Journal of Bacteriology, March 2004, p . 1556-1564, Vol . 186, No . 5

Induction of Plantaricin Production in Lactobacillus plantarum NC8 after Coculture with Specific Gram-Positive Bacteria Is Mediated by an Autoinduction Mechanism

Antonio Maldonado, Rufino Jiménez-Díaz, and José Luis Ruiz-Barba^*

Departamento de Biotecnología de Alimentos, Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, 41012 Seville, Spain

Received 7 November 2003/ Accepted 17 November 2003

 
Plantaricin NC8 [PLNC8], a coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8, has recently been purified and genetically characterized . Analysis of an 8.1-kb NC8 DNA region downstream of the PLNC8 operon revealed the presenceof at least four operons involved in bacteriocin production,showing high homology to the plantaricin cluster in L . plantarumC11 . However, we found a three-component regulatory operon involvinga quorum-sensing mechanism . Two of these components, the induction factor [PLNC8IF] and the histidine kinase, are novel, whilethe response regulator is identical to PlnD from C11 . Homologous expression of plNC8IF in NC8 allowed constitutive bacteriocin production . Heterologous expression of this gene in Lactococcus lactis MG1363 produced supernatants which promoted bacteriocin production in NC8 . Reverse transcription-PCR studies indicatedthat cocultivation of NC8 with inducing cells promoted transcriptionof the bacteriocin and regulatory operons in NC8 . An identicalresult was obtained after addition of an external source ofPLNC8IF . We propose that the presence of specific bacteria couldact as an environmental signal that is able to switch on bacteriocinproduction in L . plantarum NC8 via a quorum-sensing mechanismmediated by PLNC8IF.

 
Lactic acid bacteria [LAB] can produce a wide range of substances with antimicrobial activity [24, 27, 48] . Among these, the proteinaceouscompounds called bacteriocins have been a focus of researchbecause of their potential use as natural food preservatives[7, 8] . Bacteriocins are peptides or proteins with antimicrobial activity directed against related species [24, 27, 48] . Bacteriocinsproduced by LAB can be classified into four main groups accordingto their biochemical and genetic properties [17, 27, 35] . ClassII bacteriocins are small, heat-stable, cationic, and hydrophobicpeptides, which are synthesized as precursor molecules containing,in most cases, a leader peptide of the so-called double-glycinetype [17, 35] . This leader peptide is recognized and cleaved by a dedicated ABC transporter, resulting in translocation ofmature, active bacteriocin to the medium [21] . The class IIb subgroup contains bacteriocins whose antimicrobial activity depends on the complementary action of two different peptides[17, 35].

Production and export of class II bacteriocins require several genes, which are usually organized into two or more operons[17, 35] . The bacteriocin synthesis operon is composed of one or two [in class IIb] structural genes followed by a gene encoding the immunity protein . A second operon encoding the machinery necessary for the processing, transport, and secretion of the bacteriocins is usually located in the vicinity of the bacteriocin genes . This operon is formed by two or more genes encoding anABC transporter and its accessory protein [17, 35].

Production of several class II bacteriocins is regulated bya so-called three component regulatory system [17, 28, 29, 35, 36] . In these cases, a regulatory operon has been found to be involved in bacteriocin production . This operon is composedof a gene encoding an induction factor [IF] or peptide pheromone[Pph] followed by two genes encoding a histidine kinase protein[HK] and a response regulator [RR] . Such an IF acts as an indicatorof the cell density, which is sensed by the corresponding HK,resulting in activation of the RR, which then activates expressionof all operons necessary for bacteriocin synthesis, transport,and regulation . This quorum-sensing or autoinduction mechanismmediated by inducer peptides has been found in Carnobacteriumpiscicola [3, 30, 40, 45], Lactobacillus plantarum [4, 11], Lactobacillus sakei [4, 12], and Enterococcus faecium [37, 38]. In most of these systems, however, bacteriocin production isan unstable phenotype [10, 12, 16, 37, 44] . This instability has been attributed to reduced synthesis of the IF under experimental conditions [28, 36] . Therefore, it has been suggested that environmentalfactors may play an important role in the regulation of bacteriocinproduction, by means of increasing the basal levels of the IFs[4, 36, 42] . Thus, sakacin A production by L . sakei Lb706 andLactobacillus curvatus LTH1174 was found to be a temperature-sensitiveprocess [12] . Nevertheless, how the environment interacts withthe regulation of bacteriocin production is still poorly understood.

Recently, a novel class IIb bacteriocin, plantaricin NC8 [PLNC8], has been biochemically and genetically characterized [33] . Interestingly,this bacteriocin was produced by L . plantarum NC8 only aftercocultivation with specific gram-positive strains or the additionof heat-killed cells from some of the inducing strains [33, 34] . However, no cell-free supernatants [CFS] from any of theinducing strains had any effect [34] . Along with the inducedbacteriocin PLNC8, an autoinducing activity was also producedafter exposure of NC8 cultures to the inducing conditions . Inthis case, the CFS from a previously induced [bac⁺] NC8 culturewas able to induce bacteriocin production in a fresh NC8 singleculture [34] . This observation was confirmed by sequence analysisof the promoter region of the plNC8BAC operon [33], which maintainsthe consensus of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism [36, 42].

In this study we have gained insight on the autoinduction phenomenon observed after coculture induction of bacteriocin productionin L . plantarum NC8 . Thus, molecular analysis of an 8.1-kb DNA sequence downstream of the PLNC8 operon has revealed the presenceof at least four operons involved in bacteriocin synthesis,transport, and regulation . Although these operons are very similarto those for bacteriocin production in L . plantarum C11, a newregulatory operon of the three-component type has been found.Homologous and heterologous expression of the novel inducerpeptide [PLNC8IF] of this operon supports our previous observationsin the sense that an autoinduction mechanism is activated inNC8 as a response to the presence of specific bacteria in itsown environment [34] . This observation has been confirmed byreverse transcription-PCR [RT-PCR] analysis of the expressionof the bacteriocin and regulatory operons found in NC8 . To ourknowledge, this is the first report on a quorum-sensing mechanismmediating induction of bacteriocin production after cocultivation.

 
Bacterial strains, media, and growth conditions. L . plantarum NC8, kindly provided by Lars Axelsson [MATFORSK, Norwegian Food Research Institute, Osloveien, Norway], has been described recently as producing an inducible two-peptide bacteriocin named PLNC8 in response to cocultivation with specific gram-positive bacteria [33, 34] . It was propagated in MRS broth [Oxoid, Basingstoke,Hampshire, England] at 30°C without shaking . Lactococccuslactis MG1363 was grown in M17 broth [Oxoid] plus 1% [wt/vol]glucose [GM17] at 30°C without shaking . Where appropriate,erythromycin [Fluka Chemie GmbH, Buchs, Switzerland] was addedto the culture medium at 10 µg/ml [final concentration].

Escherichia coli DH5 was grown in Luria-Bertani broth [43] at37°C with vigorous agitation . E . coli DH5 transformant cellsharboring the recombinant plasmid pBluescript II KS[+] [StratageneEurope, Amsterdam, The Netherlands] or pSIG306 [this work] wereselected on Luria-Bertani agar plates supplemented with 150µg of ampicillin [Fluka] per ml or 200 µg of erythromycin per ml [final concentration], respectively, 16 µl of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside [X-Gal; 50 mg/ml; Promega Co., Madison, Wis.] per plate, and4 µl of isopropyl ß-D-thiogalactoside [IPTG;200 mg/ml; Gibco BRL, Basel, Switzerland] per plate.

DNA isolation and transformation procedures. Total genomic DNA from L . plantarum NC8 was isolated by themethod of Cathcart [5] . Plasmid DNAs from L . plantarum and L. lactis were isolated by the method of Anderson and McKay [1]. Plasmid DNA from E . coli was extracted as described previously [43] . L . plantarum NC8 and L . lactis MG1363 were electroporatedaccording to the methods of Aukrust and Blom [2] and Holo andNes [22], respectively . E . coli DH5 was electroporated by themethod of Dower et al . [15].

Molecular cloning and Southern and colony hybridizations. Restriction enzymes, T4 DNA ligase, and other DNA-modifyingenzymes were used as recommended by the manufacturer [BoehringerMannheim, Indianapolis, Ind.].

Chromosomal DNA from L . plantarum NC8 was digested with several restriction enzymes, and the resulting fragments were separated on a 0.7% agarose gel and then blotted onto a nylon filter [Amersham Biosciences Europe GmbH, Freiburg, Germany] . After double digestion with AccI and ClaI of a previously cloned 2.6-kb AccI fragmentcontaining the plNC8BAC operon [33], an 898-bp double-strandedDNA fragment was generated [Fig. 1] . This fragment was labeledwith fluorescein 11-dUTP by using the Gene Images Random Primekit [Amersham] and subsequently used as a probe in Southernhybridization experiments . Labeling, hybridization, washing,and detection were performed as recommended by the manufacturer[Amersham].

 
In order to extend the known sequence downstream of the plNC8BAC operon, total genomic DNA from L . plantarum NC8 was digested with BclI and separated by size on 0.7% agarose gels, and DNA fragments ranging from 5 to 7 kb were ligated to the dephosphorylated pBluescript II KS[+] cloning vector digested with the same enzyme. The ligation mixture was used to transform E . coli DH5 . Thegenomic minilibrary generated in this way was screened with the double-stranded AccI/ClaI DNA probe described above by thecolony blot technique [43] . Hybridization, washing, and detectionwere performed as described above for the Southern blot . Positiveclones were identified as carrying the plasmid construct pSIG303[Table 1], their plasmid DNA was extracted, and the BclI insertwas sequenced as described below.

 
PCR and DNA sequencing. All primers used in PCRs [Table 2] were synthesized by MWG Biotech[Ebersberg, Germany] . Primers NC8-7 and NC8-10 [33] were used to amplify an L . plantarum NC8 DNA fragment containing the plNC8A gene plus part of the plNC8B gene, encoding PLNC8 . Primer PlnM-for was designed from the end part of the cloned 6.2-kb BclI fragment of NC8 chromosomal DNA [Fig . 1] . Primer NC8-17 was designedfrom the end part of the 3.7-kb PlnM-for-PlnI-rev PCR-amplifiedfragment from NC8 [Fig . 1] . Primers KpnNC8IF-for and EcoNC8IF-revwere used to amplify a 189-bp NC8 DNA fragment correspondingto the plNC8IF structural gene including a putative ribosome-bindingsite [RBS] [5'-GGAG-3'] located 12 bp upstream of that gene.To facilitate subsequent cloning, KpnI and EcoRI sites wereintroduced at the 5' and 3' ends of these primers, respectively.Primers PlnA-for, PlnA-rev, PlnH-rev, PlnI-rev, and PlnU-revwere designed based on the published DNA sequence of the plnABCDEFGHIJKLMNOPlocus of L . plantarum C11 [EMBL accession number X94434] [9,11].

 
For amplification of DNA fragments up to 3 kb, 100-µlreaction mixtures containing 2.5 mM MgCl₂, 1x reaction buffer,100 µM each deoxynucleoside triphosphate [dNTP], 100 pmolof each primer, 5 U of Taq DNA polymerase [Promega], and 250ng of genomic L . plantarum NC8 DNA as the template were usedwith a GeneAmp PCR system 2400 thermal cycler [Perkin-Elmer Corporation, Norwalk, Conn.] . Amplification included denaturation at 94°C for 4 min, followed by 30 cycles of denaturationat 94°C for 30 s, annealing at 60°C for 1 min, and polymerizationat 72°C for 1 min . For amplification of DNA fragments largerthan 3 kb, the Expand Long Template PCR system [Roche AppliedScience, Barcelona, Spain] was used according to the manufacturer's instructions . PCR amplifications used for sequencing, cloning,or gene expression were performed by using the High-FidelityPCR system [Roche] under the conditions recommended by the manufacturer.

DNA sequencing was performed by the Servicio de Secuenciación Automática de DNA, Centro de Investigaciones Biológicas,Consejo Superior de Investigaciones Cientificas [CSIC], Madrid,Spain, with an ABI PRISM 377 DNA sequencer [Perkin-Elmer AppliedBiosystems].

Plasmid construction and gene expression. Construction of pSIG303 is described above . The plNC8IF structuralgene, including its putative RBS but not its putative promoter,was amplified by PCR as described above . The corresponding 189-bp amplified product was digested with KpnI and EcoRI and ligated to KpnI-EcoRI-digested pVE3514 [Table 1], just downstream ofthe lactococcal promoter P₅₉ [49], to yield pSIG306 [Fig . 2],which was electroporated into E . coli DH5 . A 301-bp P₅₉::plNC8IFcassette was obtained by double digestion of pSIG306 with BamHIand EcoRI, and this cassette was then cloned into BamHI-EcoRI-digested pIL252 to yield pSIG308 [Fig . 2] . This plasmid was transformed into L . plantarum NC8 and L . lactis MG1363 by electroporationas described above.

 
Induction, autoinduction, and bacteriocin assays. Induction of bacteriocin production in L . plantarum NC8 canbe accomplished either [i] by coculturing with specific gram-positivebacteria [such as L . lactis MG1363], [ii] by addition of heat-killedcells from some of these inducing bacteria, or [iii] by additionof CFS showing inducing activity [33, 34].

Bacteriocin production was induced by cocultivation as follows:a 1% inoculum of L . plantarum NC8 was cultured with a 0.5% inoculum of the inducer strain L . lactis MG1363 in MRS broth, the mixed culture was held at 30°C for 6 to 8 h, and then the inhibitory activity in the CFS was assayed . Bacteriocin induction by heat-killed cells was performed by adding 0.5% of an autoclaved overnightculture of L . lactis MG1363 to a culture of L . plantarum NC8 that had just been started with a 1% inoculum of an overnightculture of this strain . The culture was incubated at 30°Cfor 6 to 8 h, and then the bacteriocin activity in the CFS wasquantitatively tested . To check for inducing activity of CFS,samples for testing, typically 10 to 50 µl, were addedto 1 ml of MRS broth containing ca . 10⁸ cells of an overnightculture of L . plantarum NC8, and the mixture was incubated for6 to 8 h at 30°C; then the resulting CFS were examined forbacteriocin activity . For a quantitative assay of the bacteriocinactivity in the CFS, we used the microtiter plate system asdescribed previously [19], with L . plantarum 128/2 as the indicatorstrain [26].

A quantitative assay of the inducing [autoinducing] activityof a CFS was carried out as described previously [34] . In all induction experiments, L . plantarum NC8 cultures were used as negative controls for both bacteriocin and inducer activities.

For gene expression studies, L . plantarum NC8 was induced as described above with either living or heat-killed cells of L. lactis MG1363 [100-ml cultures] or by addition of CFS from L. lactis MG1363[pSIG308] cultures . For the latter purpose, 300 µl of a 10-fold-concentrated CFS of MG1363[pSIG308] [seebelow] was added to a 100-ml MRS broth culture of L . plantarumNC8 [optical density at 600 nm, 0.1; ca . 10⁸ CFU/ml] and incubated at 30°C for 8 h . As a control, L . plantarum NC8 cultures without any addition [uninduced] were carried out in parallel. Samples from both induced and uninduced NC8 cultures were collected at different points along the growth curve, and the respectiveCFS were used for induction and bacteriocin assays as describedabove, while the cells were used to isolate total RNA for usein RT-PCR experiments.

The 10-fold-concentrated CFS of L . lactis MG1363[pSIG308] was obtained from a 110-ml GM17 overnight culture . Briefly, theCFS was treated with solid ammonium sulfate [476 g of culturesupernatant per liter; 75% saturation] at 4°C for 16 h withgentle agitation . The protein precipitates were collected bycentrifugation at 10,000 x g for 15 min at 4°C, and the resulting pellets were solubilized in 7.5 ml of citrate-phosphate buffer [50 mM; pH 5.0] . These 7.5-ml samples were desalted through PD10 gel filtration columns [Amersham] equilibrated with the citrate-phosphate buffer [final volume of the samples, 10.5ml] . The same protocol was used in parallel to obtain the correspondingCFS for control experiments involving pure cultures of L . plantarum NC8 or L . lactis MG1363.

Partial purification of PLNC8IF from CFS of constitutive transformant strains. In order to verify that the autoinducer peptide PLNC8IF is secretedinto the culture media of both L . plantarum NC8[pSIG308] andL . lactis MG1363[pSIG308], partial purification and mass spectrometricanalysis of the secreted peptide were carried out . For thispurpose, 2-liter cultures of each transformant strain were processedby a protocol similar to that used to purify bacteriocins fromLAB [18, 25, 33], but selecting fractions that exhibited inductionof bacteriocin production in NC8 . After several runs on a C₂-C₁₈reverse-phase column [Pharmacia Biotech, Uppsala, Sweden], sampleswere analyzed by matrix-assisted laser desorption ionization-time-of-flight [MALDI-TOF] mass spectrometry by F . Canals, Institut de Biologia Fonamental Vicent Villar Palasí, University of Barcelona,Barcelona, Spain.

RNA isolation and RT-PCR. Samples from L . plantarum or L . lactis cultures were collectedat different times along the growth curve, and RNA was extractedfrom the cell pellets by an adaptation of the method of Rayaet al . [41] . For reverse transcription, total RNA was treatedwith RNase-free DNase I [Amersham] as recommended by the manufacturer.cDNA synthesis was carried out in a 20-µl reaction volumecontaining 1 µg of total RNA, 12.5 U of avian myeloblastosisvirus reverse transcriptase [AMV-RT; Roche, Mannheim, Germany],1x AMV-RT reaction buffer, 10 pmol of antisense primer, 2 mMeach dNTP, and 5 U of RNAguard [Amersham] at 42°C for 1h . For first-strand DNA synthesis of the plNC8BA, plnEF, plnJK,and plNC8IF-plNC8HK-plnD operons, the antisense primers usedwere NC8-10, PlnF-for, PlnK-for, and NC8-22, respectively [Table 2] . After inactivation of the enzyme at 95°C for 5 min, 1 µl of each cDNA was used for PCR analysis using theprimer pairs NC8-7-NC8-10, PlnF-for-PlnE-rev, PlnK-for-PlnJ-rev,and IFNC8-for-EcoIFNC8 to detect the plNC8BA, plnEF, plnJK, and plNC8IF-plNC8HK-plnD operons, respectively . The annealing temperature was 58°C for all PCRs . The resulting amplifiedDNA fragments were resolved on 10% acrylamide gels [43] . Control reactions using the same primer pairs prior to reverse transcription were used to check for the absence of contaminating chromosomal DNA.

Nucleotide sequence accession number. The contiguous 8,107-bp DNA sequence downstream of the PLNC8operon has been assigned GenBank accession number AF522077.

 
DNA sequence and genetic analysis of the PLNC8 locus in L . plantarum NC8. To extend the known DNA sequence downstream of the PLNC8 operon [33], we cloned and sequenced a 6.2-kb BclI L . plantarum NC8chromosomal DNA fragment [Fig . 1] that hybridized with a labeledprobe containing the plnc8BAC operon, thus obtaining as muchas 2.84 kb of novel sequence . Analysis of this sequence revealedfive open reading frames [ORFs], three of which were identicalto the plnR, plnK, and plnJ genes from the plantaricin locusof L . plantarum C11, while the other two showed high homology,respectively, with the plnL and plnM genes from the same locus[Table 3] . On the basis of this knowledge, several primers weredesigned either from the novel NC8 DNA sequence or from thepublished C11 sequence [Table 2] . Hence, to scan for possible correspondences between the plantaricin C11 and PLNC8 loci,primer plnM-for was used in PCRs in combination with other primersdesigned by us from the DNA sequence of the plantaricin locus[plnABCDEFGHIJKLMNOP] of L . plantarum C11 . As a result, a 3.7-kbDNA fragment, which overlapped with the end of the BclI fragment,was amplified with primer pair plnM-for-plnI-rev and entirelysequenced [Fig. 1] . On the basis of this new sequence, we designed the NC8-17 primer [Table 2], which was combined with primer plnH-rev in new PCRs . These PCRs resulted in an amplified DNA fragment of 5 kb [Fig . 1], from which 1,681 bp was sequenced. In summary, we have obtained a contiguous 8,107-bp DNA sequence downstream of the PLNC8 operon which has been assigned GenBank accession number AF522077 . Furthermore, several other PCRs werecarried out to check for correspondence with the C11 operon.From these PCRs we concluded that the plnG, plnH, plnS, plnT, and plnU genes from C11 were also present in NC8, as shown in Fig . 1.

 
The extended L . plantarum NC8 DNA sequence of the PLNC8 operon revealed the presence of 12 ORFs which seem to be organizedinto four putative operons [Fig . 1] . Despite the high degree of homology of most of these ORFs with genes in the plantaricinlocus in L . plantarum C11, two major differences between the bacteriocin operons in C11 and NC8 are noteworthy: [i] a 1.6-kb deletion in L . plantarum NC8 between plnM and plnP and [ii] a 3.5-kb deletion in NC8 between plnP and plnD [Fig. 1] . Consequently,plnN, plnO, plnA, plnB, plnC, and orf1 from C11 are not presentin NC8 . On the other hand, two novel ORFs between plnP and plnD were found in NC8 and were designated plNC8IF and plNC8HK [Fig.1] . These two genes plus plnD [Fig. 1] seem to be organizedas a three-component regulatory operon equivalent to the plnABCDregulatory operon in C11 [9], as discussed below.

The regulatory operon in L . plantarum NC8. In NC8, the regulatory operon for bacteriocin production startswith plNC8IF, which encodes a putative peptide of 49 amino acid residues, showing all the features described for inducer peptides[36] . As with bacteriocin-like peptides, the product of plNC8IF possesses a leader sequence of the double-glycine type that,once processed, gives rise to a mature peptide of 28 amino acidresidues, with a theoretical pI of 13.0 and a molecular weight[MW] of 3,015 . No homology with any known protein in the databaseswas found for the mature PLNC8IF.

Just 14 bp downstream of plNC8IF we found plNC8HK, which encodesa putative protein of 446 amino acid residues with a predictedpI of 5.34 and an MW of 51,053, showing significant homologywith the family of the HKs [20] . Actually, the highest similarityfor PLNC8HK [34% identity] was obtained with PlnB, the HK ofthe plantaricin locus from L . plantarum C11 [9] . Immediatelydownstream of plNC8HK, we found plnD . The putative protein encodedby plnD was 98% identical to the RR PlnD of L . plantarum C11 [9] . Therefore, based on homology and relative position, the plNC8IF-plNC8HK-plnD gene cluster seems to form a regulatory operon of the so-called three-component type, comprising an autoinducer peptide [PLNC8IF], an HK [PLNC8HK], and an RR [PLND]. Interestingly, this regulatory operon is located between plnP and plnI in L . plantarum NC8, in a position analogous to that of the regulatory operon plnABCD in L . plantarum C11 [Fig. 1].In L . plantarum NC8, however, the RR PlnC is not present.

Analysis of the DNA region upstream of plNC8IF revealed the presence of a putative promoter that resembles the regulatedpromoter of plnA [Fig . 3] as well as other bacteriocin promoters from LAB whose expression is regulated via a three-component regulatory system [11, 36] . Therefore, certain DNA motifs areconserved: the L and R repeats are virtually identical to thoseof plnA, as is the -10 region [Fig. 3] . Downstream of plnD,a rho-independent transcription terminator was found, suggestingthat the plNC8IF, plNC8HK, and plnD genes would be cotranscribed,as reported below.

 
Homologous and heterologous expression of plNC8IF. In order to determine whether the product of plNC8IF is in fact an IF for bacteriocin production in L . plantarum NC8, homologous and heterologous expression of this gene was performed . For this purpose, we fused the plNC8IF gene [including its putative RBS] to the constitutive lactococcal promoter P₅₉, producing plasmid pSIG306 [Fig . 2] . This fusion was subsequently transferredinto pIL252 to yield pSIG308 [Fig . 2], which was then establishedin L . plantarum NC8 . When NC8 transformant cells were testedfor bacteriocin production in broth cultures, all of them producedbacteriocin constitutively [Table 4] . In contrast, in controlexperiments, L . plantarum NC8 with or without pIL252 did notproduce bacteriocin at all under the same conditions [Table4] . These findings suggested that the product of plNC8IF induces bacteriocin production in NC8 and that its expression must be regulated . MALDI-TOF mass spectrometry analysis of partiallypurified PLNC8IF from L . plantarum NC8[pSIG308] CFS showed apeak corresponding to a peptide of 3,029.66 Da, i.e., 16 Damore than the theoretical molecular size of the processed peptidePLNC8IF [3,013.71 Da] . This difference can be explained by anoxidized methionine in the mature PLNC8IF . These results, togetherwith the fact that no signal corresponding to the unprocessedPLNC8IF peptide was detected in MALDI-TOF mass spectrometryanalysis [theoretical molecular size, 5,401.0 Da], stronglyindicate that PLNC8IF is effectively exported out of the pSIG308-transformedNC8 cells in its mature form after cleavage at the double-glycinemotif.

 
Transformants of L . lactis MG1363 carrying pSIG308 were analyzed for their ability to produce and export PLNC8IF to the culture medium . Addition of CFS from L . lactis MG1363[pSIG308] to L. plantarum NC8 cultures resulted in induction of bacteriocin production [Table 4], indicating that PLNC8IF was being producedand exported . In order to corroborate the presence of PLNC8IFin the L . lactis MG1363[pSIG308] CFS and to check whether theproduct of the plNC8IF gene had been processed in such a heterologousstrain, MALDI-TOF mass spectrometry analysis of fractions exhibitinginduction of bacteriocin production in NC8 was carried out.Two peaks corresponding to peptides of 3,013.70 and 3,029.70Da were detected, corresponding to the PLNC8IF mature peptideand to the same peptide with an oxidized methionine, respectively.This finding indicates that L . lactis MG1363 was able to processand export mature PLNC8IF to the culture medium, as was theoriginal NC8 strain . No bacteriocin activity was detected in L . lactis MG1363[pSIG308] CFS [Table 4] . Finally, CFS from MG1363or MG1363[pIL252] cultures did not induce bacteriocin productionin L . plantarum NC8 or show any bacteriocin activity [Table4].

Coculture with inducing cells induces transcription of the bacteriocin and regulatory operons in L . plantarum NC8. To confirm at the molecular level that exposure of NC8 to inducingcells results in expression of its bacteriocin genes and ofthe PLNC8IF [regulatory] operon, we studied the expression ofall these genes . Total RNA was isolated from samples collectedat different points of the growth phase from NC8 cultures whichhad been cocultured with the inducer strain L . lactis MG1363.As shown in Fig. 4, cocultivation with MG1363 induced in NC8the expression of the structural genes for the three two-peptide bacteriocins detected in the NC8 chromosome—PLNC8 [plNC8Aand plNC8B [Fig . 4A]], plantaricin EF [plnE and plnF [Fig . 4B]],and plantaricin JK [plnJ and plnK [Fig . 4C]]-at the early-exponential phase of growth . Bacteriocin activity was detected in the CFSof all these samples, with titers ranging from 640 to 1,280bacteriocin units [BU]/ml . Control experiments involving purecultures of NC8 showed no transcription of these genes [Fig.4], and no bacteriocin activity was found in their CFS at anytime . On the other hand, cocultivation with MG1363 resultedin induction of the putative regulatory operon plNC8IF-plNC8HK-plnDin L . plantarum NC8 [Fig . 4D] . In contrast to the bacteriocin operons, a basal level of transcription for this regulatory operon was detected in some samples of NC8 growing as a pureculture [Fig . 4D].

 
The same transcriptional analysis was carried out using autoclaved L . lactis MG1363 cells to induce bacteriocin production in NC8, and virtually the same transcriptional pattern as that for induction with living MG1363 cultures was detected [Fig . 4].

Finally, PCR experiments involving relevant primer pairs failedto detect in L . lactis MG1363 DNA any of those operons expressed in L . plantarum NC8.

PLNC8IF induces transcription of all the bacteriocin operons in NC8 as well as its own [regulatory] operon. The role of PLNC8IF as an inducer of bacteriocin productionin NC8 as well as an autoinducer of its own synthesis was shownat the molecular level by studying the expression of the relevantoperons when exposed to an external source of PLNC8IF . For thispurpose, total RNAs were isolated from samples collected atdifferent points of the growth phase from NC8 cultures thathad been induced by addition of a 10-fold-concentrated CFS from an L . lactis MG1363[pSIG308] culture, containing PLNC8IF at 10,240 IU/ml . As shown in Fig . 4, addition of PLNC8IF to NC8cultures results in induction of the expression of all the bacteriocinstructural genes in NC8 [plNC8A and plNC8B [Fig . 4A], plnE andplnF [Fig. 4B], and plnJ and plnK [Fig . 4C]] during the exponentialphase of growth, as expected from the induction by cocultivationdescribed above . Moreover, a PLNC8IF-rich CFS was able to inducethe expression of the plNC8IF-plNC8HK-plnD operon, i.e., itsown synthesis, in NC8 cultures . These findings constitute evidencethat the product of plNC8IF is an autoinducer molecule.

 
With this work we have gained a better knowledge of the regulationof bacteriocin production genes whose expression is dependenton an external stimulus, such as cocultivation with other strains.Thus, for L . plantarum NC8, a quorum-sensing mechanism is predicted to be the actual mediator between the external stimulus providedby the presence of specific bacterial strains in the same culturemedium and the production of bacteriocins.

According to our results, the presence of either living or heat-killed inducing cells promotes expression of the three bacteriocin operons in NC8 [plNC8BA, plnEF, and plnJK] [Fig. 4A to C] . Thisis readily detected by the bacteriocin activity found in thecorresponding CFS . Actually, from induced NC8 CFS, we purifiedand determined the amino acid sequence of plantaricin F as wellas MALDI-TOF chromatographic peaks corresponding to peptideswith MWs identical to those of the mature proteins encoded byplnE, plnJ, and plnK [data not shown] . Therefore, to our knowledge,NC8 is the strain of LAB naturally producing the largest numberof different bacteriocin peptides [PLNC8, PLNC8ß,PlnE, PlnF, PlnJ, and PlnK] described to date.

Concomitantly with bacteriocin production, the same stimulus[the inducing cells] is able to activate the expression of anoperon [plNC8IF-plNC8HK-plnD] that exhibits all the featuresof a three-component regulatory system operating via a quorum-sensingmechanism . Actually, a similar regulatory operon [plnABCD] hasbeen described for the bacteriocin cluster in L . plantarum C11,whose expression is dependent on the inoculum size [10, 11]. Interestingly, though, the two regulatory operons possess completely different IFs [the products of plnA and plNC8IF for C11 and NC8, respectively], which have in common only certain conserved motifs at the leader sequences and conserved regions at the promoters: L and R repeats [Fig . 3] . In fact, these direct repeatshave been shown to play an essential role in the activation-repressionof the regulatory promoter itself by specific RRs encoded inthe same operon: plnC and plnD in C11 [13, 14] . However, onlyplnD is present in NC8 . Overexpression of plnD in its homologoushost C11 had a repressive effect on both bacteriocin productionand its own [plnABCD] transcription [14] . However, this repressive effect could be quite different on a different genetic background,in a nonoverexpressed dose, and in the absence of the relatedgene plnC, as is the case in L . plantarum NC8 . On the other hand, it is known that in order to obtain the [auto]induction phenomenon, the IF interacts with its specific HK, which subsequently activates its cognate RR [4, 17, 36].

The putative gene for HK in NC8 [plNC8HK] shows quite different homologies at the amino- and carboxy-terminal sequences: while the N terminus shows just 21% identity with the HKs from other bacteriocin regulatory operons [plnB for plantaricin C11 [9] and sppK for sakacin P [23]], the carboxy terminus shows upto 47% identity with other bacteriocin HKs and kinases fromthe HPK10 group [20] . Prediction models for the topology ofthe HKs suggest that they possess two structural domains [36].The external domain [located at the N-terminal part] would beresponsible for recognizing the specific IF, while the internaldomain [C-terminal part] would be able to activate the RR byphosphorylation . Hence, it is not surprising that the N-terminalpart of the protein encoded by plNC8HK is unrelated, becauseit has to recognize a specific molecule, its own autoinducer[PLNC8IF] . On the other hand, its C-terminal part is quite conserved,because it has a common function: to phosphorylate an RR [PlnD].In turn, this RR has to recognize specific sequences at theregulated promoters, including the promoter governing its ownsynthesis [14, 39, 42, 47] . Accordingly, conserved L and R repeatscan be found at the promoter regions of all the bacteriocin and regulatory operons in NC8 . This constitutes evidence that those genes are regulated by the expression of plnD . In summary, this is the first report of interchangeable IFs [PLNC8IF and PlnA] acting through the same RR and bacteriocin genes.

Constitutive expression of PLNC8IF by L . plantarum NC8 or addition of a PLNC8IF-containing CFS to an NC8 culture results in bacteriocin and autoinducer production by this strain, in agreement with the model that PLNC8IF mediates the induction provided by the inducing cells . This observation was confirmed by analysis ofthe RT-PCR experiments . Thus, addition of purified PLNC8IF promotedthe expression of the bacteriocin and regulatory operons inNC8 in the same way as the presence of either living or heat-killedinducer cells does . A basal level of transcription was detectedfor the plNC8IF-plNC8HK-plnD regulatory operon, in agreementwith previous observations regarding the need for maintenanceof a minimum level of regulatory proteins so that the cell cansense the external IF concentration and respond to it at once[3].

On the other hand, as predicted by the DNA sequence analysis, mature PLNC8IF results after cleavage at the double-glycinemotif in L . plantarum NC8, as well as in the heterologous hostL . lactis MG1363, according to the MALDI-TOF analyses . This observation corroborates our previous results, for during aminoacid sequencing of PLNC8 by the Edman degradation method, someof the samples assayed showed sequences corresponding to maturePLNC8IF . In addition, it has been demonstrated that PLNC8IFdoes not exhibit any bacteriocin activity by itself.

In conclusion, we propose that the presence of specific bacteria acts as an environmental signal to switch bacteriocin productionon in L . plantarum NC8 . A quorum-sensing mechanism mediatedby PLNC8IF appears to be involved in this process . In addition,L . plantarum NC8 offered the exceptional opportunity of studyinghow a particular IF [i.e., PLNC8IF] is able to autoinduce aregulatory system as well as to regulate the expression of aseries of bacteriocin operons which had previously been describedas being governed by a different autoinducer [plantaricin A,in C11] . Both PLNC8IF and PlnA need specific HKs [encoded byplNC8HK and plnK in NC8 and C11, respectively] due to theirhigh ligand specificities, but they both act through the sameRR [plnD], provided that consensus motifs [L and R repeats]are present at the promoter sequences of the operons to be regulated.

 
This work was supported by the Spanish Government through MCYT project AGL2000-1611-CO3-01 . A.M . was the recipient of a grantfrom MCYT, Madrid, Spain.

We thank Belén Caballero-Guerrero for excellent technical assistance.

 
* Corresponding author . Mailing address: Departamento de Biotecnología de Alimentos, Instituto de la Grasa [CSIC], Avda . Padre García Tejero, 4, Aptdo . 1078, 41012 Seville, Spain . Phone: 34 54 69 08 50 . Fax: 34 54 69 12 62 . E-mail: jlruiz@cica.es.

1. Anderson, D . G., and L . L . McKay. 1983 . Simple and rapid method for isolating large plasmid DNA from lactic streptococci . Appl . Environ . Microbiol . 46:549-552.
2. Aukrust, T., and H . Blom. 1992 . Transformation of Lactobacillus strains used in meat and vegetable fermentations . Food Res . Int . 25:253-261.
3. Axelsson, L., and A . Holck. 1995 . The genes involved in production of and immunity to sakacin A, a bacteriocin from Lactobacillus sake Lb706 . J . Bacteriol . 177:2125-2137.
4. Brurberg, M . B., I . F . Nes, and V . G . H . Eijsink. 1997 . Pheromone-induced production of antimicrobial peptides in Lactobacillus. Mol . Microbiol . 26:347-360.
5. Cathcart, D . P. 1995 . Purification, characterization and molecular analysis of plantaricin S, a two-peptide bacteriocin from olive fermenting Lactobacillus plantarum strains . Ph.D . thesis . Cranfield University, Bedford, United Kingdom.
6. Chen, J.-D., and D . A . Morrison. 1988 . Construction and properties of a new insertion vector, pJDC9, that is protected by transcriptional terminators and useful for cloning of DNA from Streptococcus pneumoniae. Gene 64:155-164.
7. Daeschel, M . A. 1993 . Applications and interactions of bacteriocins from lactic acid bacteria in foods and beverages, p . 63-91 . In D . G . Hoover and L . R . Steenson [ed.], Bacteriocins of lactic acid bacteria . Academic Press, Inc., New York, N.Y.
8. de Vuyst, L., and E . J . Vandamme. 1994 . Antimicrobial potential of lactic acid bacteria, p . 91-142 . In L . de Vuyst and E . J . Vandamme [ed.], Bacteriocins of lactic acid bacteria: microbiology, genetics and applications . Blackie Academic & Professional, London, United Kingdom.
9. Diep, D . B., L . S . Håvarstein, J . Nissen-Meyer, and I . F . Nes. 1994 . The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an agr-like regulatory system . Appl . Environ . Microbiol . 60:160-166.
10. Diep, D . B., L . S . Håvarstein, and I . F . Nes. 1995 . A bacteriocin-like peptide induces bacteriocin synthesis in Lactobacillus plantarum C11 . Mol . Microbiol . 18:631-639.
11. Diep, D . B., L . S . Håvarstein, and I . F . Nes. 1996 . Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11 . J . Bacteriol . 178:4472-4483.
12. Diep, D . B., L . Axelsson, C . Grefsli, and I . F . Nes. 2000 . The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component regulatory system . Microbiology 146:2155-2160 .
13. Diep, D . B., O . Johnsborg, P . A . Risøen, and I . F . Nes. 2001 . Evidence for dual functionality of the operon plnABCD in the regulation of bacteriocin production in Lactobacillus plantarum. Mol . Microbiol . 41:633-644.
14. Diep, D . B., R . Myhre, O . Johnsborg, Å . Aakra, and I . F . Nes. 2003 . Inducible bacteriocin production in Lactobacillus is regulated by differential expression of the pln operons and by two antagonizing response regulators, the activity of which is enhanced upon phosphorylation . Mol . Microbiol . 47:483-494.
15. Dower, W . J., F . J . Miller, and W . C . Ragsdale. 1998 . High efficiency transformation of E . coli by high voltage electroporation . Nucleic Acids Res . 16:6127-6145.
16. Eijsink, V . G . H., M . B . Brurberg, P . H . Middelhoven, and I . F . Nes. 1996 . Induction of bacteriocin production in Lactobacillus sake by a secreted peptide . J . Bacteriol . 178:2232-2237.
17. Eijsink, V . G . H., L . Axelsson, D . B . Diep, L . S . Håvarstein, H . Holo, and I . F . Nes. 2002 . Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication . Antonie Leeuwenhoek 81:639-654.
18. Floriano, B., J . L . Ruiz-Barba, and R . Jiménez-Díaz. 1998 . Purification and genetic characterization of enterocin I from Enterococcus faecium 6T1a, a novel antilisterial plasmid-encoded bacteriocin which does not belong to the pediocin family of bacteriocins . Appl . Environ . Microbiol . 64:4883-4890 .
19. Geis, A., J . Singh, and M . Teuber. 1983 . Potential of lactic streptococci to produce bacteriocin . Appl . Environ . Microbiol . 45:205-211.
20. Grebe, T . W., and J . B . Stock. 1999 . The histidine protein kinase superfamily . Adv . Microb . Physiol . 41:139-227.
21. Håvarstein, L . S., D . B . Diep, and I . F . Nes. 1995 . A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export . Mol . Microbiol . 16:229-240.
22. Holo, H., and I . F . Nes. 1995 . Transformation of Lactococcus by electroporation . Methods Mol . Biol . 47:195-199.
23. Hühne, K., A . Holck, L . Axelsson, and L . Krockel. 1996 . Analysis of the sakacin P gene cluster from Lactobacillus sake Lb674 and its expression in sakacin-negative Lb . sake strains . Microbiology 142:1437-1448.
24. Jack, R., J . R . Tagg, and B . Ray. 1995 . Bacteriocins of Gram-positive bacteria . Microbiol . Rev . 59:171-200.
25. Jiménez-Díaz, R., R . M . Rios-Sánchez, M . Desmazeaud, J . L . Ruiz-Barba, and J . C . Piard. 1993 . Plantaricin S and T, two new bacteriocins produced by Lactobacillus plantarum LPCO10 isolated from a green olive fermentation . Appl . Environ . Microbiol . 59:1416-1424.
26. Jiménez-Díaz, R., J . L . Ruiz-Barba, D . P . Cathcart, H . Holo, I . F . Nes, K . H . Sletten, and P . J . Warner. 1995 . Purification and partial amino acid sequence of plantaricin S, a bacteriocin produced by Lactobacillus plantarum LPCO10, the activity of which depends on the complementary action of two peptides . Appl . Environ . Microbiol . 61:4459-4463.
27. Klaenhammer, T . R. 1993 . Genetics of bacteriocins produced by lactic acid bacteria . FEMS Microbiol . Rev . 12:39-86.
28. Kleerebezem, M., L . E . N . Quadri, O . P . Kuipers, and W . M . de Vos. 1997 . Quorum sensing by peptide pheromones and two-component signal-transduction systems in gram-positive bacteria . Mol . Microbiol . 24:895-904.
29. Kleerebezem, M., and L . E . N . Quadri. 2001 . Peptide pheromone-dependent regulation of antimicrobial peptide production in Gram-positive bacteria: a case of multicellular behavior . Peptides 22:1579-1596.
30. Kleerebezem, M., O . P . Kuipers, W . M . de Vos, M . E . Stiles, and L . E . N . Quadri. 2001 . A two-component signal transduction cascade in Carnobacterium piscicola LV17B: two signaling peptides and one sensor-transmitter . Peptides 22:1597-1601.
31. Kleerebezem, M., J . Boekhorst, R . van Kranenburg, D . Molenaar, O . P . Kuipers, R . Leer, R . Tarchini, S . A . Peters, H . M . Sandbrink, M . W . Fiers, W . Stiekema, R . M . Lankhorst, P . A . Bron, S . M . Hoffer, M . N . Groot, R . Kerkhoven, M . de Vries, B . Ursing, W . M . de Vos, and R . J . Siezen. 2003 . Complete genome sequence of Lactobacillus plantarum WCFS1 . Proc . Natl . Acad . Sci . USA 100:1990-1995 .
32. Le Loir, Y., A . Gruss, S . D . Ehrlich, and P . Langella. 1998 . A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis. J . Bacteriol . 180:1895-1903 .
33. Maldonado, A., J . L . Ruiz-Barba, and R . Jiménez-Díaz. 2003 . Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8 . Appl . Environ . Microbiol . 69:383-389 .
34. Maldonado, A., R . Jiménez-Díaz, and J . L . Ruiz-Barba. 2004 . Production of plantaricin NC8 by Lactobacillus plantarum NC8 is induced in the presence of different types of Gram-positive bacteria . Arch . Microbiol . 181:8-16.
35. Nes, I . F., D . B . Diep, L . S . Håvarstein, M . B . Brurberg, V . Eijsink, and H . Holo. 1996 . Biosynthesis of bacteriocins in lactic acid bacteria . Antonie Leeuwenhoek 70:113-128.
36. Nes, I . F., and V . G . H . Eijsink. 1999 . Regulation of group II peptide bacteriocin synthesis by quorum sensing mechanisms, p . 175-192 . In G . M . Dunny and S . C . Winans [ed.], Cell-cell signaling in bacteria . American Society for Microbiology, Washington, D.C.
37. Nilsen, T., I . F . Nes, and H . Holo. 1998 . An exported inducer peptide regulates bacteriocin production in Enterococcus faecium CTC492 . J . Bacteriol . 180:1848-1854 .
38. O'Keeffe, T., C . Hill, and R . P . Ross. 1999 . Characterization and heterologous expression of the genes encoding enterocin A production, immunity, and regulation in Enterococcus faecium DPC1146 . Appl . Environ . Microbiol . 65:1506-1515 .
39. Parkinson, J . S. 1993 . Signal transduction schemes in bacteria . Cell 73:857-871.
40. Quadri, L . E . N., M . Kleerebezem, O . P . Kuipers, W . M . De Vos, K . L . Roy, J . C . Vederas, and M . E . Stiles. 1997 . Characterization of a locus from Carnobacterium piscicola LV17B involved in bacteriocin production and immunity: evidence for global inducer-mediated transcriptional regulation . J . Bacteriol . 179:6163-6171.
41. Raya, R., J . Bardowski, P . S . Andersen, S . D . Ehrlich, and A . Chopin. 1998 . Multiple transcriptional control of the Lactococcus lactis trp operon . J . Bacteriol . 180:3174-3180.
42. Risøen, P . A., M . B . Brurberg, V . G . H . Eijsink, and I . F . Nes. 2000 . Functional analysis of promoters involved in quorum sensing-based regulation of bacteriocin production in Lactobacillus. Mol . Microbiol . 37:619-628.
43. Sambrook, J., E . F . Fritsch, and T . Maniatis. 1989 . Molecular cloning: a laboratory manual, 2nd ed . Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
44. Saucier, L., A . Poon, and M . E . Stiles. 1995 . Induction of bacteriocin in Carnobacterium piscicola LV17 . J . Appl . Bacteriol . 78:684-690.
45. Saucier, L., A . S . Paradkar, L . S . Frost, S . E . Jensen, and M . E . Stiles. 1997 . Transcriptional analysis and regulation of carnobacteriocin production in Carnobacterium piscicola LV17 . Gene 188:271-277.
46. Simon, D., and A . Chopin. 1988 . Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis . Biochimie 70:559-566.
47. Stock, J . B., A . M . Stock, and J . M . Mottonen. 1990 . Signal transduction in bacteria . Nature 334:395-400.
48. Tagg, J . R., A . S . Dajani, and L . W . Wannamaker. 1976 . Bacteriocins of gram-positive bacteria . Bacteriol . Rev . 40:722-756.
49. van der Vossen, J . M . B . M., D . van der Lelie, and G . Venema. 1987 . Isolation and characterization of Streptococcus cremoris Wg2-specific promoters . Appl . Environ . Microbiol . 53:2452-2457.

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