluctuations, proton exchange), ligand binding, multimeric states, and effects of competitive receptors (11,15,26).
Using CD and NMR spectroscopy, we have determined the three-dimensional structures of two signaling peptides, UA159sp from S. mutans UA159 and a C-terminally truncated peptide TPC3 from JH1005 defective in genetic competence (10). Initial NMR data for both peptides were recorded in aqueous buffer to provide an assessment of the structural nature of the peptides. In the presence of water, both peptides were not soluble enough for extensive NMR studies. In TFE, however, these peptides formed well-defined α-helices from residues Leu4-Gly20 of UA159sp and Ser5-Thr16 of TPC3 (Fig. 2). Both UA159sp and TPC3 formed a similar structure in TFE and DPC-d 38 micelles, although the Cterminal three residues of TCP3 were truncated. The structural coordinates for two peptides have been deposited in the RCSB Protein Data Bank (http://www.pdb.org/) with RCSB ID code rcsb039055 or PDB ID code 2I2J for UA159sp and RCSB ID code rcsb039053 or PDB ID code 212H for TPC3. An important feature observed from structural analyses was that both UA159sp and TPC3 formed amphipathic α-helices with a well-defined hydrophobic face characterized by a row of four highly hydrophobic phenylalanines (F7, F8, F11, and F15) on one side and hydrophilic residues on another side (Fig. 2). The hydrophobic face comprised about 40% of the surface of the helix, whereas hydrophilic and charged residues comprised the remainder of the surface. A major structural difference between UA159sp and TPC3 was that UA159sp showed a clear C-terminal motif of slightly more motional freedom than TPC3. From structures of other similar binding peptides, a well-defined row of hydrophobic residues on an amphipathic α-helix is common and necessary for ligand binding to the receptor protein (12,19). Our work suggests that the quorum-sensing signaling peptides from S. mutans, probably from other naturally transformable streptococci, fall into the FXXFF motif family, a general protein-protein interaction motif, where the interaction is made through a hydrophobic face formed by the hydrophobic residues packed into a hydrophobic pocket of the receptor (11,12,26). The fact that the amphipathic helical structure is reflected in the binding and induction of quorum-sensing suggests that the peptide may be helical within the active site of the receptor. The membrane environment may help to form the helical structure, facilitating binding to the receptor. We have chosen to dissolve the peptides in widely used solvent TFE because it improves structural stability of peptides and is considered to be biologically relevant (27). TFE is also thought to mimic a protein receptor environment by stabilizing helices in regions with intrinsic α-helical propensity that are likely to form helices when binding to their protein partner.

Signaling Peptide-Receptor Activation in Newly Constructed Strains
In previous studies, we exclusively used S. mutans UA159 and its derivatives to assay QS activation in response to signaling peptides (2,10). However, this genome sequence reference strain is not a typical producer for CSP-induced bacteriocins (7,24,25). To construct a comC mutant that allowed us to assay CSP-dependent bacteoricin production, we transformed the previously generated comC deletion construct (20) into S. mutans GS5, a wild-type strain that was demonstrated to produce all known QS-controlled bacteriocins (6,7,22). The new mutant, named SMdC, was confirmed to have the internal region of comC replaced by a spectinomycin (Spec r ) resistance cassette. The mutant was confirmed to be defective in quorum sensing for induction of genetic competence (Fig. 3) and bacteriocin production (Fig. 4), unless CSP or a peptide agonist was added into the culture. Our work confirmed that this mutant provided an excellent negative background, enabling the assay of peptide-dependent QS activation without interference by endogenous CSP.
We also used this mutant (SMdC) as a host to generate two types of lacZ transcriptional reporter fusion strains to assay the promoter activity of QS-controlled target genes, comDE and nlmAB, in response to addition of CSP. The lacZ reporter strain, SMdC-pYH2 (PcomDE::lacZ, comC − , Spec r , Kan r ), along with its background control strain SMdC-pSL (comC − , Spec r , Kan r ) were then assayed for β-galactosidase activity. The results confirmed that upon addition of CSP strain SMdC-pYH2 was induced to express predicted β-gal activity at similar levels to those of the previously constructed lacZ reporter strain in UA159 (10). In contrast, the background control strain SMdC-pSL did not show significant difference in the expression of β-gal activity. To assay QS activation of the downstream genes controlled by the ComCDE signaling transduction pathway, we also constructed two nlmAB-lacZ fusion strains by transforming a previously generated fusion construct PnlmAB::lacZ (6) into SMdC-GS5 and parent GS5. These strains were genetically confirmed and named SMdC-PnlmAB (PnlmAB::lacZ, comC − , Kan r , Spec r ) and SMGS5-PnlmAB (PnlmAB::lacZ, Kan r ), respectively. The β-gal activity assay showed that prior to addition of CSP SMdC-PnlmAB was defective in expressing a normal level of β-gal activity (Fig. 4a). Upon addition of CSP, however, SMdC-PnlmAB was rapidly induced to express an increasing level of β-gal activity and reached to the highest level around 90 min after addition of CSP (Fig. 4b). Interestingly, SMdC-PnlmAB still maintained such a high level of the activity for at least 2 h. Clearly, such expression of β-gal activity appeared to be a major difference between this reporter strain and SMdC-pYH2, in which the expres- sion of β-gal activity declined around 40 min after the addition of CSP (10). However, the expression of β-gal activity by SMdC-PnlmAB was highly inconsistent with those of other reports (6,7), suggesting that the expression of nlmAB gene was likely enhanced by an unknown factor following initial activation by CSP. Thus, both lacZ reporter strains representing two levels of gene regulation provided us with a sensitive detecting system to assay and identify signaling peptide agonists.

Application of the Methods to Identify Signaling Peptide Agonists
Based on the three-dimensional structures of S. mutans wild-type signaling peptide UA159sp (CSP) and C-terminally truncated peptide TPC3 from mutant JH1005 defective in genetic competence, we designed and synthesized a series of truncated peptides and peptides with amino acid substitutions (10). By functional analysis of these peptides, we found that CSP from S. mutans displayed two functional domains. The C-terminal structural motif consisting of a sequence of polar hydrophobic charged residues is crucial for activating the signal transduction pathway, while the core α-helical structure extending from residue 5 through the end of the CSP is required for receptor binding. The AC 50 of CSP (UA159sp) was determined to be about 25 nM (10). With these data, we developed a rationale to design and synthesize several new peptide agonists ( Table 1). By assaying their activity in quorum-sensing activation using the methods described here, we found that hydrophobic residues, leucine (L) or phenylalanine (F), could replace the hydrophilic residues at positions 9, 12, or 13 of the core α-helix of CSP without significant change of its activity ( Table 1). In contrast, peptides, F7Q, F11Q, and F15Q that had hydrophobic phenylalanines at positions 7, 11, or 15 replaced by a hydrophilic residue, glutamine (G), failed to activate quorum sensing (10). In addition, we found that alanine (A) at position 18 appeared to be important for the activity, since a deletion or replacement of this residue abolished the peptide activity. The evidence strongly suggests that the maintenance of the hydrophobic face of the signaling peptide is crucial for receptor recognition. These results are consistent with the results of Havarstein and colleagues (9), who have found that the hydrophobic patch of CSP1 and CSP2 of S. pneumoniae mainly contributes to the receptor recognition and the specificity. Interestingly, the hydrophobic patch in the S. pneumoniae CSP1 also consists of three highly hydrophobic phenylalanines at positions 7, 8, and 11 and an isoleucine at position 12. In contrast to S. pneumoniae, however, CSP receptor recognition in S. mutans appears to be less specific among strains. For example, the peptides with a truncation of N-terminal residues, such as TPN1 or TPN2, or C-terminal residue TPC1, also functioned as agonists with AC 50 values similar to CSP (10). These appear to be consistent with the report of Allan et al. (28), who have found no strict correlation between the CSP genotype and the ability to induce quorum sensing among S. mutans strains. Together, these findings suggest that the S. mutans CSP and its variants may have less recognition specificity of interacting with its receptor protein. Unlike S. pneumoniae that requires two independent signaling systems, the ComCDE and BlpRH, to regulate genetic competence and bacteriocin production, S. mutans only uses one signaling system with more flexible peptide-receptor interaction to regulate these phenotypes. We speculate that more peptide variants may be found in S. mutans isolates, and they likely function in the same fashion as CSP (UA159sp) to activate quorum sensing for bacteriocin production and genetic competence. To support this speculation, we designed a shorter peptide agonist, SPA-18, to assay its activity. We found that this shorter peptide could completely replace CSP (UA159sp) in activating quorum sensing (Fig. 4), genetic competence (data not shown), and bacteriocin production (Fig. 5).

Conclusions
Quorum-sensing signaling systems involving the interaction between a signaling peptide and its cognate histidine kinase receptor are widely distributed in Gram-positive bacteria. Structure-activity analysis of signaling peptides and their analogs may provide clues for understanding the specificity of peptide-receptor interactions in these bacteria. In this report, we have used the S. mutans CSP as an example to describe the methods for structure-activity analysis of quorum-sensing signaling peptides by combining activity assays with CD and NMR spectroscopy. By analyzing these peptides, we have identified several signaling peptide agonists that activate quorum-sensing cascade for induction of bacteriocin production and genetic competence in S. mutans. The methods described herein may provide a platform for analyzing structure-activity relationships of signaling peptides from many other naturally transformable streptococci.
4. The cultures are incubated at 37°C for 15 min before added with a transforming DNA (plasmid pVA-gtfA) at the final concentration of 1 µg/mL. The cultures are incubated at 37°C for additional 2 h.

5.
A 100-µL cell suspension from each culture is spread on a THYE agar plate plus erythromycin (10µg/mL) after gentle vortexing.
6. An aliquot of the cell suspension is taken, diluted, and inoculated on THYE agar plates with no antibiotic to determine total viable cell counts.
7. All the plates are incubated at 37°C for 2 days before assessment of transformation frequency, which is expressed as percentages of transformants (erythromycin-resistant colonies) against total recipient cells per millimeter cell suspension.
8. An aliquot of the cell suspension prior to addition of the transforming DNA is also taken and inoculated on THYE plates supplemented with the same antibiotic to determine spontaneous mutation.
of 0.1% sodium dodecyl sulfate (SDS). The samples are immediately centrifuged at 10,000×g at 4°C for 5 min.
7. An aliquot (100µL) of the supernatant from each sample is added in triplicate onto a 96-well microtiter plate. Then, 50µL of O-nitrophenyl-D-galactopyranoside is added into each well at a final concentration of 80µM.
8. The reactions are incubated for 1 h, stopped by adding 50µL of 1 M Na 2 CO 3 into each well, and then quantified by a multi-detection micro-plate reader (Synergy) at 420 nm. The reading results are saved for calculation of specific βgal activity.
9. An aliquot (100µL) of the supernatants is also transferred into a microtiter plate for determination of the concentration of proteins using Bio-Rad protein assay (Bio-Rad).
10. Specific β-gal activity of each sample is calculated from triplicate samples from two independent experiments and the data are then normalized against negative or background controls. Specific β-gal activity is expressed as Miller units (A 420 per min −1 mL −1 mg −1 protein).
11. The data from specific β-gal activity are then plotted as percentages of maximal activation versus log peptide concentrations. The half maximal activation concentration (AC 50 ) is determined from Sigmoidal dose-response curves using Prism 4 (Graphpad).