Effect of a BlpC-Based Quorum-Sensing Induction Peptide on Bacteriocin Production in Streptococcus thermophilus

Bacteriocin synthesis in some Streptococcus thermophilus strains is under the control of a complex blp locus but bacteriocin is produced only when a quorum-sensing regulatory mechanism is activated by the protein product of the blpC component. To demonstrate the regulatory effect of BlpC in S. thermophilus ST110 (NRRL-B59671), which naturally produces bacteriocin, the effect of the 30mer quorum-sensing induction peptide (QSIP) embedded in BlpC was tested in a knockout mutant in which the blpC gene was eliminated and was devoid of antimicrobial activity. Between concentrations of 30 and 250 ng/ml, the addition of synthetic QSIP to cultures at several points of the growth curve resulted in the accumulation of up to 3,200 units/ml of bacteriocin after 8 h of growth at 37 oC. Addition of QSIP to the culture in late log phase (OD660 ≥ 1.0) when the medium pH is already 4.8 or lower, failed to trigger bacteriocin production. We used synthetic QSIP to survey its impact on 35 strains of S. thermophilus that do not display bacteriocin activity by agar diffusion assays. The addition of QSIP (250 ng/ml) to S. thermophilus cultures in the early or mid-log phase induced bacteriocin production in two strains that could be re-classified as bacteriocin producers. The results demonstrated the involvement of a quorum-sensing regulatory mechanism in bacteriocin synthesis in S. thermophilus ST110 and also demonstrated the utility of the 30 mer QSIP in discovering bacteriocins with potentially novel antimicrobial spectra by enhancing bacteriocin production in other strains of S. thermophilus that ordinarily display a bacteriocin-negative phenotype.


Introduction
Streptococcus thermophilus belongs to a group of food grade lactic acid bacteria (LAB) that are essential starter cultures in the production of yogurt and cheeses.Several strains of S. thermophilus have been shown to naturally produce bacteriocins that are small, ribosomally produced peptides with narrow and/or broad spectrum antimicrobial activity (Villani et al., 1995;Ward & Somkuti, 1995;Marciset et al., 1997;Aktypis et al., 1998;Ivanova et al., 1998;Mathot et al., 2003;Gilbreth & Somkuti, 2005;Fontaine & Hols, 2008).Genome sequencing of Streptococcus thermophilus strains LMD-9, CNRZ1066 and LMG18311 revealed a cluster of genes encoding potential bacteriocin-like peptides (blp) based on genetic similarities to the class II bacteriocin locus described for S. pneumoniae (deSaizieu et al., 2000).Genes encoding potential bacteriocins were identified based on the presence of peptides containing a double glycine leader sequence: including BlpD, U, E, and F in LMD-9; BlpU and K in LMG18311; and BlpK in CNRZ1066; however under standard culturing conditions all three strains failed to produce active bacteriocins (Hols et al., 2005).In addition, in all three strains the blp locus was also shown to contain genes encoding for a three-component quorum sensing (QS) system, with blpH and blpR encoding a putative histidine kinase and response regulator respectively, and blpC encoding the precursor of the quorum sensing induction peptide (QSIP) (Hols et al., 2005).Overexpression of BlpC in S. thermophilus LMD-9 resulted in the identification of a 30-mer secreted peptide (3403.7 Da) representing the mature pheromone, that plays a key role in the display of bacteriocin activity (Fontaine et al., 2007).Production of an active bacteriocin could also be induced in strain LMD-9 when the 30-mer peptide was added to early log phase cultures.However, overexpression of BlpC or extracellular addition of the synthetic peptide had no effect on bacteriocin production in S. thermophilus strains CNRZ1066 and LMG18311 due to mutations in blpB which encodes BlpB, an accessory transport protein required for secretion of the QSIP and bacteriocin peptides (Fontaine et al., 2007).Further analysis of the blp locus in strain LMD-9 showed that expression of BlpD alone was sufficient to inhibit the growth of most target bacteria, but the overall broad spectrum of activity may require expression of multiple peptides (BlpD, U, E, F) and the presence of a thiol-disulfide oxidase encoded by blpG (Fontaine & Hols, 2008).In addition, immunity to the bacteriocin was dependent on the products of orf 1 and orf2, located immediately downstream of blpD; and orf 7, located immediately downstream of blpE (Fontaine & Hols, 2008).
Although we previously reported the apparently constitutive production of bacteriocins in two S. thermophilus strains (Ward & Somkuti, 1995;Gilbreth & Somkuti, 2005), information was still needed on the role of various components of the blp cluster of genes in bacteriocin synthesis.The purpose of this study was to test the involvement of the blpC subunit in bacteriocin production by a mutant culture (blpC -) of S. thermophilus strain ST110 and to evaluate the response of other, apparently non-bacteriocin producing strains to the addition of synthetic 30-mer QSIP that was identical to the active peptide present in BlpC of strain LMD-9.

Synthesis of Quorum-Sensing Induction Peptide (QSIP)
The 30-mer QSIP (N'-SGWMDYINGFLKGFGGQRTLPTKDYNIPQA-C') present within the gene product of blpC in S. thermophilus LMD-9 (Fontaine et al., 2007) was prepared by microwave-assisted solid phase peptide synthesis with Fmoc (N-(9-fluorenyl) methoxycarbonyl)-protected amino acids, using a CEM Liberty synthesizer, according to the manufacturer's recommended protocol (CEM Corp., Matthews, NC).The peptide was cleaved in the CEM microwave chamber using a 95:2.5:2.5 mix of TFA:triisopropyl-silane:H 2 O for 30 min at 35 ºC and 10 W power setting.After repeated precipitations in ice cold ether, the peptide was dissolved in H 2 O and analyzed by HPLC (C18 peptide column).The molecular mass of the peptide product (3,403.7Da) was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS).

Bacterial Strains
S. thermophilus strains were maintained in tryptone-yeast extract-lactose (TYL) medium at 37 ºC (Somkuti & Steinberg, 1986).Escherichia coli DH5α, used as interim host for subcloning plasmid constructs, was grown in BHI medium.When needed, media was supplemented with erythromycin (Em) at 150 µg/ml (E.coli) or 15 µg/ml (S. thermophilus) or kanamycin (Km) at 150 µg/ml.Pediococcus acidilactici F (gift from B. Ray, University of Wyoming) was maintained in deMan, Rogosa and Sharpe (MRS) broth and inoculated into MRS agar when used as the target organism.

Antimicrobial Assays
Bacteriocin activity of cell-free culture fluids of S. thermophilus ST110 (NRRL-B59671, Agricultural Research Service Culture Collection, NCAUR-USDA, Peoria, IL, http://nrrl.ncaur.usda.gov),and its blpC -knockout mutant were tested by an agar diffusion method with Pediococcus acidilactici F as the target organism in MRS agar, in which each well was filled with 50 µl of sample (Gilbreth & Somkuti, 2005).The amount of bacteriocin produced was estimated by the spot-on-the-lawn method (Henderson et al., 1996), after a twofold dilution of each sample with sterile distilled H 2 O and depositing 5 µl on the surface of 2-mm deep MRS agar plates inoculated with P. acidilactici F. The highest dilution showing an inhibition zone was designated as 1 bacteriocin activity unit (AU).Total bacteriocin activity was calculated as the reciprocal of the highest dilution multiplied by a factor of 200 and expressed as AU/ml.In evaluating the effect of QSIP on S. thermophilus strains with blp -phenotype, filtrates of cultures grown for 16 h without or with added QSIP were tested for bacteriocin activity against S. thermophilus ST113 (NRRL-B59386) which was previously noted for high sensitivity to bacteriocins produced by other S. thermophilus strains (Ward & Somkuti, 1995).

Conditions for Evaluating Synthetic QSIP
The presence of the various components of the blp gene cluster in S. thermophilus ST110 with possible role in bacteriocin production was checked by PCR (Perkin-Elmer Thermal Cycler, Model 9700), cycling conditions: 95 ºC for 5 min, followed by 30 cycles of (95 ºC for 1 min, 50 ºC, 55 ºC or 60 ºC for 1 min, 74 ºC for 1 min, followed by 1 cycle of 74 ºC for 7 min and a 4 ºC soak.Forward and reverse primers for each blp component tested (Table 1) were designed using the blp locus in S. thermophilus LMD-9 (ATCC BAA-491, American Type Culture Collection, Rockville, MD) as the model (Hols et al., 2005).PCR products were cleaned up for sequencing using Ampure reagent (Agencourt, www.agencourt.com).
Sequencing was performed in an ABI 3730 DNA Analyzer (Applied Biosystems), and sequences were trimmed and aligned using Sequencher software (Genecodes Corp., Ann Arbor, MI).Nucleotide sequences were compared to sequences in the NCBI database using BLASTn at http://www.ncbi.nlm.nih.gov/.
A control strain of S. thermophilus ST110 lacking blpC (knockout mutant) was developed by a gene inactivation technique using the integrative vector pKS1, which contains a temperature-sensitive origin of replication and genes encoding for erythromycin and kanamycin resistance (Shatalin & Neyfakh, 2005).DNA fragments corresponding to the 3' ends of blpB (708 bp) and blpH (701 bp) were amplified by PCR followed by the purification of fragments using the QIAquick PCR purification kit (Qiagen Inc, Valencia, CA) and cloned into pKS1 at the corresponding restriction endonuclease recognition sites.The resulting vector pSTKOC was transformed into freshly prepared E. coli competent cells by a standard heat-shock method (Sambrook et al., 1989) and propagated at 37 ºC.Plasmid was recovered from E. coli by alkaline lysis followed by CsCl/ethidium bromide ultracentrifugation (Stougaard & Molin, 1981) and introduced by electrotransformation into S. thermophilus ST110 according to a previously described protocol (Somkuti & Steinberg, 1988).Chromosomal integration of the vector was accomplished by a two-step homologous-recombination protocol (Renye & Somkuti, 2012).
The effect of QSIP on bacteriocin production was tested by adding QSIP (250 ng/ml) at different time intervals to cultures of S. thermophilus ST110 and its knockout mutant growing in TYL.QSIP was also added to a growing culture of the knockout mutant to evaluate the effect of QSIP concentration (30-250 ng/ml) on bacteriocin production with Pediococcus acidilactici F as the test organism.
The effect of QSIP was also tested in a group of 35 laboratory strains of S. thermophilus with blp -phenotype with no antimicrobial activity against S. thermophilus ST113.QSIP was added at 250 ng/ml final concentration at different times during growth and after 16 h at 37 ºC, culture filtrates were tested by the agar diffusion assay for activity against S. thermophilus ST113.

Real-time PCR Analysis
S. thermophilus strains were grown in TYL medium until the culture reached an OD 660 between 0.8 and 1.0.RNA was extracted using the RiboPure TM -Bacteria kit (Ambion-Life Technologies, Grand Island, NY).Residual DNA was removed with DNase I treatment (Ambion) for 30 minutes at 37 ºC.RT-PCR was performed using an Applied Biosystems 7500 Fast RT-PCR system (Life Technologies).Cycling conditions were: 40 cycles of 95 ºC for 30 s, 45 ºC for 30 s and 60 ºC for 30 sec., and a melt curve analysis was performed from 60-95 ºC with fluorescence readings taken continuously after a 1% increase in temperature.cDNA synthesis and RT-PCR were carried out in a single step using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Life Technologies) with a total RNA concentration of 1 µg for amplification of blp components, and 10 ng for 16 S rRNA as the reference.The ABI 7500 Fast software was used to determined C T values, and the relative quantification of gene expression was determined using the 2 -ΔΔCT method (Livak & Schmittgen, 2001), where ΔC T = C T (target) -C T (reference), and ΔΔC T = C T (+QSIP) -C T (-QSIP).Results are reported from a minimum of three independent RT-PCR reactions (±standard deviation).

Detection of Blp Components in S. thermophilus ST110
Bacteriocin production in S. thermophilus is dependent on the presence of the blp gene cluster that was previously described in several strains, including LMD-9, LMG18311 and CNRZ1066 (Hols et al., 2005) and the functionality of its various components was studied in several strains (Fontaine et al., 2007).Based on PCR analysis and nucleotide sequence data, gene products containing a double glycine leader sequence and qualifying as potential bacteriocins include BlpD, U, E and F in strain LMD-9 (ATCC, BAA-491), BlpU and K in strains LMG18311 (ATCC, BAA-250) and S. thermophilus ST110 (NRRL-B59671), and BlpK in strain CNRZ1066 (Hols et al., 2005)(Table 2).Based on research on the functionality of various blp components in knockout mutants of S. thermophilus LMD-9, it was previously concluded that the blpD component alone may be responsible for bacteriocin activity (Fontaine & Hols, 2008).However, the absence of blpD in S. thermophilus ST110 as shown by PCR analysis indicated that bacteriocin synthesis in this strain is controlled by other components of the blp locus.
According to the LMD-9 model (Fontaine et al., 2007), the product of blpC, a 53-mer protein (BlpC) includes the 30-mer QSIP peptide N'-SGWMDYINGFLKGFGGQRTLP-TKDYNIPQA-C' that plays a key role in activating the cascade of events leading to bacteriocin synthesis.It was also found that although some S. thermophilus strains (e.g.LMD-9) have a functional blpC component, the amount of BlpC gene product is not adequate to activate bacteriocin synthesis (Fontaine & Hols, 2008) but externally supplied QSIP induced bacteriocin production.

Restoration of Bacteriocin Production in S. thermophilus ST110 blpC -knockout mutant
Following electrotransformation with the pSTKOC integrative vector, S. thermophilus ST110 transformants were resistant to both erythromycin and kanamycin when grown at 32 ºC (permissible temperature for plasmid replication).Subsequent transfers at 42 ºC resulted in a single homologous recombination event with the entire plasmid inserted into the blp gene cluster.Clones confirmed by PCR to have the single crossover within the blp gene cluster were further transferred at 30 ºC which allowed for the second recombination event to occur, replacing blpC with the kanamycin marker gene.Removal of blpC from the blp gene cluster resulted in the loss of bacteriocin activity as indicated by the absence of an inhibition zone in agar diffusion assays.However, when the mutant culture lacking blpC was supplemented at various time intervals with QSIP at 250 ng/ml, bacteriocin production was restored (Figure 1 and Figure 2).The optimum time of QSIP addition to induce bacteriocin production was between 0 and 4 h after the start of incubation and the amount of bacteriocin produced after 8 h of incubation was estimated at 3,200 AU/ml (Figure 1).

Impact
We tested strains of QSIP were at 37 ºC, a each cultu supernatan strain ST1 The result additional  Induced expression of blpK was also tested since it may encode a unique bacteriocin component in strains ST106 and ST118, and was shown to increase by 198 (±15) and 24 (±3) fold, respectively.The high level of expression observed for blpK in ST106 suggests that it may be required for optimal production of the ST106 bacteriocin(s).Expression of the blpRH operon was also tested in all three strains, but as reported previously its expression was not significantly altered (≤2) in response to the addition of exogenous QSIP (Fontaine et al., 2007).

Conclusions
The results confirmed the pivotal role of blpC in the production of bacteriocins in S. thermophilus ST110 (NRRL-B59671).Since S. thermophilus is designated as food grade and various strains are used as essential biocatalysts in the industrial production of fermented dairy foods, the capacity to produce peptide(s) imparts them added value and may improve the level of protection against bacterial contamination.The results also demonstrated that QSIP is a valuable screening tool in identifying S. thermophilus strains that may produce potentially novel bacteriocins by restoring or improving the functionality of a compromised blpC component of the blp cluster of genes.
Figur knockout ng/ml to a Figure negative s

Table 1 .
Primers used to detect components of the blp locus in S. thermophilus a sequences determined by primer walking.

Table 2 .
Blp components with gly-gly leader peptides in S. thermophilus strains