Journal of Applied Microbiology 1998, 84, 1099–1103

Detection of specific bacteriocin-producing lactic acidbacteria by colony hybridization

M.I. Martınez1, E. Rodrı guez2, M. Medina 2, P.E. Hernandez1 and J.M. Rodrı´guez1

1Departamento de Nutricion y Bromatologıa III, Facultad de Veterinaria, Universidad Complutense, and2Departamento de Tecnologıa de los Alimentos, CIT-INIA, Madrid, Spain

6400/09/97: received 15 September 1997, revised 5 November 1997 and accepted 11 November 1997

M.I . MARTINEZ, E. RODRIGUEZ, M. MEDINA, P.E. HERNANDEZ AND J.M. RODRIGUEZ. 1998. Acolony hybridization method for detecting lactic acid bacteria encoding specificbacteriocins was developed. Specific PCR-generated probes were used to detect coloniesof pediocin PA-1, lactococcin A, enterocin AS-48, nisin A and lacticin 481producing strains. The probes were shown to be sensitive and specific for sequencesbelonging to the structural genes of the respective bacteriocins.

INTRODUCTION

Bacteriocins of lactic acid bacteria (LAB) are attractive to thefood industry because they may be used as natural biop-reservatives and contribute to the improvement of the mic-robiological quality of foods (Jack et al. 1995). Also, theseantimicrobials would allow a significant reduction in the levelof chemical additives and/or in the intensity of the physicaltreatments currently employed during food processing.Therefore, they could also help to provide healthier foods.

Although bacteriocin activity can easily be assayed (Dae-schel 1992), purification and amino acid sequencing is theonly method available to determine exactly which bacteriocinis being expressed by a particular strain. Obviously, such anapproach cannot be used for routine screenings because ofthe specialized equipment and personnel required.

DNA techniques, such as the polymerase chain reaction(PCR) or DNA hybridization, are widely used to detect spec-ific sequences and therefore, they could be applied for theidentification of genes belonging to the operon or gene clusterof a specific bacteriocin. Although the detection of such genesdoes not guarantee its expression, the development of specificprobes and/or primers would be very useful when searchingfor a novel bacteriocin. Those strains with the potential toproduce bacteriocins whose biosynthesis is encoded in genesalready sequenced could easily be discarded. In addition,specific probes and primers would be a valuable tool forstudying the behaviour of bacteriocinogenic LAB used asstarter cultures in food fermentations, especially when study-

Correspondence to: Dr Juan M. Rodrıguez, Dpto. Nutricion y BromatologıaIII, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040Madrid, Spain (e-mail : jmrodrig@eucmax.sim.ucm.es).

© 1998 The Society for Applied Microbiology

ing combinations of strains that produce different bacteri-ocins.

Although colony hybridization has been successfully usedto identify LAB species or even strains (Betzl et al. 1990 ;Cocconcelli et al. 1991), this technique has not been appliedfor the detection of potential bacteriocin-producing strains.In this work, the use of specific DNA probes is reported forthe detection of pediocin PA-1, lactococcin A, enterocin AS-48, nisin A or lactocin 481 producing organisms by a colonyhybridization method.

MATERIALS AND METHODS

Bacterial strains

The LAB strains used in this study are listed in Table 1.All the strains were cultured in MRS medium (Oxoid Ltd,Basingstoke, UK) at 32 °C.

Preparation and labelling of the DNA probes

Probes consisted of PCR products obtained using colonies ofreference strains as templates. The oligonucleotides used asprimers (Fig. 1) were made on a DNA synthesizer model381A (Applied Biosystems, Foster City, CA, USA) using thesupplier’s reagents and protocols. A search was made inthe GenBank database, using the Genetics Computer Groupprogram (Devereux et al. 1984), to ensure that the oli-gonucleotides did not share homologies with the sequencesof other bacteriocins available in the database. Amplificationswere carried out in an Omnigene thermal reactor (HybaidLtd, Teddington, UK) under the conditions described by

1100 M.I. MARTINEZ ET AL.

Table 1 Lactic acid bacteria strainsused in this study

—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Strain Sourcea Bacteriocin Reference—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Enterococcus faecalis INIA 4 INIA enterocin AS-48 Joosten et al. (1995)Lactococcus lactis

subsp. lactis ATCC 11454 ATCC nisin A Steen et al. (1991)subsp. lactis WM4 FSN lactococcin A Stoddard et al. (1992)subsp. lactis CNRZ 481 INRA lacticin 481 Piard et al. (1990)

Pediococcus acidilactici 347 NBIII pediocin PA-1 Rodrıguez et al. (1997)—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––a Abbreviations : ATCC, American Type Culture Collection (Rockville, MD, USA) ; FSN,Department of Food Science and Nutrition, University of Minnesota (St Paul, MN,USA) ; INIA, Instituto Nacional de Investigacion y Technologıa Agraria y Alimentaria(Madrid, Spain) ; INRA, Institut National de la Recherche Agronomique, Station deRecherches Laitieres (Jou-en-Josas Cedex, France) ; NBIII, Departamento de Nutriciony Bromatologıa III, Facultad de Veterinaria, Universidad Complutense (Madrid, Spain).

Horn et al. (1991). The amplified fragments were purifiedusing the QIAquick spin PCR purification kit (QiagenGmbH, Hilden, Germany) and labelled using the directnucleic acid labelling system (Amersham International,Amersham, UK) according to the instructions of the manu-facturer.

Preparation of membranes and hybridization

Two kinds of trials were conducted. First, colony hybrid-ization of pure isolates was carried out to verify the sensitivityof the DNA probes. Once isolated colonies of the micro-organisms had grown on MRS agar plates after incubation at32 °C for 72 h, nylon membranes Hybond-N+ (AmershamInternational) were laid directly on the culture surfaces andkept for at least 1 min. Then, membranes were removed,placed on filter papers soaked with a 0·01 mol l−1 Tris-HCl,0·25 mol l−1 sucrose, pH 7·5 solution supplemented withlysozyme (5 mg ml−1) and mutanolysin (30 units ml−1) toachieve cell lysis, and processed according to the methoddescribed by Cocconcelli et al. (1991). The enzymic treatmentis required to lyse LAB because of their particular cell wallstructure (Lonvaud-Funel et al. 1991). Hybridizations werecarried out at 42 °C according to the instructions of theAmersham International nucleic acid labelling and detectionsystem. Filters were washed twice in 0·5×SSC (1×SSC is0·15 mol l−1 NaCl plus 0·015 mol l−1 sodium acetate)–0·4%SDS for 10 min at 42 °C, and then washed twice in 2×SSCfor 5 min at room temperature.

Alternatively, a second kind of trial was used to test thespecificity of the probes. For easier differentiation of thebacteriocin producers, the cells were inoculated on MRS agar

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 1099–1103

plates in such a way that the colonies appeared as letters, eachletter representing a different bacteriocin-producing strain(Fig. 3). After incubation at 32 °C for 24 h, nylon membraneswere applied to the cultures and processed as stated above.In this format, cultures of strains producing different bac-teriocins were simultaneously tested with successive probesspecific for DNA sequences belonging to each bacteriocin.Hybridizations were performed as described above.

RESULTS AND DISCUSSION

After designing probes theoretically specific for their respec-tive bacteriocin structural genes, colony hybridizations werecarried out using plates containing pure cultures of the dif-ferent bacteriocin-producing strains. Each probe developedto recognize a DNA sequence specific for one bacteriocingenerated a positive signal from the culture lysate of a strainthat produced such a bacteriocin (Fig. 2). Similar results wereobtained when Enterococcus faecalis TAB21, TAB28, TAB41and TAB70 (enterocin AS-48 producers) (Joosten et al. 1995,1997), Lactococcus lactis BB24 and G18 (nisin producers)(Rodrıguez et al. 1995a), and Pediococcus acidilactici X13, P20and Z102 (pediocin PA-1 producers) (Rodrıguez et al. 1997)were tested with their respective specific probes (results notshown). The signals obtained demonstrate that DNA of pedi-ococci, lactococci and enterococci are accessible to PCR-generated probes in colony hybridization assays.

In order to test the specificity of the probes, colonies ofthe different bacteriocin-producing strains were grown onthe same plate. Then, the resulting membrane was testedsuccessively with the different probes. As can be seen in Fig.

DETECTION OF BAC+ LACTIC ACID BACTERIA 1101

Fig. 1 Location and sequences of the primers used for PCR generation of the probes. Genes are indicated by black boxes. The primersare shown as numbered arrows below the map. Lines connecting primers represent the amplified fragments with sizes given in base pairs

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 1099–1103

1102 M.I. MARTINEZ ET AL.

Fig. 2 Colony hybridization of pure cultures of (a) Pediococcus acidilactici 347, using probe P ; (b) Lactococcus lactis subsp. lactis WM4,using probe A ; (c), Enterococcus faecalis INIA, using probe I ; (d) Lactococcus lactis subsp. lactis ATCC 11454, using probe N ;(e) Lactococcus lactis subsp. lactis CNRZ 481, using probe 4

3, the probe for each bacteriocin did not hybridize with DNAfrom the micro-organisms producing other bacteriocins, thusshowing a high specificity.

The identification of bacteriocin-producing micro-organ-isms has significant importance in basic as well as appliedresearch on food microbiology. In the past few years, it hasbeen shown that nucleic acid-based techniques such as PCRand Southern hybridization are excellent tools for identifyingstrains encoding a specific bacteriocin (Harris et al. 1992 ;Rodrıguez et al. 1995a,b ; Joosten et al. 1997 ; Rodrıguezet al. 1997). The contribution of DNA techniques to theidentification of bacteria with specific traits can be increasedby colony hybridization. This technique was first used toidentify recombinant clones of Escherichia coli and laterapplied for the detection of food-borne pathogens with probescharacteristic of genes coding for toxins or other virulencefactors (Hill and Keasler 1991). Colony hybridization has alsobeen used for the identification of enterococci, pediococciand lactococci (Betzl et al. 1990 ; Cocconcelli et al. 1991 ;Rodtong et al. 1993). However, it has never been applied to

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 1099–1103

the bacteriocin field. In contrast with other related tech-niques, colony hybridization does not require previous iso-lation of DNA and allows the direct analysis of mixedcultures. Thus, it can be very useful in initial screeningswhen searching for bacteria which produce novel bacteriocinsor studying the prevalence of specific bacteriocin producersin food systems.

In this study, we have shown that when suitable probesare used, colony hybridization can be a sensitive and specificmethod of detecting strains that encode a particular bac-teriocin.

ACKNOWLEDGEMENTS

This work was partially supported by grants ALI94-1026from the Comision Interministerial de Ciencia y Tecnologıa(CICYT, Spain) and SC95-089 from the Instituto Nacionalde Investigacion y Tecnologıa Agraria y Alimentaria (INIA,Spain).

DETECTION OF BAC+ LACTIC ACID BACTERIA 1103

Fig. 3 Colony hybridization of Pediococcus acidilactici 347 (P),Lactococcus lactis subsp. lactis WM4 (A), Enterococcus faecalis INIA4 (I), Lactococcus lactis subsp. lactis ATCC 11454 (N), andLactococcus lactis subsp. lactis CNRZ 481(4). (a) Array of the letter-shaped colonies. Hybridization to the specific probes P (b), A(c), I (d), N (e), and 4 (f)

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