Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Development of a PCR-based assay for rapid detection of class IIa bacteriocin genes
Micha Wieckowicz, Marcin Schmidt, Anna Sip and Wodzimierz Grajek
Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, Poznan, Poland

Keywords artisanal cheese, class IIa bacteriocins, PCR, primer design, sequence alignment. Correspondence Micha Wieckowicz, Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, 60-627 Poznan, Poland. E-mail: wieckom@up.poznan.pl

Abstract Aims: We have developed a PCR-based assay using custom designed panel of primers which allows rapid detection of class IIa bacteriocin-coding genes. To demonstrate the applicability of the developed assay, the method was applied on 40 metagenomic DNA preparations isolated from native microbiota of Polish artisanal cheeses produced in the Tatra Mountains. Methods and Results: The developed assay was designed on the basis of a large scale alignment of class IIa bacteriocin-coding genes. A panel of seven primer pairs with conrmed ability to detect class IIa bacteriocin-coding sequences was obtained. The following study has revealed a superb bacteriocinogenic potential of all forty analysed cheese samples. Conclusions: The majority of obtained sequences were lactic acid bacteria (LAB) related, although some sequences showed signicant similarity to bacteriocin-coding sequences present in non-LAB bacteriocin producers. The results suggest that several potentially new bacteriocin-coding sequences were found. Signicance and Impact of the Study: The developed assay can be extremely helpful in establishing whether isolates from the environment of interest have a potential of synthesizing antilisterial class IIa bacteriocins. Application of the approach may represent a useful tool contributing to ecological studies looking for valuable probiotic, bacteriocinogenic microbiota developing in foods.

2010 1467: received 25 August 2010, revised 17 December 2010 and accepted 19 December 2010
doi:10.1111/j.1472-765X.2010.02999.x

Introduction Bacteriocins are proteinaceous compounds exerting bactericidal or bacteriostatic mode of action against bacteria usually closely related to the producer organism (Rodrguez et al. 1995). In fermented foods, lactic acid bacteria (LAB) display numerous antimicrobial activities triggered by generally recognized as natural compounds, able to inuence the safety and quality of foods. LAB play an essential role in the majority of food fermentations, and a wide variety of strains are routinely employed as starter cultures in the manufacture of dairy, meat, vegetable and bakery products. This is mainly due to the production of organic acids, but also of other compounds, such as bacteriocins and antifungal peptides. Several bacteriocins with an industrial potential have been puried and characterized (De Vuyst and Leroy 2007). The major classes of bacteriocins produced by LAB include: (I) lantibiotics, (II) small heat stable peptides,

(III) large heat labile proteins and (IV) complex proteins whose activity requires the association of carbohydrate or lipid moieties (Klaenhammer 1993). The continuous search for bacteriocin-producing LAB has then been directed towards exploration of substances targeting Listeria monocytogenes because of recurring and serious listeriosis outbreaks observed in the recent years (Farber and Peterkin 1991). Bacteriocins with such activity are classied mainly in the class II, with reports clearly showing that they are predominantly class IIa bacteriocins. The class IIa consists of peptides characterized by strong similarity of amino acid sequence within their N-terminal region and, what has been proved by many results, with a characteristic strong antilisterial activity. Particularly all class IIa bacteriocins identied so far are highly active against Listeria strains (Heng et al. 2007b), while only part of bacteriocins of other categories are antilisterial (Ennahar and Deschamps 2000). The highly promising results of many studies underline the impor281

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tant role of functional, bacteriocinogenic LAB strains which they may play in the food industry as starter cultures, co-cultures or bioprotective cultures, to improve food quality and safety (Abee et al. 1995). Furthermore, there is increasing interest in isolating new bacteriocinproducing strains of foodborne origin that could be developed for bioprotective and probiotic effects. We have developed a PCR-based assay using a custom designed panel of bacteriocin-specic degenerate primers, which allow the detection of class IIa bacteriocin coding genes by targeting a conserved sequence motif, typical for all antilisterial bacteriocin coding genes. This nucleotide motif corresponds to the conserved N-terminal amino acid sequence, present in mature bacteriocin molecules (peptides). Initially, we have compared the nucleotide sequences of all antilisterial bacteriocin coding genes from several bacterial species available in Entrez Nucleotide Database (http://www.ncbi.nlm.nih.gov/nuccore) and designed PCR primers derived from the highly conserved regions. The presented assay allows examination of either starter cultures, co-cultures or consortia of strains isolated from food products for the presence of class IIa bacteriocincoding genes using PCR analysis of total DNA extracts. The presented approach can be applied prior to performing the antimicrobial activity detection using the agar-spot method and further subsequent testing by the welldiffusion assay, especially when little is known about the microbiota inhabiting a particular ecosystem of interest. To demonstrate a potential application of the developed assay presented in this article, metagenomic DNA derived from microbiota of 40 artisanal cheese products made in Polish Tatra Mountains were subjected to screening for class IIa bacteriocin-coding genes. Our goal was to obtain information about whether the complex bacterial community that develops in Polish artisanal cheeses has a potential to produce class IIa bacteriocins. Materials and methods Bacterial strains and culture conditions Three LAB strains obtained from the American Type Culture Collection (ATCC) and one strain from the DepartTable 1 Bacteriocin-producing strains used as reference

ment of Biotechnology and Food Microbiology (Poznan University of Life Sciences) own collection were used in this study as reference strains for the analysis of the designed primer panel. All of these strains, listed in Table 1, have a documented ability to synthesize class IIa antilisterial bacteriocins. The freeze-dried cultures obtained from ATCC were regenerated and cultivated according to manufacturers instructions. The Enterococcus faecium ATCC 19434 culture was spread plated onto BHI agar and incubated at 37C for 72 h. The Leuconostoc mesenteroides ATCC 10830a, Lactobacillus plantarum ATCC 14917 and Carnobacterium divergens V41 cultures were spread plated onto MRS agar and incubated at for 72 h at 26C (Leuc. mesenteroides and Lact. plantarum) and 30C (C. divergens). Well-developed individual colonies were picked and transferred into BHI or MRS broth and incubated at appropriate temperature for 24 h. Multiple sequence alignment of class IIa bacteriocincoding genes and primers design AlignX application, a Vector NTI Advance 11 (Invitrogen) module based on the popular ClustalW algorithm was used to perform multiple sequence alignments (Lu and Moriyama 2004). Primers used in this study were designed manually regarding the multiple sequence alignment results. Nucleotide sequences of 24 bacteriocin-coding genes were obtained from Entrez Nucleotide Database and aligned using Vector NTI software. PCR optimization, cloning and sequencing of PCR products Total DNA of the four reference strains was isolated using Genomic Mini DNA Isolation Kit (A&A Biotechnology, Gdynia, Poland). The PCR reactions were set up in a total volume of 20 ll containing 05 U DyNazyme Polymerase (Finnzymes), 4 mmol l)1 magnesium chloride, 02 mmol l)1 dNTPs (Finnzymes), 1 lmol l)1 of each primer and 100 ng of the template DNA. Amplication conditions were as follows: initial denaturation 5 min at 94C, 30 cycles of denaturation 45 s at 94C, annealing 45 s at 4456C gradient and elongation 1 min at 72C

Micro-organism Carnobacterium divergens V41 Enterococcus faecium Leuconostoc mesenteroides ssp. mesenteroides FR52 Lactobacillus plantarum

Collection Own collection ATCC 19434 ATCC 10830a ATCC 14917

Entrez nucleotide database accession number CAA11804.1 AAC45870.1 AAP37395.1 AAL09346.1

Bacteriocin produced Divercin V41 Enterocin P Mesentericin Y105 Bacteriocin 423

Reference Metivier et al. (1998) Herranz et al. (2001) Aucher et al. (2004) Van Reenen et al. (2003)

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plus a nal extension step 5 min at 72C. Agarose gel electrophoresis of the PCR products was performed on a 3% high resolution agarose gel (Merck, Warsaw, Poland). To conrm the specicity of the designed primer panel and identity of PCR amplicons, the corresponding bands were excised from an agarose gel, puried by using the Gel-Out Agarose Purication Kit (A&A Biotechnology), cloned into pGEM-T Easy Vector (Promega) and subjected to sequencing analysis (performed by Genomed, Poland). Homologies of the obtained sequences were searched in the NCBI database using Blast service (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Preparation of cheese samples Forty cheese samples obtained from different producers located in Polish Tatra Mountains were used in this study to evaluate the bacteriocinogenic potential of their native microbiota. Detailed characteristics of the cheeses used in this study is provided in Table 2. Each sample of cheese (10 g) was aseptically grated and emulsied in sterile 2% (w v) trisodium citrate. Dilutions were made in TE buffer and stored until isolation of metagenomic DNA. Metagenomic DNA isolation, PCR analysis and cloning Forty metagenomic DNA isolates of the microbiota derived from the analysed cheese samples were screened for the presence of class IIa bacteriocin-coding genes using a PCR-based assay developed by the authors. This experiment involved seven primer pairs which specicity was conrmed earlier. Prior to DNA isolation, cheese microbiota were cultivated overnight in basal broth to enrich the cultures. Total DNA was isolated using Genomic Mini DNA Isolation Kit (A&A Biotechnology). The

PCR reactions were set up as previously described with a primer annealing temperature estimated at 44C. Agarose gel electrophoresis of the obtained PCR products was performed on a 3% high resolution agarose gel (Merck). The corresponding bands were excised from the gel, puried and cloned as described previously. Sequence analysis and search for homologies of the obtained sequences were performed as described above. Results Primers design Initially, 44 DNA sequences of class IIa bacteriocin-coding genes were obtained from Entrez Nucleotide Database and processed with the Vector NTI Molecular Viewer module for alignment purposes. The results of the general alignment revealed that many identical sequences are deposited in the database under different denotations and accession numbers. The analysis of phylogenetic trees generated by AlignX software allowed us to select 24 unique sequences of class IIa bacteriocin-coding genes with relevant sequence dissimilarities and those sequences were further analysed. After establishing regions of high sequence similarity on both ends of the aligned sequences, a panel of highly specic PCR primers was designed on these regions (Table 3). When required, the primers contained degeneracies at one or more variable positions. Both forward and reverse primers were custom designed internally to the bacteriocin-coding gene encoding the bacteriocins to ensure that any variations in the anking DNA sequence will not affect identication of strains possessing the target gene sequence. The forward primer was designed in a way allowing specic hybridization within the 5 end

Table 2 Detailed characteristics of polish artisanal cheeses used in the study

Samples used for study Total number of samples 1 11 10 10 2 12 8 8 3 13 2 2 4 14 5 5 5 15 5 5 6 16 10 10

Cheese type Sheep milk goka Bryndza

Production method Semi-hard, made from unpasteurized sheep milk, spontaneously fermented, ripened in wooden forms, smoked Spreadable cheese, made from unpasteurized sheep milk or a mixture (1 : 1) of fermented fresh sheep cheese and fermented cow cheese, ripened for 14 days, ground and mixed with salt and water Rennet curd cheese made from unpasteurized sheep milk, cottage-type with mild avour Semi-hard, made from unpasteurized cow milk, spontaneously fermented, ripened in wooden forms, smoked Semi-hard, made from unpasteurized sheep milk, spontaneously fermented, ripened in wooden forms, nonsmoked Semi-hard, made from mixed unpasteurized sheep and cow milk, spontaneously fermented, ripened in wooden forms, smoked

Bundz Cow milk goka Non-smoked cow milk goka Mixed goka

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Table 3 Characteristics of the oligonucleotide sequences used for PCR analysis

Oligonucleotide designation Forward BCgr1NSf BCgr1R1K BCgr1R2D BCgr2NSf BCgr2R1K BCgr2R2D BCgr3ZDf BCgr3R1K BCgr3R2D BCgr4NSf1K BCgr4NSf2D BCgr4R1K BCgr4R2D BCgr4R1K BCgr4R2D BCgr5R BCgr5NSf1K BCgr5NSf2D Reverse Sequence (5 to 3) GGT GGT AAA TAC TAT GGT AA CC CCA GTT AAC AGA GCA G CTA AAT GAT DTA CNC CAC AAG ATG AAA AAG AAA KTA KTW AA TT ACA ATA MAS ACC ATT TCC CC AAT YTA CCC AAC ATT TTT TAT GGT AAT GGT GTT TAT TG AAT ATT TTC TTT AG AAG CCC AGC CAC TAA TAA AAA ATA TTA TGG AAA TGG AGT A GAC ATT CCT GCA ATA CAA TTA GCA CTT CCC TGG AAT ATG AAA CAT TTA AAA A GAC ATT CCT GCA ATA CAA TTA GCA CTT CCC TGG AAT TT CCA ACC AGC DGC TCC GAT TGG GGA AAA GCT ATT GGT GGA AAA TAC TAC GGT AA Approximate product size (bp) Sequences used for primer design Clade 1 62 94 Clade 2 123 168 Clade 3 72 112 Clade 4 88 130 159 201 Clade 5 69 131

region, which corresponds to the highly conserved sequence translated into N-terminal YGNGVxCxxxxC motif of the mature bacteriocin peptide. Binding sites of the reverse primers were selected internally to the gene sequence, which allows obtaining a product of c. 50250 bp during the PCR reaction. class IIa bacteriocin genes share a highly homologous sequence motif on the 5 end. By contrast, the 3 end of the gene sequence is a region of high diversity within this class of bacteriocins and therefore it lacks such a region of high consensus. This is because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Thus, it was merely impossible to design one set of primers with a satisfactory degeneracy level for all of the compared sequences. Therefore, 24 class IIa bacteriocin-coding gene sequences were divided into ve groups on the basis of the sequence similarity analysis (Figure 1). This resulted in obtaining a region of satisfactory consensus on the 3 end of the analysed gene sequences as well. PCR optimization The specicity of the PCR-based assay was veried by the use of a genomic DNA isolated from four reference strains with a known ability to synthesize class IIa bacteriocins. The protocol conditions for the detection and specic amplication of class IIa bacteriocin-coding gene fragment were optimized. During PCR optimization, the specicity of 18 designed degenerate primers was tested
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and genomic DNA isolated from bacteria with a conrmed bacteriocin activity was used as a template. During the rst step of our investigation, a series of PCR reactions with an annealing temperature gradient was performed (44, 48, 52 and 56C respectively). The results proved general specicity of the assay. Furthermore, because of a low yield of PCR products during subsequent amplications, an addition of selected PCR enhancing agents was tested to achieve a satisfactory amount of PCR product (2% DMSO (Chakrabarti and Schutt 2001) or 1 mmol l)1 spermidine (Fiedorow and Szweykowska-Kulinska 1997) was added to a PCR mixture). As a result of the optimization steps, the best annealing temperature was established at the level of 44C for all primer pairs. The addition of PCR enhancing agents was regarded as necessary during amplication with primers designed for Clade 1, Clade 2 and Clade 5 bacteriocin-coding sequences. As a result of primer degeneracies, additional non-specic amplicons differing in size from the anticipated ones were observed. Attempts were made to eliminate the nonspecic amplication by changing the PCR reaction parameters; however, this resulted in reduced levels of amplication of the desired amplicon. In few cases, a slight discrepancy between PCR product sizes, those predicted on the basis of sequence alignment and those observed on the gel, occurred (as with Clade 1 and Clade 5 primers). As the length of the sequence located in between primer annealing sites can somehow vary in different bacteriocin-coding genes, such amplicons

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Bacteriocin423-AF304384 (01184) PediocinAcH-S74PEDACH (01105) SakacinG-EU570253 (01981) LeucocinA-LEULAIP (01084) MesentericinY105-AY286003 (01174)
BacteriocinMC4-1-EU047916 (01809)

Clade 1

HiracinJM79-DQ664500 (01848) CarnobacteriocinB2-CPU76763 (01829) CarnobacteriocinBM1-CABCABM (01327) EnterocinP-AF005726 (00288) EnterocinP-FJ416487 (00213) EnterocinP-DQ867124 (00039) EnterocinP-DQ867125 (00004) EnterocinP-like-AB075741 (00195) EnterocinP-AY728265 (00783) SakacinA-Z46867 (01539) DivercinV41-AJ224003 (01633) EnterocinA-AF240561 (00031) EnterocinA-X94181 (00019) EnterocinCRL35-AY398693 (00043) MundticinKS-AB066267 (00023) SakacinX-AY206863 (01337) Piscicolin126-AY812745 (01238) SakacinP-former-bavaricinA-AF526262 (01685)

Clade 2

Clade 3

Clade 4

Clade 5

Figure 1 Phylogenetic tree of sequences used for alignment.

were also extracted from the gel and submitted to sequence analysis. Optimization experiments using DNA from four reference strains resulted in a selection of 18 PCR products, which were submitted for sequence analysis. The sequence analysis of obtained PCR products resulted in acquiring seven particular primer pairs with conrmed ability to amplify the class IIa bacteriocin coding gene fragments. The sequences of the seven PCR products resulting from performed amplications were identical or revealed signicant similarities to bacteriocincoding gene sequences deposited in Entrez Nucleotide Database. Sequences of the remaining PCR products appeared to be non-bacteriocin-related (Figure S1). Screening for a class IIa bacteriocin production potential in metagenomic DNA isolates Metagenomic DNA preparations isolated from the native cheese microbiota were screened for the presence of class IIa bacteriocin-coding genes. The results of this analysis are presented in Table 4. The study has revealed that during PCR analysis all the forty metagenomic DNA isolates derived from analysed cheese samples yielded a product representing the expected gene sequence with the use of at least one of the designed primer pairs. An example of the class IIa-coding gene detection among metagenomic DNA isolated from selected cheese microbiota is shown in Figure 2. Twelve PCR products amplied with the highest efciency were selected for sequence analysis and database mining purposes. Sequences of all of the selected amplicons showed signicant similarity to bacteriocin sequences deposited in the database resources. The majority of obtained sequences were LAB related, although some sequences showed signicant similarity to bacterio-

cin-coding sequences present in non-LAB bacteriocin producers. Noteworthy, primers designed for Clade 2 of bacteriocin sequences did not yield a bacteriocin-related sequence during PCR with C. divergens reference template DNA; however, positive results were obtained with BCgr2NSf and BCgr2R1K primers during analysis of metagenomic DNA isolated from mixed goka cheese (Figure S2). Discussion As a result of a growing demand for new foods produced under milder treatment conditions, the food industry requires new technologies of preventing growth of the foodborne pathogenic bacteria. The search for new GRAS bacteriocin-producers with the desired potency and target specicity for industrial applications strongly depends on the discovery of novel bacteriocins. Simultaneously, there is a strong need for developing new techniques of genome mining for identication of new antimicrobial peptides as well as pathways involved in the production of these antimicrobials (Nes and Johnsborg 2004). It is reasonable to assume that numerous bacteriocins exist in the environment, but they can be difcult to detect and identify. Until recently, detection of new bacteriocins relied on functional assays, in which putative producer organisms were puried and screened for the production of antimicrobial activity against a selection of indicator organisms. The advantages of using PCR analysis with bacteriocinspecic primers instead of the common approach of bacteriocin identication, by culture-dependent techniques, have been emphasized many times. However, until now this approach has usually been used in a way that was both time- and labour consuming. PCR reactions aiming
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Table 4 Presence of class IIa bacteriocin-coding genes among microbiota isolated from analysed cheese products as determined using PCR
Sheep milk goka Primer pair BCgr1NSf1 and BCgr1R1K BCgr2NSf and BCgr2R1K BCgr4NSf1K and BCgr4R1K BCgr4NSf1K and BCgr4R2D BCgr4NSf2D and BCgr4R1K BCgr4NSf2D and BCgr4R2D BCgr5NSf2D and BCgr5R Cow milk goka Nonsmoked cow milk goka Mixed goka

Bryndza

Bundz

Number of samples with class IIa bacteriocin-coding gene detected 10 10 100% 8 10 80% 9 10 90% 3 10 30% 0 10 0% 1 10 10% 2 10 20% 88 100% 88 100% 88 100% 18 125% 08 0% 18 125% 08 0% 22 100% 22 100% 22 100% 22 100% 12 50% 02 0% 02 0% 55 100% 15 20% 55 100% 05 0% 05 0% 05 0% 05 0% 05 0% 05 0% 05 0% 05 0% 05 0% 05 0% 45 80% 10 10 100% 9 10 90% 9 10 90% 0 10 0% 0 10 0% 0 10 0% 1 10 10%

1/1 200 bp 150 bp 100 bp 75 bp

1/2 1/3 1/4 1/5 1/6 1/7 1/8 1/9 1/10 2/1 2/2 2/3 2/4 2/5 2/6 2/7 2/8 3/1 3/2

Figure 2 Agarose gel electrophoresis of PCR amplicons generated from metagenomic DNA isolated from sheep milk goka cheese (lanes 1 1 1 10), bryndza (lanes 2 12 8) and bundz (lanes 3 13 2) cheese microbiota with primer set for Clade 2 bacteriocin genes. Primers BCgr2NSf and BCgr2R1K giving a c. 123 bp fragment (lanes 1 11 4, 1 61 7 and 1 92 8); Molecular mass marker shown in lane M (OGene Ruler Ultra, Fermentas).

at bacteriocin-coding gene detection used to be carried out in a series of parallel experiments, using several primer pairs specic to each individual bacteriocin. Consequently, amplication reactions targeting different bacteriocin-coding genes required performing many simultaneous or consecutive experiments (Moreno et al. 2002; Trmcic et al. 2008). Furthermore, the use of specic primers allows detection of known, already described bacteriocins (Choho et al. 2008; Belgacem et al. 2010). Our degenerate primers have an unprecedented advantage of detecting new, previously unidentied ones. To our knowledge, this is the rst PCR assay that uses primers designed on the basis of a large scale alignment of class IIa antilisterial bacteriocin genes, practically covering all referenced bacteriocin gene sequences deposited in the NCBI database. Using the assay presented in this study, the presence of a class II bacteriocin gene can be determined easily in a single amplication reaction. It has been reported that transcriptionally regulated bacteriocins have additional open reading frames (ORFs), located downstream from the prebacteriocin coding
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sequence. The downstream located ORFs constitute a three component signal system a transduction autoregulatory cassette encoding a possible induction factor, a histidine protein kinase (HPK) and a response regulator (RR) (Ennahar et al. 2000). Recent studies (Yi et al. 2010), although based entirely on the results published previously (Losteinkit et al. 2001), showed that it is possible to design a degenerate reverse primer with a binding site located within the HPK-coding region. This approach resulted in obtaining large amplicons (c. 3 kb) with high discrepancies ranging from 400 bp up to 800 bp among particular amplication reactions, depending on the template used during PCR. Therefore, it can be concluded that the applied approach is more prone to inuences from deletions or insertions in the region located between the primer binding sites, including the bacteriocin prepeptide gene. Such signicant changes that may occur in the amplicons length can be faulty interpreted as speciesdependent discrepancies. Such alterations may render the assay positive, despite the lack of some relevant genes. Although these attempts resulted in obtaining some

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positive results, they were not conrmed by sequence analysis of the amplicons, which is crucial in determining whether the PCR product actually represents the expected gene sequence (Johnson 2000). Furthermore, amplication of such large products might be inconvenient for assay specicity conrmation analyses. In general, the amplicon size should range from 100 to 1000 bp, where the upper limit results from difculties in amplifying larger sequences (Hyndman and Mitsuhashi 2003). The assay presented in this study uses primers with binding sites located within the prebacteriocin-coding region, which allows more precise targeting of the gene of interest. Hence, amplication reactions with primers presented in this article lead to obtaining more signicant results, which can be easily submitted to sequencing and database mining purposes. PCR assay aiming at class IIa bacteriocin-coding genes detection performed on isolated metagenomic DNA derived from cheese microbiota revealed their remarkable bacteriocinogenic potential. Consortia inhabiting these products are excellent candidates for further investigation of their bacteriocin production potential in relation to their safe use in food products. The results of this study constitute an interesting contribution to the knowledge on the superb bacteriocinogenic microbiota of Polish artisanal cheeses made in the Tatra Mountains. To our knowledge, this is the rst report on the assessment of bacteriocinogenic potential of microbiota isolated from these products. Sequence analysis of the selected amplicons revealed high query coverage (70100%) compared with the class IIa bacteriocin-coding sequences deposited in Entrez Nucleotide Database. All of the analysed sequences were also characterized by low Expect value (E-value), which indicates that the results are signicant, and thus, bacteriocin related. In few cases, performing the alignment resulted in obtaining high E-value. In general, E-value is a parameter that refers to a number of hits one can expect to obtain by chance when screening a database for a sequence of a particular size. The lower the E-value, or the closer it is to zero, the more signicant the match is. As the E-value calculation algorithm takes into account the length of the sequence of interest, it is important to remember that short sequence alignments have relatively high E-values. Hence, short sequence alignments with high E-values are relevant because shorter sequences have a higher probability of occurring in the database purely by chance (Altschul et al. 1990). Amplication with primer pairs designed on the basis of Clade 1, Clade 2 and Clade 4 of bacteriocin-coding sequences lead to generation of fragments with the highest similarity to bacteriocins produced by food-related LAB, such as Lact. plantarum, Ped. acidilactici or Ent. fae-

cium. Sequence analysis of amplicons obtained with primer pairs designed for Clade 4 (BCgr4NSf1K with BCgr4R1K as well as BCgr4NSf2D with BCgr4R1K) as well as Clade 5 of bacteriocin-coding sequences also gave results related to bacteriocin-producing bacteria, yet other than LAB (Staphylococcus saprophyticus subsp. saprophyticus, Staphylococcus aureus and Streptococcus dysgalactiae subsp. equisimilis). This indicates that there were no records deposited in the Entrez Nucleotide Database with higher similarity to the query sequence, suggesting that a potentially new bacteriocin-coding sequence was found. However, recently published results conrm that strains responsible for development of bovine mastitis, are able to synthesize class II bacteriocins, such as a class IIa peptide-ubericin A (Heng et al. 2007a). Strep. dysgalactiae subsp. equisimilis is a casual agent for bovine mastitis, able to produce a novel bacteriocin (Heng et al. 2006). Sequence analysis of the PCR product obtained with the primer pair designed for the Clade 5 bacteriocin-coding sequence, with metagenomic DNA isolated from nonsmoked cow milk goka, revealed relevant (43%) similarity to dysgalacticin a bacteriocin not yet fully characterized by the authors of the cited study. These results suggest that dysgalacticin shares some common features with class IIa bacteriocins; however, this has to be subjected to further investigation. Sequence analysis of the PCR product obtained with the BCgr4NSf1K and BCgr4R1K primer pair, with metagenomic DNA isolated from sheep milk goka, also showed signicant similarity to a bacteriocin produced by non-LAB strains Staph. saprophyticus subsp. saprophyticus and Staph. aureus. Among bacteriocins produced by Staphylococci, class II peptides are also found (Ceotto et al. 2009). The analysed sequence featured low E-value together with query coverage ranged between 54 and 62% which indicates that no other sequences deposited in the database had a higher similarity level than sequences derived from the aforementioned species. Such sequences are likely to be novel class IIa bacteriocins. In a few particular cases (PCR products obtained with BCgr2NSf and BCgr2R1K, BCgr4NSf1K and BCgr4R1K, BCgr4NSf2D and BCgr4R1K as well as BCgr5NSf2D and BCgr5R primer pairs), the search for sequence similarity among database records showed that the analysed sequences were characterized by low E-value with query coverage much lower than 100% at the same time. This may suggest that either the detected bacteriocin-coding sequence was not deposited in the Entrez Nucleotide Database, or it is a coding sequence of a novel, previously undescribed class IIa bacteriocin. The elucidation of the bacteriocinogenic potential of autochthonous LAB, isolated from these specic natural niches, gives an opportunity to select products that can
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be valuable in terms of making a genetically dened collection of natural LAB isolates with such activity. Starter cultures prepared with application of such strains could be used for production of fermented products of particular geographical origin. Our ndings contribute to both screening for new class IIa bacteriocins as well as bacteriocin-producing strains from traditional Polish artisanal cheeses. This can help selecting cultures that could be of considerable interest to enhance the microbiological quality in manufacture of dairy products. Thus, results of this study encourage utilization of the strains that are currently isolated and identied as antilisterial agents in traditional fermented foods. It is emphasized that Polish artisanal cheeses from the Tatra Mountains may be a rich source of bacteriocin-producing LAB with the antimicrobial potential. This novel method allows performing a rapid initial screening, before the traditional isolation of the bacteriocin-producing strains is carried out. To fully estimate the ability of potentially bacteriocinogenic strains to express and produce biologically active antimicrobial peptides, selection, based on phenotypic, biochemical and antimicrobial activity studies, must be performed as described elsewhere (Van Reenen et al. 1998). Detection of the bacteriocin-coding gene is a crucial moment in assessing the antimicrobial activity of selected strains. When putative bacteriocin genes are identied, several approaches can be employed to induce expression of the gene and to investigate the antimicrobial potency of the peptide and the culture. Technologies to obtain the peptide of interest might include peptide synthesis, expression cloning, in vitro transcription translation, and the use of dened growth conditions to trigger bacteriocin production (Nes and Johnsborg 2004). Many recent studies regarding different ongoing LAB genome sequencing projects strongly emphasize that there is strong demand for molecular tools helpful in rapid and reliable mining of LAB genomes in search of bacteriocin-coding genes (Klaenhammer et al. 2002). The assay presented in this article can be effective in searching for novel class IIa bacteriocins, when the results of the antimicrobial activity studies and the PCR results aiming at a particular bacteriocin gene imply the existence of other bacteriocin-coding genes that were not tested in such particular studies. Investigating the correlation between the presence of class IIa bacteriocin-coding genes and antimicrobial activity against specic organism is likely to offer a powerful tool in identifying novel agents exhibiting such activity. This will allow scientists to take a full advantage of studying antimicrobial potency of food-grade LAB.

Acknowledgements The primers presented in this study are patent pending, WIPO ST 10 C PL390490. International patent pending. The research presented in this study is nancially supported by the Polish Ministry of Science and Higher Education grant no 2044 B P01 2008 35. References
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 Sequence analysis of selected PCR products obtained with designed primer pairs on DNA isolated from reference strains. Figure S2 Sequence analysis of selected PCR products obtained with designed primers on metagenomic DNA preparations isolated from cheese microbiota. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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