System. Appl. Microbial. 23, 400-408 (2000) SYSTErvt4TIC AND © Urban & Fischer Verlag _htt-,-p:_Ilw_w_w_.ur_ba_nf_isc_h_er._de--,-/jo_u_rna_ls_/s_am ____________ APPLIED MICROBIOLOGY

Development of Molecular RAPD Marker for the Identification of Pediococcus acidilactici Strains

DIEGO MORA, CARLO PARINI, MARIA GRAZIA FORTINA, and PIER LUIGI MANACHINI

Department of Food Science and Microbiology, Industrial Microbiology section, University of Milano, Milano, Italy

Received July 1,2000

Summary

A RAPD analysis performed using a single primer targeted to the pediocin AcHlPA-1 gene was carried out on several P. acidilactici strains and on some related species of lactic acid bacteria. The high degree of genetic variability detected in P. acidilactici strains did not allow the selection of a common RAPD fragment that could be chosen as a potential species-specific DNA marker. Nevertheless a 700 bp frag­ment, that was found to be peculiar of all potential pediocin producer strains analyzed, was cloned and sequenced with the aim to develop a species specific PCR marker. Sequence analysis of the cloned 700 bp fragment showed one putative small open reading frame (ORF1), with no significant homology with known genes, and a partial putative second coding region (ORF2) with a high degree of similarity with several methionyl tRNA synthetasis (metS) genes. The two coding regions were separated by a short spacer region. Primers targeted to ORF2 plus part of the spacer region and primers designed for the am­plification of the entire cloned RAPD fragment were found to be species-specific for the detection of P. acidilactici strains. Furthermore primers designed on the ORFl sequence allowed the amplification of a 439 bp fragment only in some P. acidilactici strains, including pediocin producing strains.

Key words: Pediococcus acidilactici - Pediococcus pentosaceus - RAPD - PCR-based identification -species-specific probes

I ntrod uction

Pediococcus acidilactici and Pediococcus pentosaceus are homo fermentative vegetable associated lactic acid bacteria (LAB) commonly used in several fermented products (McKAY and BALDWIN, 1990). These species are involved in the preparation of starter cultures in meat and in vegetable fermented products, and are present as secondary flora in different types of cheese (BHOWMIK and MARTH, 1989; BHOWMIK et a!., 1990). Recently, P. acidilactici strains were isolated from chili bo (a non fer­mented traditional Malaysian vegetable food ingredient) (LEISNER et a!., 1999) and from crops silage (CAl et a!., 1999). The importance of these species in the food indus­try is also related to their potential use as biopreservation tools when pediocin producer strains are involved (GOFF et a!., 1996; STILES, 1996; VESCOVO et a!., 1996). Pedio­coccus acidilactici and Pediococcus pentosaceus are phe­notypically quite similar (GARVIE, 1986) and can be dif­ferentiated by the determination of DNA-DNA homolo­gy, the G+C content, the 16S rRNA sequencing and more rapidly by 16S rRNA and IdhD gene-targeted multiplex PCR assay (GARVIE, 1986; COLLINS et a!., 1990; MORA et

0723-2020/00/23/03-400 $ 15.00/0

a!., 1997). The problem of accurate definition and char­acterization of bacterial species and strains is of great rel­evance in microbial ecology, in the determination of tax­onomic identity, in clinical diagnosis and in food analy­sis. Nevertheless, in some cases, the correct taxonomic localization of strains belonging to the species P. acidilac­tici is problematic as demonstrated, for example, by the identification of an Enterococcus faecium bacteriocin producing strains as P. acidilactici (CINTAS et a!., 1995; CINTAS et a!., 1998), or by the presence in the American Type Culture Collection catalog of P. acidilactici strains ATCC 33314 and ATCC 8081 that are registered as P. pentosaceus in the DSMZ catalog (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). Furthermore, it was recently underlined the concrete human pathogenic role of P. acidilactici strains as agent of pneumonitis and bacteremia (SARMA and Mo­HANTY, 1998), but an exhaustive phenotypic and geno­typic characterization of these strains was not reported.

The aim of this study was to propose a new PCR marker for the identification of P. acidilactici strains.

RAPD Marker for the Identification of P. acidilactici Strains 401

Considering that these species are little known at the molecular level because only five genes have been com­pletely or partially sequenced (COLLINS et ai., 1990; GARMYN et ai., 1995a; GARMYN et ai., 1995b; MORSE et ai., 1996; GROISSILLIER and LONVAUD-FuNEL, 1999), we choose the RAPD technique (WELSH and MCCLELLAND, 1990) as tool for generating species/strain specific DNA probe (BAZZICALUPO and FANI, 1995) for the detection and identification of the P. acidilactici strains.

Table 1. Tested strains, their origin and characteristics.

Strains

Pediococcus acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilactici • P. acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilactici P. acidilacticilpentosaceus P. acidilacticilpentosaceus P. pentosaceus P. pentosaceus P. pentosaceus P. pentosaceus P. pentosaceus P. pentosaceus P. pentosaceus P. damnosus P. parvulus P. parvulus Lactobacillus casei subsp. casei L. rhamnosus L. delbrueckii subsp. bulgaricus L. helveticus Streptococcus thermophilus

DSM 20284T DSM 20238 ATCC 8042 ATCC 12697 ATCC25740 Pdill (a)

PG (a)

PAC1.0 (h )

PAC750F (h )

Psp2 (a)

F (e)

JDI-23 (d)

UL5(e) LMG 17674 (f) LMG 17680 (f) LMG 17687 (f) LMG 17689 (f )

LMG 17690(0 LMG 17692 (f)

ATCC 33314 ATCC 8081 ATCC 33316 ATCC 10791 ATCC 25745 DSMZ20283 DSMZ20333 DSMZ 20336 FBB-61 (h)

DSM 20331T ATO 34 (g)

ATO 77 (g)

DSM 20011T

Rahm 1 a

ATCC 11842 ATCC 15009T

NCDO 574

Materials and Methods

Bacterial strains and culture conditions Strains were routinely maintained at 4 °C after growth at

37°C for 12 or 24 h in MRS or M17 broth (Difco). For longer term maintenance, stock cultures were stored in 20% (v/v) glyc­erol, 80% (v/v) MRS at -20°C and -80 0c. The strains of LAB used in this work, their origin and some relevant characteristics are shown in Table 1.

Origin and relevant Characteristics

isolated from sourdough isolated from sourdough pediocin AcH/PA-1, producer strain PAC1.0 cured strain, pediocin AcHIPA-1 non-producer strain pediocin AcH/PA-l, producer strain pediocin AcH/PA-l, producer strain pediocin JDI-23, producer strain pediocin 5, producer strain isolated from chili bo isolated from chili bo isolated from chili bo isolated from chili bo isolated from chili bo isolated from chili bo

pediocin AcHlPA-1 producer strain pediocin AcH/PA-l producer strain

(a) Strains kindly provided by Prof. A. Galli Volonterio, Department of Food Science and Microbiology, Agricultural, Food and Eco­logical Microbiology section (MAAE), University of Milano, Italy; (h ) Strains kindly provided by Dr T.R. Klaenhammer, Department of Food Science, College of Agriculture and Life Sciences, North Carolina State University, obtained by Dr G. Giraffa, Experimental Dairy Institute, Lodi, Italy; (e) Strain kindly provided by Prof. Bibek Ray, Department of Animal Science, Food Microbiology Laboratory, University of Wyoming; (d) Strain kindly provided by Prof. Bob Hutkins, Department of Food Science and Technology, University of Nebraska-Lincoln, Lin­coln, USA; (e) Strain kindly provided by Dr. Eric Emond, Stela Research Centre, Department of Food Science and Nutrition, Laval University, Quebec, Canada; (f) Belgian Co-ordinated Collections of Micro-organisms (BCCMTM), Laboratorium voor Microbiologie Universistait Gent (LMG); strains kindly provided by Dr J. Leisner, Department of Veterinary Microbiology Royal Veterinary and Agricultural University, Fred­eriksberg, Denmark.

402 D. MORA et al.

DNA extraction For the PCR reaction 100 pI of an overnight culture in MRS

broth were added to 400 pI of TE IX buffer (10 mM Tris-HCI, 1 mM NazEDTA, pH 8) containing 0.45 mg/ml of lysozyme. This suspension was incubated for 30 min at 37 DC and then SDS and proteinase K were added respectively at a final concen­tration of 0.6% (wt/vol) and 7 U/ml. After incubation for 30 min, the solution was extracted with an equal volume of phenol. The DNA was then precipitated by adding 1110 vol­umes of sodium acetate and 2 volumes of 95% ethanol. The DNA pellet was air dried and subsequently dissolved in 50 pI of sterilized water (HPLC grade). For large amount DNA, cells from an overnight culture in 200 ml of MRS broth were pro­cessed as previously described (MANACHINI et al., 1985). All the DNA solutions obtained were stored at -20 DC.

RAPD experiment RAPD experiments were performed in a final volume of 25 pI

using 1 pI of bacterial DNA solution obtained as above; 1110 volume of lOX reaction buffer (Amersham Pharmacia Biotech, Milano, Italy); 200 pM of each deoxynucleoside triphosphate (dNTP); 2.5 mM of MgCI2; 1 M of primer pedAF (5'-ATAC­TACGGTAATGGGGT-3'); and 0.02 U/pl of Taq polymerase (Amersham Pharmacia Biotech, Milano, Italy). Temperature profile was carried out with a primary DNA denaturation step at 94 DC for 2 min followed by 5 cycles of 45 sec at 94 DC, 45 sec at 31 DC and 2 min at 72 DC; additional 30 cycles were carried out increasing the annealing temperature to 40 DC. The final extension was continued for 7 min at 72 DC. Amplification reactions were performed in a Gene Amp PCR System 2400 (Perkin-Elmer, Monza, Italy). After the amplification,S pI of product were electrophoresed at 5 V/cm (1.5% agarose gel, 0.2 mg/ml of ethidium bromide) in TAE buffer and photo­graphed in UV light.

Cloning of RAPD fragment. sequence determination and PCR experiments

The RAPD amplified products obtained for the strain PAC 1.0 were visualized by agarose gel electrophoresis and the 700 bp fragment was excised from the gel using the Qiagen ex­traction kit (Qiagen GmbH). The excised fragment was cloned into the pMOSBlue vector according to the manufacturer's rec­ommendations (Amersham Pharmacia Biotech, Milano, Italy) and the sequence of both the strands was determined with the dydeoxy chain termination principle (SANGER et al., 1977), using the ABI Prism BigDye™ terminators technology in a ABI Prism™ 310 DNA sequencer (Perkin Elmer, Monza, Italy). T7 (5'-TAATACGACTCACTATAGGG-3') and U19 (5'-GTTTTC­CCAGTCACGTT-3') were used as sequencing primers. The obtained sequence was analyzed using DNASIS software (Hi­tachi Software Engineering) for the presence of open reading frames and it was compared with published sequences in the

EMBL database using the Wu-blastn service of the National Center of Biotechnology Information. The sequence of the RAPD cloned fragment was used to design four internal primers, OrflF, Or£1R, Orf2F and Orf2R (Figure 2A; Table 2) that were tested for their strain/species specificity using the following PCR conditions: reactions were performed in 25 pI of volume containing 1 pI of bacterial DNA solution obtained as above, 1110 volume of lOX reaction buffer (Amersham Pharmacia Biotech, Milano, Italy), 200 pM of each deoxy­nucleoside triphosphate (dNTP), 2.5 mM of MgClz and 0.5 pM of each primers. Temperature profile was carried out with a primary DNA denaturation step at 94 DC for 2 min followed by 30 cycles of 45 sec at 94 DC, 45 sec at 65 DC and 1 min at 72 DC, the final extension was continued for 7 min at 72 DC; after amplification 5 pI of product were electrophoresed and photographed as above.

DNA restriction analysis and Southern hybridization ex­periment

Restriction analysis of total DNA obtained as above was carried out for 18 h at 37 DC in 20 pI reaction mixture contain­ing 1-5 pg of DNA preparation, 2 pI of incubation buffer and 15-20 U of Sail as restriction enzyme. Restriction digests were analyzed by electrophoresis at 5 V/cm in TAE buffer (0.7% wt/vol agarose gel), stained in 0.5 pg/ml of ethidium bromide solution, photographed as above, and transferred to nylon membranes (Boehringer, Milano Italy) by Southern blot (SAM­BROOK et al., 1985). Amplified 712( fragment (Table 2) from P. acidilactici PAC 1.0 strain was DIG-dUTP labelled by random priming with the Labelling and dectection Kit (Boehringer, Milano, Italy) and used as probe in hybridization experiment. Hybridization, was performed according to the manufacturer's recommendations with pre-hybridization and hybridization steps in 50% (wt/vol) formamide at 42 DC and with stringent washes in O.lx SSC, at 60 DC.

Results

RAPD analysis

RAPD analysis was carried out using the pedAF primer targeted to the plasmid gene pedA coding the pe­diocin AcH/PA-1 (MARUGG et al., 1992; MOTLAGH et al., 1994). Analyzing the obtained pattern profiles (Fig. lA, 1B; Table 3) it was possible to separate P. acidilactici strains from P. pentosaceus and from all other reference species of LAB. P. acidilactici strains were characterized by the presence of a main fragment of 450 bp that was present in all strains with the exception of the strain

Table 2. Sequence, position and description of the primers used in the polymerase chain reaction.

Primers

OrflF OrflR Orf2F Orf2R

Sequence (5' --7 3')

ATGATGGGGAAACTGCCAAT CTAATTGCATCGGGCCCA CCGTTTTTCCGCGTGCTATA AAAAGAAGACGTCCTTGCCT

Position la)

17-36 438-455 479-498 668-687

la) The position of the primers is according to the sequence showed in Figure 2A; Ib) Genes target, dimension of the amplified fragments in base pairs (bp) and their designation.

Description Ib)

Or£1, 439 bp, Or(l£

Orf2, 209 bp, metSf; (712 bp, 712f) Ie)

Ie) Dimension and designation of the amplified fragment obtained using OrflF and Orf2R as primers.

RAPD Marker for the Identification of P. acidilactici Strains 403

Table 3. PCR and hybridization results on P. acidilactici, P. pentosaceus strains and related species of lactic acid bacteria. Letters codes indicate different RAPD pattern types.

Strain RAPD PCR results 712f probe (e) Species Pattern assignment type IdhDf (,I 712f (bl Orflf (el metSf (dl

P. acidilactici DSM 20284T A + + + + P. acidilactici P. acidilactici DSM 20238 A + + + + P. acidilactici P. acidilactici ATCC 8042 A + + + nd P. acidilactici P. acidilactici ATCC 12697 A + + + nd P. acidilactici P. acidilactici ATCC 25740 B + + + + P. acidilactici P. acidilactici Pdi 11 C + + + + + P. acidilactici P. acidilactici PG C + + + + nd P. acidilactici P. acidilactici PAC 1.0 D + + + + + P. acidilactici P. acidilactici PAC 750F D + + + + nd P. acidilactici P. acidilactici Psp2 D + + + + nd P. acidilactici P. acidilactici F D + + + + nd P. acidilactici P. acidilactici JD 1-23 D + + + + nd P. acidilactici P. acidilactici UL5 D + + + + nd P. acidilactici P. acidilactici LMG 17674 E + + + + P. acidilactici P. acidilactici LMG 17687 E + + + + P. acidilactici P. acidilactici LMG 17689 E + + + + P. acidilactici P. acidilactici LMG 17680 F + + + + + P. acidilactici P. acidilactici LMG 17692 F + + + + nd P. acidilactici P. acidilactici LMG 17690 G nd P. pentosaceus P. acidilacticilpentosaceus G P. pentosaceus

ATCC 33314 P. acidilacticilpentosaceus G P. pentosaceus

ATCC 8081 P. acidilacticilpentosaceus G nd P. pentosaceus

DSMZ20206 P. pentosaceus ATCC 33316 G P. pentosaceus P. pentosaceus ATCC 10791 G P. pentosaceus P. pentosaceus ATCC 25745 G P. pentosaceus P. pentosaceus DSMZ 20283 G nd P. pentosaceus P. pentosaceus DSMZ 20336 G nd P. pentosaceus P. pentosaceus FBB-61 G nd P. pentosaceus P. damnosus DSM 20331 T H nd P. parvulus ATO 34 I nd P. parvulus ATO 77 I nd Lactobacillus casei sub.casei nd nd

DSM 20011T

Lactobacillus rhamnosus Rahm 1 nd nd Streptococcus thermophilus nd nd

NCD0574 Lactobacillus delbrueckii sub.

bulgaricus ATCC 11842 nd nd Lactobacillus helveticus nd nd

ATCC 15009T

(a) PCR experiments carried out using primers IdhDF and IdhDR (Mora et ai, 1998); (bl PCR experiments carried out using primers OrflF and Orf2R; (el PCR experiments carried out using primers OrflF and Orf1R; (d) PCR experiments carried out using primers Orf2F and Orf2R; (e) Hybridization experiment carried out using 712f as probe. + = positive to PCR or hybridization experiments; - = negative to PCR or hybridization experiments; nd = not determined.

ATCC 25740. A typical pattern profile, with a main am­plification fragment at about 700 bp, was peculiar of all pediocin producer strains and of the strain PAC750F (PACl.O cured strain, non-pediocin producer strain). The latter result suggested that the pediocin plasmid was not involved in the RAPD amplification despite the use of a primer targeted to the plasmid gene pedA. Because of the high degree of polymorphism present among the P. acidi-

lactici strains, it was not possible to detect a common RAPD fragment. On the contrary P. pentosaceus strains showed a very homogeneous pattern profile character­ized by two main amplified fragments at about 1500 and 2200 bp. This last RAPD profile was also shown by P. acidilactici strains ATCC 8081, ATCC 33314, and LMG 17690 that, as a consequence, were clustered in the P. pentosaceus group.

404 D. MORA et al.

M123456 7 8 9 10 11 M M 12 13 14 15

A

M' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 M'

Fig. 1. A) RAPD patterns of P. acidilactici, P. pentosaceus strains and related species of lac­tic acid bacteria. Lanes 1 to lane 5, P. acidilac­tici strains DSMZ 20284T, DSMZ 20238, Pdill, PG and Psp2; lanes 6 to 7, P. acidilacti­cilP. pentosaceus strains ATCC 8081, ATCC 33314; lanes 8 strain LMG 17690; lanes 9 to

13 P. pentosaceus strains ATCC 33316, ATCC 25745, ATCC 10791, DSMZ 20336, DSMZ 20283; lane 14 P. damnosus strain DSMZ 2033F; lane 15 P. parvulus strain ATO 34; M = Molecular weight marker VI (Boehringer, Milano, Italy): 2176, 1766, 1230, 1033, 653, 517, 453, 394, 298, 234, 220, 154 bp. 8) RAPD patterns of P. acidilac­tici strains. Lanes 1 to 4, strains DSMZ 20284T, DSMZ 20238, ATCC 8042, ATCC 12697; lane 5 strain ATCC 25740; lane 6 and 7 strains Pdill and PG; lanes 8 to12 strains F, Psp2, PAC 1.0, PAC 750F and UL5; lanes 13 to 15 strains LMG 17674, LMG 17687 and LMG 17689; lanes 16 and 17 strains LMG 17680 and LMG 17692; M'= Molecular weight marker 100 bp ladder (Amersham­Pharmacia Biotech, Milano Italy).

B

Cloning and sequencing of RAPD fragment

The 700 bp amplified fragment that clearly differenti­ated potential pediocin producer strains from all other strains (Fig. IB; Table 3), was excised from the gel, cloned and sequenced. Analysis of the nucleotide se­quence revealed that the fragment was 712 bp with a G+C content of 45.4% (accession number A]250099). Surprisingly, the nucleotide sequence of the pedAF primer, used in the RAPD experiment, was not found at the termini of the cloned 712 bp fragment, suggesting that a rearrangement probably occurred during the am­plification or the cloning step. Computer analysis of the sequence showed the presence of a small putative open reading frame, ORFl, of 450 bp and the first 170 bp of a second open reading frame, ORF2; the two ORF were separated by a spacer region of 76 bp (Fig. 2A). The 712 bp nucleotide sequence was compared with those con­tained in other databases using the Wu-blastn program and a strong similarity, P(N) 1.3-21 and 4.2-2°, was found among the ORF2 and the gene coding for the methionyl­tRNA synthetase (metS) of Bacillus subtilis and B. stearothermophilus respectively, while no significant ho­mology with known sequence was found for the ORF1. The first 55 amino acid sequence of the putative ORF2 protein was compared with the sequences available in other databases using the blastp program, which gave an high similarity, P(N) 1.5-9 and 6.8-8, with the amino acid sequences of methionyl-tRNA synthetase of Bacillus sub­tilis and B. stearothermophilus respectively. Moreover the amino acid sequences of the putative ORF2 protein was aligned with those of methionyl-tRNA synthetase from Bacillus subtilis and B. stearothermophilus and Es­cherichia coli (Fig. 2B), and a small conserved stretch HIGH like, typical of the class I tRNA synthetases, was found (ERIANI et al., 1990; CUSACK et al., 1990).

PCR experiments

Two sets of primers were designed on the obtained se­quence of the 712 bp RAPD fragment. One set (OrflF, OrflR) was targeted to the ORFI region and the second set (Orf2F, Orf2R) was designed for the specific amplifi­cation of ORF2 region plus 64 bp of the spacer region between the two ORE PCR experiments performed using primers OrflF and OrflR gave amplification of the desired fragment of 439 bp (Or(l£) only when DNA from strains Pdill, PG, LMG 17680, LMG 17692 and from all pediocin producer strains (PAC 1.0, Psp2, F, ]DI-23, UL5) was used (Table 3, Fig. 3A). On the con­trary when Orf2F and Orf2R primers were used, the ex­pected amplified fragment of 209 bp (MetSf) was present in all P. acidilactici strains, with the exception of strains ATCC 8081, ATCC 33314, and LMG 17690, and it was absent when DNA from P. pentosaceus strains and relat­ed LAB species was used (Table 3, Fig. 3B). Identical re­sults were obtained when PCR experiments were carried out using OrflF and Orf2R as forward and reverse primers respectively; the 712 bp fragment (712f) was present only when DNA from P. acidilactici strains was used, and was absent when DNA from strains ATCC 8081, ATCC 33314 and LMG 17690 was subjected to the amplification (Table 3, Fig. 3C). These three last strains were also found negative when tested with primers targeted to the IdhD gene, that were previously reported to be species-specific for P. acidilactici strains (MORA et al. 1997).

Hybridization experiments

A Hybridization experiment, using the 712f fragment as probe, was carried out against total DNA digested with SaLI of P. acidilactici DSMZ 20284T, DSMZ 20238,

A 1

61

121

181

241

301

361

421

481

541

601

661

B

RAPD Marker for the Identification of P. acidilactici Strains 405

OrOF AACATGTGTTCGTCAAATGATGGGGAAACTGCCAATTGCCGTGGAATTCCGTAACGCTAG 60

M M G K L P AVEF RN A

TTGGTTTACTGATGCGGTGACTGAAGATACCTTATCTTATTTACAACGCTTAAAAATGAT 120 SWFTD AV TE D T LSYLQR LKM

TAACGTGACGGTTGATGAACCCTTTGATGGGAACCAAGGGATGCCGTTTGTTTTACAAGT 180 N VT V D EPFD GNQG MPF V LQ

CACCAGCGCAAAACAGGCGTTTTTCCGGCTACACGGGCGCAATGCCAGCGGCTGGTTCAG 240 VTS A K Q A FFRLHG R N A SG WF

TAGCGGCAAGAACTGGCGGCGCGAACGGACCAACTACCGGTACTCGTCTGCAGAACTGAA 300 S S GKNWR RE RT NYRYSSAEL

AGAGTTGGCAGAATCCATCAAGGCGGTCGCGGAATCAGTCCAAGACGTCATGGTGATTTT 360 K E L A E S KA V AE SV QD VMV

TAACAACAATGGGAATCACGATGCGGTAGCCAACGCTAAAGAATTGCAAGAACTCCTAGG 390 FN N NGN HD A V A NA KE L QE LL

OrOR AATTCATTTTACGGGACTGGGCCCGATGCAATTAGACTTATTTTAGCCCGGGAAGCGCCC 480 G HFTGL G PMQLD L F

OrflF GTTTTTCCGCGTGCTATATGGTACAATAACAGTAATTATCTTTAGACTAGAATTGAGGAA 54 0

CCATGATGGCAGAAAATAATACTTATTACATTACAACACCGATTTATTATCCATCCGGCA 600 MMA E NN TYY T T P Y Y P SG

AATTGCACATTGGTAATTCCTATACCACGATTGCTTGCGATGCGGAAGCCCGTTTTCAAC 660 KLH GN Y T T ACD AE ARFQ

OrflR GGTTACAAGGCAAGGACGTCTTCTTTTTAACCGGTACTGACGAACACATGTT . ....... 720 RLQ GK D U F F L TG T D E HM

P. acidilactici PAC 1.0 1 M - - M - - - - - AENNTYYITTPIYYPSGKL HIGN SYTT IACDAEARFQRLQGKDU FFLTGTDEHM 55

B. subtilis 1 M-------PQENNTFYITTPIYYPSGKL HIGH AYTTVAGDAMARYKRLKGFDVRYLTGTDEHG 56

B. stearothermophilus 1 M---------EKKTFYLTTPIYYPSDKL HIGH AYTTVAGDAMARYKRLRGYDVMYLTGTDEHG 54

E. coli 1 MPTM----TQVAKKILVTCALPYANGSI HLGH MLEHIQADVWVRYQRMRGHEVNFICADDAHG 59

Fig. 2. A) Nucleotide sequence of the 712f RAPD fragment from P. acidilactici strain PAC1.0. Regions underlined represent the tar­get sequences of primer OrflF, OrflR, Orf2F and Orf2R. Amino acid sequences translation from ORFl and ORF2 were showed under the nucleotide sequence in the single-letter code. B) Alignment of Bacillus subtilis, Bacillus stearothermophilus and Esche­richia coli methionyl-tRNA synthetase partial amino acid sequences and the deduced amino acid sequence from ORF2 from P. acidi­lactici PAC 1.0. Identical residue among the amino acid sequence of the three Gram positive bacteria were typed in bold. The HIGH motives, typical of the class I aminoacyl-tRNA synthetase (ERIANI et al. 1990; CUSACK et al. 1990), are boxed.

ATCC 25740, Pdill, PAC 1.0, LMG 17674, LMG 17680, LMG 17687, LMG 17689, ATCC 33314, ATCC8081 and P. pentosaceus ATCC 25745, ATCC 10791, ATCC 33316. Hybridization signals were detect­ed only in P. acidilactici strains with the exception of strains ATCC 8081 and ATCC 33314 (data not shown). Interestingly, hybridization signals were detected at dif­ferent molecular weights ranging from 6.5 kb to 13.5 kb approximately (Fig. 4). Strains DSMZ 20284T, ATCC 25740, LMG 17674, LMG 17687, and LMG 17689 showed a common profile with hybridization signal at about 11.7 kb, strain LMG 17680 and Pdi11 were char­acterize by signal at 13.5 kb while a 7.9 kb fragment was peculiar of strain DSMZ 20238 . Strain PAC1.0, the rep­resentative of all pediocin producer strains, showed a pe­culiar hybridization signal at about 6.1 kb.

Discussion

Random Amplified Polymorphic DNA fingerprinting analysis (RAPD) is often used to develop strain or species-specific DNA molecular marker, particularly when the bacterial species analyzed is little known at the molecular level (BAZZICALUPO and FANI, 1995), as in the case of Pediococcus acidilactici.

In this study RAPD analysis was performed on several strains of P. acidilactici, P. pentosaceus and other related LAB. The analysis of the amplification patterns profiles allowed to separate P. acidilactici strains from the closest related P. pentosaceus and from all other reference species of LAB. While P. acidilactici strains were charac­terized by a high degree of polymorphism, homogeneous and different RAPD profiles were shown by all P. pento-

406 D. MORA et al.

M 1 2 3 4

A

M 1 2 3 4

B

M 1 2 3 4

C

1 2 3 4 S

5 6 7

5 6 7

5 6 7

.-13.5

.-11.7

.- 7.9

.- 6.1

8 9 10 11

8 9 10 11

8 9 10 11

Fig. 4. Hybridization experiment using the 712f fragment as probe against total DNA digested with Sail of P. acidilactici strains. Lane 1 strains DSMZ 20284T; lane 2 strains DSMZ 20238; lane 3 strain ATCC 25740; lane 4 strain Pdill; lane 5 strain PAC 1.0. M = molecular weights (kb).

12 M Fig. 3. A) Agarose gel electrophoresis showing the specificity of the amplification of the fragment Or(l£ in P. acidilactici, P. pentosaceus and related lactic acid bacteria. Lanes 1 and 2 strains P. acidilactici DSMZ

Orflf 20284T and ATCC 25740; lanes 3 and 4 strains P. acidilactici PAC 1.0 and PG; lane 5 P. acidilactici strain LMG 17674; lane 6 P. acidilactici strain LMG 17680; lanes 7 to 9 P. acidilactici strains LMG 17690, ATCC 8042 and ATCC 12697; lane 10 P. pen-tosaceus strain ATCC 10791; lane 11 and 12 P. damnosus strain DSMZ 20331 and P. parvulus strain ATO 34.

12 M' B) Agarose gel electrophoresis showing the specificity of the amplification of the frag-ment MetSf in P. acidilactici, P. pentosaceus and related lactic acid bacteria. Lanes 1 to 7 P. acidilactici strains DSMZ 20284\ ATCC 25740, Pdi11, PAC 1.0, LMG 17674, LMG 17680, LMG 17687; lanes 8 P. acidilactici

metSf strain LMG 17690; lane 8 P. acidilactici strain LMG 17690; lane 9 P. acidilactici strain LMG 17692; lanes 10 to 12 P. acidi-lactici strain ATCC 8081, P. pentosaceus strain DSMZ 20283, P. damnosus strain DSMZ20331. C) Agarose gel electrophoresis showing the specificity of the amplification of the frag-

12 M' ment 712f in P. acidilactici, P. pentosaceus and related lactic acid bacteria. Lanes 1 to 5 P. acidilactici strains DSMZ 20284T, ATCC 25740, PAC 1.0, LMG 17674, LMG 17680; lane 6 P. acidilactici strain LMG 17690; lane 7 to 10 P. acidilactici strain

712f ATCC 33314, P. pentosaceus strain DSMZ 20283, P. damnosus strain DSMZ 20331, P. parvulus strain ATO 34, and Lactobacil-lus helveticus strain ATCC 15009T•

For all the pictures, M = molecular weight marker 100 bp ladder (Amersham-Pharma-cia Biotech, Milano, Italy).

saceus strains, by two strains of uncertain taxonomic po­sition (ATCC 8081, ATCC 33314) (Table 3) and by strain LMG 17690 previously identified as P. acidilactici (LEISNER et al. 1999). Because it is reported the discrimi­nation power of the RAPD technique between these two closely related species of pediococci (NIGATU et al. 1998), strains ATCC 8081, ATCC 33314, and LMG 17690 were considered as P. pentosaceus .

Unfortunately the high degree of genetic variability detected in P. acidilactici strains did not allow the selec­tion of a common RAPD fragment that could be chosen as a potential species-specific DNA marker. Nevertheless a 700 bp fragment, that was found to be peculiar of all potential pediocin producer strains analyzed, was excised from the gel, cloned and sequenced to verify its potential role as strain or species-specific DNA marker. The com­puter analysis of the obtained sequence showed one pu­tative small open reading frame (ORF1), with no signifi-

RAPD Marker for the Identification of P. acidilactici Strains 407

cant homology with known genes, and a partial putative second coding region (ORF2) with a high degree of simi­larity with several methionyl tRNA synthetasi (metS) genes. Furthermore the deduced amino acid sequence from ORF2 of P. acidilactici PAC 1.0 showed a small conserved stretch HIGH like, that strongly supported the hypothesis that ORF2 codifies for methionyl-tRNA syn­thetase or for a class I aminoacil tRNA synthetase (ERI­ANI et al., 1990; CUSACK et al., 1990).

With the aim to verify the strain or species-specificity of the sequenced DNA region, two sets of primers were designed and tested in PCR experiments, using DNA from P. acidilactici, P. pentosaceus and from all the other related LAB. PCR assays carried out using the primer sets Orf2F, Orf2R or OrflF, Orf2R should be considered a useful tool for the identification of P. acidilactici strains, while primer set OrflF-OrflR, should be consid­ered specific only for a restricted group of strains com­prising pediocin producer, sour dough and chili bo isolat­ed. Despite the results obtained, at the moment, there are not common phenotypic characteristics among the posi­tive strains to the primer set targeted to ORF1 that could justify its use in taxonomic analysis, but further study are in progress to characterize these strains phenotypically. Nevertheless, the primer set OrflF and OrflR could be useful in a preliminary screening aimed to the identifica­tion of pediocin producing strains. Strains ATCC 8081, ATCC 33314 and LMG 17690, identified previously as P. pentosacues by RAPD analysis, were always found negative when tested with the primer sets OrflF-OrflR Orf2F-Orf2R and also when tested with P. acidilactici species-specific primers targeted to the IdhD gene (MORA et al. 1997). These results were according to the RAPD identification that also confirm the ascription to the species P. pentosaceus of the strains ATCC 8081, ATCC 33314 as it is reported by the DSMZ catalog where they were registered as P. pentosaceus strains DSMZ 20206 and 20333 respectively.

Moreover, Southern hybridization experiment showed that the 712f fragment could be also used as species-spe­cific probe in hybridization assays, and revealed the pres­ence of consistent sequence diversity between the puta­tive metS plus its upstream region of P. acidilactici and the closest P. pentosaceus. Furthermore, the use of 712f probe in hybridization assays revealed its potential role as tool in P. acidilactici strains typing analysis because of the presence of hybridization signals at different molecu­lar weights for the several strains analyzed, where pe­diocin producer strains seemed to show a peculiar pro­file.

Recently, a certain degree of genetic and phenotypic polymorphism within the species P. acidilactici was re­ported also among crops silage and chili bo isolates (CAl et al., 1999; LEISNER et al., 1999). This intra-species vari­ability could be, in some case, the reason of misidentifi­cation of strains belonging to this species, and the devel­opment of different molecular markers, targeted to sever­al chromosomal regions, should be a substantial contri­bution towards the evaluation of their correct taxonomic position.

Acknowledgment This research was supported by a grant of the Ministry of

the University and Technological and Scientific Research (MURSTex40%).

We would like to thank Dr. E. EMOND, Prof. B. HUUTKINS, Prof. T. R. KLAENHAMMER, Prof. GALLI VOLONTERIO, Dr. G. GI­RAFFA, Dr J. LEISNER and Prof. B. RAY for providing P. acidilac­tici strains.

References

BAZZICALUPO, M., FANI, R. The use of RAPD for generating spe­cific DNA probes for microorganisms In Methods in Mole­cular Biology, vol. 50: 155-175 (Clapp, J.P., ed.), Humana Press Inc., Totowa, NJ, 1995.

BHOWMIK, T., MARTH, E.: Role of Micrococcus and Pediococcus species in cheese ripening: a review. J. Dairy Sci. 73, 859-866 (1989).

BHOWMIK, T., RIESTERER R., VAN BOEKEL, M. A. J. S., MARTH, E. H.: Characteristics of low-fat Cheddar cheese made with added Micrococcus or Pediococcus species. Milchwis­senschaft 45,230-235 (1990).

CAl, Y., KUMAI, S., OGAWA, M., BENNO, Y., NAKASE, T.: Charac­terization and identification of Pediococcus species from for­age crops and their application for silage preparation. Appl. Environ. Microbiol. 65,2901-2906 (1999).

CiNTAS, L. M., RODRIGUEZ, J. M., FERNANDEZ, M. E, SLETTEN, K., NES, 1. E, HERNANDEZ, P. E., HOLO, H.: Isolation and characterization of pediocin L50, a new bacteriocin from P. acidilactici with a broad inhibitory spectrum. Appl. Environ. Microbial. 61,2643-2648 (1995).

CiNTAS, L. M., CASAUS, P., FERNANDEZ, M. E, HERNANDEZ P. E.:. Comparative antimicrobial activity of enterocin L50, pe­diocin PA-l, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria. Food Microbiology 15, 289-298 (1998).

COLLINS, M. D., WILLIAMS, A. M., WALL BANKS, S.: The phyloge­ny of Aerococcus and Pediococcus as determined by 16S rRNA sequence analysis: description of Tetragenococcus gen. nov. FEMS Microbial. Lett. 70,255-262 (1990).

CUSACK, S., BERTHET-COLOMINAS, c., HARTLEIN, M., NASSAR, N., LEBERMAN, R. A second class of synthetase structure re­vealed by X-ray analysis of Escherichia coli seryl-tRNA syn­thetase at 2.5 A. Nature 347,249-255 (1990).

ERIANI, G., DELARVE, M., POCH, 0., GANGLOff, J. MORAS, D.: Partition of RRNA synthetases into two classes based on mu­tually exclusive sets of sequence motifs. Nature 347, 203-206 (1990).

GARMYN " D., FERAIN, T., BERNARD, N., HOLS, P., DELCOUR, J.: Cloning, nucleotide sequence, and transcriptional analysis of the Pediococcus acidilactici L-( + )-Lactate dehydrogenase gene. App!. Environ. Microbial. 61, 266-272 (1995).

GARMYN b, D., FERAIN, T., BERNARD, N., HOLS, P., DELPLACE, B., DELCOUR, J.: Pediococcus acidilactici IdhD gene: cloning, nu­cleotide sequence, and transcriptional analysis. J. Bacterial. 177,3427-3437 (1995).

GARVIE, E. I.: Genus Pediococcus, pp. 1075-1079. In: Bergey's Manual of Systematic Bacteriology (P.H.A., SNEATH, N.S., MAIR, M.E., SHARPE, J.G., HOLT, eds.) Baltimore, Williams e Wilkins 1986.

GOFF, J. H., BHUNIA A. K., JOHNSON, M. G.: Complete inhibi­tion of low levels of Listeria monocytogenes on refrigerated chicken meat with pediocin AcH bound to heat-killed Pedio­coccus acidilactici cells. J. Food Prot. 59, 1187-1192 (1996).

GROISILLIER, A., LONVAUD-FuNEL, A.: Comparison of partial malolactic enzyme gene sequences for phylogenetic analysis

408 D. MORA et al.

of some lactic acid bacteria species and relationships with the malic enzyme. Int.]. Syst. Bacteriol. 49,1409 (1999).

LEISNER, J. J., POT, B., CHRISTENSEN, H., RUSUL, G., OLSEN, J. E., WEE, B. W., MUHAMAD, K., GHAZALI, H. M.: Identification of lactic acid bacteria from chili bo, a malaysian food ingredi­ent. Appl. Environ. Microbiol. 65, 599-605 (1999).

MANACHINI, P. L., FORTINA, M. G., PARINI, C. CRAVERI, R.: Bacillus thermoruber sp. nov., nom. rev., a red pigmented thermophilic bacterium. Int. J., Syst. Bacteriol. 35, 493-496 (1985).

MARUGG, J. D., GONZALEZ, C. E, KUNKA, B. S., LEBEBOER, A. M., PUCCI, M. J., TOONEN, M. Y., WALKER, S. A., ZOET­MUDLER, L. C. M., VANDERBERGH, P. A.: Cloning, expression, and nucleotide sequence of genes involved in the production of pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC 1.0. Appl. Environ. Microbiol. 58,2360-2367 (1992).

McKAY, L. L., BALDWIN, K. A.: Application for biotechnology: present and future improvements in lactic acid bacteria. FEMS Microbiol. Rev. 87,3-14 (1990).

MORA, D., FORTINA, M. G., PARINI, c., MANACHINI, P. L.: Iden­tification of Pediococcus acidilactici and Pediococcus pen­tosaceus based on 16S rRNA and IdhD gene-targeted multi­plex PCR analysis. FEMS Microbial. Lett. 151: 231-236 (1997).

MORSE, R., COLLINS, M. D., BALSDON, J. T., WALLBANKS, S., RICHARDSON, P. T.: Nucleotide sequence of part of the rpoC gene encoding the' subunit of DNA-dependent RNA poly­merase from some Gram-positive bacteria and comparative amino acids sequence analysis. Syst. Appl. Microbiol. 19, 150-157 (1996).

MOTLAGH, A., BUKHTIYAROVA, M., RAY, B.: Complete nu-· cleotide sequence of pSMB 74, a plasmid encoding the pro-

duction of pediocin AcH in Pediococcus acidilactici. Lett. Appl. Microbiol. 18,305-312 (1994).

NIGATU, A., AHRNE, S., GASHE, B. A., MOLIN, G.: Randomply amplified polymorphic DNA (RAPD) for discrimination of Pediococcus pentosaceus and Ped. acidilactici and rapid grouping of Pediococcus isolates. Lett. Appl. Microbiol. 26, 412-416 (1998).

SAMBROOK, J., FRITSCH, E. E, MANIATIS, T.: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratoty, New York 1989.

SANGER, E, NICKLEN, S., COULSON, A. R.: DNA sequencing with chain-terminating inhibitors. P.N.A.S. USA 74, 5463-5467 (1977).

SARMA, P. S., MOHANTY, S.: Pediococcus acidilactici Pneumoni­tis and Bacteremia in a Pregnant Woman. ]. Clin. Microbiol. 36,2392-2393 (1998).

STILES, M. E.: Biopreservation by lactic acid bacteria. Antonie van Leewenhoek 70, 331-345 (1996).

VESCOVO, M., TORRIANI, S., ORSI C., MACCHIAROLO, E, Sco­LARI, G.: Application of antimicrobial-producing lactic acid bacteria to control pathogens in ready-to-use vegetables. J. Appl. Bacteriol. 81, 113-119 (1996).

WELSH, ]., MCCLELLAND, M.: Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213-7218 (1990).

Corresponding author: Dr DIEGO MORA, DI.S.T.A.M. - Sezione Microbiologia Indus­triale, Via Celoria, 2, 20133, Milano, Italy. Tel.: 0039 02 23955849; Fax: 0039 02 70630829; E-mail: diego.mora@unimi.it