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Autolytic activity and pediocin-induced lysis in Pediococcus acidilactici and Pediococcus pentosaceus strains
D. Mora, F. Musacchio, M.G. Fortina, L. Senini and P.L. Manachini
Department of Food Science and Microbiology, University of Milano, Milano, Italy
2002/340: received 3 September 2002 and accepted 22 November 2002
D . M O R A , F . M U S A C C H I O , M . G . F O R T I N A , L . S E N I N I A N D P . L . M A N A C H I N I . 2003.
Aims: To evaluate the autolytic phenotype of Pediococcus acidilactici and P. pentosaceus, the peptidoglycan hydrolases content and the effect of pediocin AcH/PA-1 and autolysins on cell lysis. Methods and Results: The autolytic phenotype of Pediococcus strains was evaluated under starvation conditions in potassium phosphate buffer. The strains tested showed an extent of autolysis ranging between 40 and 90% after 48 h of starvation at 37°C. Peptidoglycan hydrolase content was evaluated by renaturing sodium dodecyl sulphate– polyacrylamide gel electrophoresis (SDS–PAGE) using cells of Micrococcus lysodeikticus as a target for the enzymatic activity and a major activity band migrating at about 116 kDa was detected. Additional secondary lytic bands migrating in a range of molecular weight between 45 and 110 kDa were also detected. The lytic activity, evaluated in the presence of different chemicals, was retained in 15 mM CaCl2 and in a range of pH between 5 and 9Æ5 but was strongly reduced in presence of 8% NaCl and in the presence of protease inhibitors. The substrate speciﬁcity of peptidoglycan hydrolases of Pediococcus strains was evaluated in renaturing SDS–PAGE incorporating cells of different bacterial species. Lytic activity was detected against cells of Lactococcus lactis subsp. lactis, L. delbrueckii subsp. bulgaricus, Lactobacillus helveticus and Listeria monocytogenes. The interaction between pediocin AcH/PA-1 and autolysis was evaluated and a relevant effect of bacteriocin in cell-induced lysis was observed. Conclusions: The autolytic phenotype is widely distributed among P. acidilactici and P. pentosaceus and the rate of autolysis is high in the majority of the analysed strains. Several autolytic bands, detected by renaturing SDS–PAGE, retained their activity against several lactic acid bacteria and L. monocytogenes. Signiﬁcance and Impact of the Study: The characterization of the autolytic phenotype of Pediococcus acidilactici and P. pentosaceus strains should expand the knowledge of their role in fermentation processes where these species occur as primary or secondary bacterial population. Keywords: autolysis, Pediococcus acidilactici, Pediococcus pentosaceus, pediocin AcH/PA-1.
INTRODUCTION Pediococcus acidilactici and P. pentosaceus are homofermentative vegetable associated lactic acid bacteria involved in the preparation of starter culture in meat and in vegetable fermented products (McKay and Baldwin 1990; Onno and
Correspondence to: Diego Mora, Department of Food Science and Microbiology, Via Celoria, 2, 20133, Milano, Italy (e-mail email@example.com).
Roussel 1994), and present as secondary ﬂora in different types of cheese (Bhowmik and Marth 1989; Bhowmik et al. 1990; Giraffa et al. 1997; Neviani et al. 1998). These two species were also investigated for pediocins production and their possible utilization in biopreservation processes (Marugg et al. 1992; Stiles 1996; Vafopoulou et al. 1999). Despite the commercial importance of P. acidilactici and P. pentosaceus in fermented products, the autolytic phenotype of these species have never been investigated. Autolysis
ª 2003 The Society for Applied Microbiology
P. Denmark. pedS Bac). R. P. stock cultures were stored in 20% (v/v) glycerol. P. Bac+. their origin and relevant characteristics and other bacterial strains Strain P. ª 2003 The Society for Applied Microbiology. Giraffa. pedS MATERIAL AND METHODS Bacterial strains and growth conditions Strains of P. NCK 537 were grown in MRS broth at 37°C. P. Lactobacillus helveticus ATCC 15009T and Lactobacillus sp. Bac). P. pedS Plants. pH 6Æ5) and resuspended in the same buffer or in MES buffer (2-N-Morpholino-ethanesulfonic acid) (50 mmol l)1. pedR Bac).600 nm 0Æ8–1) grown in MRS. Department of Veterinary Microbiology. Bac). N-acetylmuramyll-alanine amidase and endopeptidases) that are classiﬁed with the generic name of peptidoglycan hydrolases (ChapotChartier 1996). pedS Vegetable. pedS Bac). pedR derived from strain PPE1Æ0 Bac). pedS Chili Bo.600 nm of 0Æ6–0Æ8 and incubated at 37°C. brain heart infusion broth and reinforced clostridial medium respectively. 2000). pentosaceus were routinely maintained at 4°C after growth at 37°C for 12 or 24 h in MRS broth (Difco). Lactococcus lactis subsp. Dipartimento di scienze e Tecnologie Alimentari e Microbiologiche. P. 1995. Sour dough. Bac). North Carolina State University. Medizinische Fakultat der Humboldt. Universitat zu Berlin. The aim of this study is to evaluate the autolytic phenotype in P. Bac). Molekularbiologisch-Diagnostisches Labor. Bac). 1992. Department of food Science. pedS. Univeritatsklinkum. Universita – Strains kindly provided by Dr R. The degree of autolysis was expressed as the percentage decrease of the O. pedS Mutant Bac). Frederiksberg. P. Staphylococcus aureus DSM 799. Bac). pedS Chili Bo. 561–570 . the effect of spontaneous/induced cell lysis in meat or vegetable fermented product have never been investigated.D. Pillidge et al. 2000). pedS Mutant Bac). pedS. 80% (v/v) MRS at )20 °C. Bac). pentosaceus. P. acidilactici acidilactici acidilactici acidilactici DSM 20284T DSM 20238 MI20238R ATCC 8042 ATCC 12697 ATCC 25740 WB 1104/99* Origin and relevant characteristic Barley. Klaenhammer. Husson-Kao et al. While the importance of autolysis in accelerating cheese ripening because of the release of intracellular enzymes is well known and several studies have been carried out to detect and characterize autolytic enzymes (Buist et al. Lodi. ` degli Studi di Milano. Italy. Italy. is a complex process involving different enzymes (N-acetylmuramidases. acidilactici acidilactici acidilactici acidilactici acidilactici acidilactici acidilactici acidilactici pentosaceus pentosaceus pentosaceus pentosaceus pentosaceus pentosaceus PG PAC1Æ0 PAC750F BCCM 17674à BCCM 17680à BCCM 17689à BCCM 17687à BCCM 17692à DSM 20283 DSM 20282 ATCC 8081 ATCC 33314 PPE1Æ0 MIPPE1Æ0R Lactobacillus sp. Milano. pentosaceus strains isolated from vegetable. Strains of Lactobacillus delbrueckii subsp. M O R A ET AL. Cibik and Chapot-Chartier 2000. P. Bacteriocin negative strain. à Strains isolated from a non-fermented traditional Malaysian vegetable food ingredient (Chili Bo) and kindly provided by Dr J. Strains kindly provided by Dr T. Centro Nazioonale Ricerca. Leisner. Moreover. 94.D. Table 1 Pediococcus acidilactici and P.600 nm after 48 h. Journal of Applied Microbiology. Milano. Royal Veterinary and Agricultural University. Motlagh et al. pedS Isolated from male patient with septicemia (Heinz et al. P. P. College of Agricultural and Life Sciences. Istituto Sperimentale Lattiero Caseario. Mutanolysin activity Mutanolysin activity was evaluated on Pediococcus cells harvested in exponential phase and resuspended in MES P. acidilactici and P. Listeria monocytogenes MACa1 and Clostridium sporogenes CL14 were grown at 37°C in nutrient broth. pedS Bac).562 D . P. Pediocin resistant strain. § Strain kindly provided by Prof A. Bac). Autolysis of whole cells in buffer solution The autolytic phenotype of Pediococcus strains was evaluated on harvested exponential phase cells (O. bulgaricus ATCC 11842T L. NCK 537 L. P. Lodi. pedS Bac). delbrueckii subsp. Galli and Dr M. P. Bac). Bac). Scarpellini. bulgaricus ATCC 11842T. acidilactici P. helveticus ATCC 15009T Listeria monocytogenes MACa1§ Clostridium sporogenes CL14– * Strain kindly provided by Dr Michael Lefmann. lactis BMG6Æ02 was grown in M17 broth implemented with glucose (GM17) at the ﬁnal concentration of 1% (w/v) at 30°C. Pediocin sensitive strain. Bac). washed in potassium phosphate buffer (50 mmol l)1. obtained by Dr G. pH 6) to an O. Pediocin AcH/PA-1 producing strain. acidilactici P. Bromatologico e Microbiologico del Latte. acidilactici P. pedR Chili Bo. Italy. pedR derived from strain DSM 20238 Mash. pedS Bac+. 1994) on cell lysis has been evaluated. N-acetylglucosaminidases. meat and dairy products. pedS Chili Bo. P. pedR. The strains of Pediococcus used in this work and their origin are shown in Table 1. P. Centro per lo Studio Tecnologico. Bac) pedS Mash. pedS Bac). For longer term maintenance. acidilactici and P. 1998. the effect of pediocin AcH/PA-1 (Marugg et al. pedS Chili Bo. Bac).D.
L. NCK 537. 1998). L. Italy). Milano. helveticus ATCC 15009T. pH 6) supplemented with 1 mmol l)1 MgCl2 to an O. Amicon) and the crude pediocin AcH/PA-1 preparation (CPP) was stored at )20 °C. Italy) containing AEBSF (4-(2-aminoethyl)benzenesulfonyl ﬂuoride hydrochloride) (2 mmol l)1). Detection of pediocin AcH/PA-1 resistance was monitored. by overlaying P. Protein contents were determined by the Bradford method (Bradford 1976) and 3–4 lg of protein extraction were analysed in renaturing SDS-PAGE. gels were soaked in 200 ml of sterilized. 10 mmol l)1 NaCl and 2% SDS (w/v). Sample preparation was carried out mixing 40 ll of whole-cell SDS extract and 30 ll of loading buffer containing 50 mmol l)1 Tris-HCl. the plates were checked for inhibition zones. pH 7). and then 10 ml of soft agar containing about 106 CFU of the indicator strain Lactobacillus sp. Italy). Renaturing SDS-PAGE and detection of peptidoglycan hydrolases activity Renaturing SDS-PAGE electrophoresis was performed as described by Buist et al.600 nm were evaluated after 20 min at 37°C. 0Æ5% (v/v) b-mercaptoethanol and 0Æ5% (w/v) bromophenol blue. 10 mmol l)1 ethylenediaminetetraacetic acid (EDTA).D. bulgaricus ATCC 11842T. EDTA (1 mmol l)1). After incubation for 12–18 h at 37°C. Milano. The effect of pH was evaluated using renaturation solution buffered with 10 mmol l)1 sodium acetate (pH 5) or 10 mmol l)1 TrisHCl (pH 7–9Æ5). Sigma. The mutanolysin activity were expressed as the percentage decrease of the O. 2Æ5% (v/v) glycerol. sporogenes CL14 were harvested from 25 ml of culture broth grown 12–18 h in the appropriate media at 30–37°C. E-64 (14 lmol l)1). Autoclaved cells of Micrococcus lysodeikticus ATCC 4698 (Sigma) were incorporated into polyacrylamide running gels at a concentration of 0Æ2% (w/v).600 nm was evaluated. Bands of lytic activity were visualized by staining the gel with 1% (w/v) methylene blue (Sigma) in 0Æ01% (w/v) KOH and subsequent destaining in sterilized distilled water. 94. pentosaceus pediocin resistant mutants. acidilactici PAC1Æ0 producer stabbed strain with soft agar containing potential P. 1 mmol l)1 MgCl2. The growth inhibition of indicator bacteria was detected after 12 h of incubation at 37°C. NCK 537 were poured on the plates.600 nm. The bacteriocin activity of the CPP was evaluated and expressed in arbitrary units (AU) per millilitre as described previously (Bhunia et al. Antibacterial activity test and selection of pediocin AcH/PA-1 resistant mutants Pediococcus acidilactici PAC 1Æ0 was tested for the pediocin AcH/PA-1 production as previously described (Mora et al. centrifuged at 13000 ·g for 10 min and the supernatant ﬂuid containing the cell-protein extract recovered. pH 6Æ8. 2% (w/v) SDS. lactis subsp. were added to the cell suspension and the O. The obtained suspension was vigorously mixed and boiled at 100°C for 5 min.D. a 100 ll of CPP was serially diluted and 5 ll of each dilution was placed in a MRS agar plate inoculated with 107 CFU ml)1 of Lactobacillus sp. monocytogenes MACa1 and C. Pediocin resistant mutants were obtained isolating colonies from P. After electrophoresis. A volume of 3 ml of cell suspension were equilibrated at 37°C and the O. Substrate speciﬁcity of peptidoglycan hydrolases was evaluated as follows: cells of L. Mutant strains selected were then tested for their bacteriocin resistance as described above. Cell proteins extraction Cells grown in MRS broth were harvested in exponential phase. acidilactici PAC1Æ0 was stabbed onto MRS plates and incubated at 37°C for 3 h. distilled water for 30 min and then in 200 ml of 25 mmol l)1 Tris-HCl (pH 7) containing 1% (w/v) of Triton X-100 for 12–36 h. cremoris 2250. The effects of different chemicals on peptidoglycan hydrolases activity were evaluated by incubating gel slices in renaturation buffer containing CaCl2 (0Æ5–15 mmol l)1) or NaCl (0Æ5–8%) or 200 ll of protease inhibitor cocktail (PIC. 1991). Bestatin (130 lmol l)1). Subsequently 50 ll of mutanolysin solution (150 U ml)1) (Sigma) prepared in TES buffer (N-tris-Hydroxymethyl-methyl-2-aminoethanesulfonic acid) (50 mmol l)1.D. Brieﬂy. as described above.600 nm of about 0Æ5. Milano. P. L. Preparation of concentrated crude pediocin AcH/PA-1 solution and evaluation of the bacteriocin titre Concentrated pediocin AcH/PA-1 solution was obtained by ultraﬁltration of 200 ml of PAC1Æ0 MRS culture supernatant using a YM2 Diaﬂo membrane (cut-off 1000 Da.AUTOLYTIC ACTIVITY IN PEDIOCOCCUS 563 buffer (50 mmol l)1. Leupeptin (1 lmol l)1) and Aprotinin (0Æ3 lmol l)1).D. The molecular weight of the lytic bands were estimated by running in the same gel a prestained molecular weight marker (New England Biolabs. Journal of Applied Microbiology. acidilactici or P. delbrueckii subsp. 561–570 . (1995) with 12% polyacrilamide separating gel in a Mini-Protean III apparatus (Biorad Laboratories. washed and resuspended in 1 ml of sterilized distilled water and incorporated into polyacrylamide running gel. pentosaceus strains grown in the presence of 500 ll of ﬁlter sterilized PAC1Æ0 MRS culture supernatant. One AU of pediocin AcH/PA-1 was deﬁned as 5 ll of the highest ª 2003 The Society for Applied Microbiology. acidilactici or P. washed in sterile water and resuspended in 100 ll of extraction solution containing 10 mmol l)1 Tris-HCl pH 8.
The autolytic proﬁles of Pediococcus strains are shown in Fig. strain DSM 20283 and DSM 20238 showed an increase of autolytic activity of 23Æ6 and 9Æ3% respectively in phosphate buffer.D. pentosaceus PPE1Æ0.564 D . 1 Extent of autolysis of Pediococcus acidilactici and P. ranging between 40Æ5 and 90Æ7% and between 17 and 95% respectively (Fig. The extent of autolysis after starvation in potassium phosphate and MES buffer was comparable.600 nm. Cell suspensions were divided into two aliquots one of which was implemented with 128 AU ml)1 of crude pediocin AcH/PA-1 solution and incubated at 37°C for 8 h. dilution of crude preparation yielding a deﬁnite zone of growth inhibition. The autolytic levels in phosphate and in MES buffer were generally comparable with the exception of P. acidilactici DSM 20238. The prolongation of the time of starvation had no signiﬁcant effect on the extent of autolysis. and by several additional signals in a range of molecular weight between 45 and 110 kDa detectable after RESULTS Autolytic behaviour and mutanolysin activity of Pediococcus acidilactici and P. Moreover. Detection and activity spectrum of peptidoglycan hydrolases of Pediococcus strains The detection of peptidoglycan hydrolases of Pediococcus strains was carried out in renaturing SDS-PAGE containing cells of M.D. 2a). pH 6Æ5). pentosaceus PPE1Æ0. 2002) evaluating the mutanolysin activity on cells harvested in exponential growth phase. resuspended in the same buffer to an O. detectable after 1–2 h of gel renaturation (Fig. lactis BMG6Æ02 (Ouzari et al. Most of the analysed strains showed a high level of autolysis (between 65 and 95%) with the exception of P. Journal of Applied Microbiology. The degree of autolysis was monitored evaluating the decrease of the O. The results obtained showed level of mutanolysin activity ranging between 65 and 96% for the majority of the analysed strains. P. 1). Effect of pediocin AcH/PA-1 on the autolytic phenotype of Pediococcus strains The effect of pediocin on the rate of autolysis of Pediococcus strains was carried out on harvested exponential phase cells (O. The integrity of the cell wall structure and the cell osmotic sensitivity of Pediococcus strains was carried out as previously described (Ouzari et al.600 nm 0Æ8-1) grown in MRS. acidilactici and P. 2. washed in potassium phosphate buffer (50 mmol l)1. lysodeikticus as substrate for the enzymatic activity. ATCC 8081 and LMG 17680 that showed an increase of autolytic activity between 20Æ8 and 36Æ7% when the starvation was carried out in MES buffer. 2002) used as reference strain for the peptidoglycan hydrolase activity. the lowest values detected were 30% for P. WB1104/99. pentosaceus strains The extent of autolysis of Pediococcus strains was evaluated in phosphate buffer after 48 h of incubation.D. 561–570 . 94. The autolytic proﬁles were compared with that obtained in the same conditions for L. M O R A ET AL. buffer and between 40Æ5 and 44Æ5% in phosphate buffer. pentosaceus DSM 20283 chracterized by level of autolysis ranging between 17 and 53% in MES 100 100 50 50 25 25 Mutanolysin activity (%) Extent of autolysis (%) 75 75 0 DS 2 M 83 AT 20 CC 23 8 12 69 7 P W PE B 1· 11 0 AT 04/ CC 99 BC 80 81 C BC M 1 7 CM 68 0 BC 17 CM 692 AT 1 CC 76 33 87 31 4 BC CM PG DS 17 M 674 AT 202 TC 84 2 AT 57 TC 40 DS 80 M 42 BC 20 CM 282 17 6 PA 89 C1 ·0 20 0 Fig. ATCC 12697 and P.600 nm of 0Æ8–1Æ2. pentosaceus strains were both characterized by a 116 kDa autolytic signal. lactis subsp. pentosaceus strains grown in MRS broth and resuspended in potassium phosphate buffer (grey bars) or in MES buffer (white bars) and extent of mutanolysin activity in MES buffer (black diamonds) DS M ª 2003 The Society for Applied Microbiology. pentosaceus DSM 20283 and 33Æ5% for P.
acidilactici ATCC 25740. using cells of L. pentosaceus PPE1Æ0. pentosaceus PPE1Æ0 were grown until reaching the exponential phase in presence of 200 ll of PIC. acidilactici DSM 20284T. P. lactis subsp. delbrueckii subsp. The results obtained showed identical zymogram proﬁles between PIC-treated cells and control cells. lactis BMG6. acidilactici and P. The lytic pattern shown by Pediococcus strains against cells of M. lane 4: P.lactis BMG6Æ02 by adding PIC to the renaturing buffer after the SDS–PAGE. In this context. P. lactis BMG6Æ02 evaluated by renaturing SDS–PAGE containing autoclaved cells of Micrococcus lysodeikticus using standard renaturation condition (a) and using renaturation buffer implemented with 200 ll of protease inhibitor cocktail (b). acidilactici PAC1Æ0 and P. 94. lane 3: L.AUTOLYTIC ACTIVITY IN PEDIOCOCCUS 565 1 (a) 2 3 4 5 M (b) 1 2 3 4 5 M kDa 175 83 AcmA 62 47·5 AcmA Fig. lactis BMG6Æ02. lactis BMG6Æ02. pentosaceus PPE1Æ0. lane 5: P. N-acetyl muramidase (AcmA) of L. lane 2: P. monocytogenes MACa1 were used. lactis BMG6Æ02 is indicated 116 kDa 41·6 kD ª 2003 The Society for Applied Microbiology. PAC1Æ0 and P. pentosaceus PPE1Æ0 whole-cell SDS in renaturing SDS–PAGE substituting M. helveticus ATCC 15009T. lysodeikticus cells with cells of other lactic acid bacteria and cells of dairy-spoilage and pathogenic bacteria. 561–570 . lysodeikticus was maintained. 3) showed an evident decrease of intensity in the activity of peptidoglycan hydrolases in PIC-treated renaturing SDS–PAGE compared with those obtained in standard condition. additional lytic bands 2 3 (b) 1 2 3 Fig. Journal of Applied Microbiology. The whole-cell protein extraction obtained was analysed in renaturing SDS-PAGE and compared with the control samples prepared using the standard protocol. acidilactici DSM 20284T . acidilactici PAC1Æ0. Lane 1: L. lactis BMG6Æ02. L. 2 Peptidoglycan hydrolases proﬁle of Pediococcus acidilactici. N-acetyl muramidase (AcmA) of L. The activity spectrum of peptidoglycan hydrolases of Pediococcus strains was evaluated using P. ATCC 25740. bulgaricus ATCC 11842T as target. pentosaceus strains were extremely similar with some variation in the intensity of the zymogram signals. acidilactici PAC1Æ0. pentosaceus (a) 1 PPE1Æ0 and L. lactis BMG6Æ02 and L.02 is indicated. Interestingly. acidilactici PAC1. M ¼ Pre-stained molecular weight marker (New England Biolabs) 12–18 h of gel renaturation (Fig. 2b). assuming that SA bands were originated by partial proteolytic degradation of the main lytic band of 116 kDa. in the range of molecular weight between 62 and 116 kDa. Because of the unexpected number of lytic signals in the zymogram analysis of Pediococcus strains the nature of SA bands detectable after 12–18 h of gel renaturation was investigated. 3 Peptidoglycan hydrolases proﬁle of Pediococcus acidilactici. excluding the proteolytic degradation hypothesis for SA bands. lane 2: P. No species-speciﬁc zymogram proﬁles were detected. The exact number of the additional secondary autolytic (SA) bands was not easy to detect but ranged between 6 and 8. lactis BMG6Æ02 evaluated by renaturing SDS–PAGE containing autoclaved cells of Micrococcus lysodeikticus after 1 h (a) and 18 h (b) of renaturation. pentosaceus and Lactococcus lactis subsp. P. P. lane 3: P. Further experiment was carried out on whole-cell protein extraction of P. The results obtained (Fig. When cells of L. The peptidoglycan hydrolase proﬁles of P.0. pentosaceus and Lactococcus lactis subsp. Lane 1: P. the inhibition of peptidoglycan hydrolase activity by PIC treatment was evident only in the peptidoglycan hydrolases proﬁle of Pediococcus while no reduction in activity was detectable for N-Acetyl muramidase AcmA of L.
4). 561–570 . 1% of Triton X-100 with addition of: (a) NaCl 1%. the mutanolysin. 5). (d) CaCl2 1 mmol l)1 . acidilactici or P. The secondary lytic bands in the range of molecular weight from 40–50 kDa were detectable only in presence of CaCl2 (1–15 mmol l)1) and at pH 5. Lane 1: P. (a) 116 kDa (b) (c) (d) The potential effect of pediocin AcH/PA-1 on the autolytic phenotype of Pediococcus strains was carried out using two different approaches: (i) in the ﬁrst approach the effect of pediocin AcH/PA-1 on Pediococcus cell lysis was evaluated in association with a commercial available autolysin. (ii) in the second approach the effect of pediocin AcH/PA-1 was directly tested on Pediococcus strains. With regards to the interaction between (e) (f) (g) (h) 62 kDa pH 5 NaCl 1% 5% 8% 8 9·5 CaCl2 1 mmol l–1 15 mmol l–1 Fig. showed that the main lytic band of 116 kDa and the secondary lytic bands ranging from 62 to 110 kDa retained their activity in all the conditions tested. pentosaceus evaluated by renaturing SDS–PAGE containing cells of Listeria monocytogenes MACa1. The analysis of several aliquots of concentrated CPP AcH/PA-1 by renaturing SDS-PAGE did not allow to detect the presence of peptidoglycan hydrolases. M O R A ET AL. (c) NaCl 8%. using renaturing buffer at different pH values: (e) 10 mmol l)1 sodium acetate pH 5. 94. The results obtained (Fig. 25 mmol l)1 Tris-HCl (pH 7). (e) CaCl2 15 mmol l)1. lane 4: P. sporogenes CL14 and surprisingly. lane 3: P. NaCl (0Æ5–8%. used as reference strains. acidilactici PAC1Æ0. pentosaceus were used as target for peptidoglycan hydrolases activity in renaturing SDS– PAGE. no lytic activity was detected when cells of P. (f) 10 mmol l)1 Tris-HCl pH 8. Effect of pediocin AcH/PA-1 on the autolytic phenotype of Pediococcus strains 83 62 47·5 30 Fig. Standard renaturation buffer.566 D . Consequently the concentrated CPP was used in the following experiments. (g) 10 mmol l)1 Tris-HCl pH 9Æ5 ª 2003 The Society for Applied Microbiology. A signiﬁcant reduction of activity was detected in the presence of NaCl 8% (w/v). w/v) on peptidoglycan hydrolases of Pediococcus pentosaceus DSM 20284T. 1 2 3 4 kDa 175 Inﬂuence of pH and chemical compounds on the peptidoglycan hydrolases proﬁle The inﬂuence of different pH (5–9Æ5) and different concentrations of CaCl2 (1–15 mmol l)1). acidilactici ATCC 25740. 4 Peptidoglycan hydrolases proﬁle of Pediococcus acidilactici and P. pentosaceus PPE1Æ0 at low molecular weight (30–35 kDa) were detected (Fig. were evaluated changing the renaturation condition after SDS–PAGE. No activity was detected on C. (b) NaCl 5%. Journal of Applied Microbiology. acidilactici DSM 20284T. Peptidoglycan hydrolases proﬁle of Pediococcus acidilactici DSM 20284T evaluated as described above. lane 2: P. 5 Peptidoglycan hydrolases proﬁle of Pediococcus acidilactici DSM 20284T evaluated against autoclaved cells of Micrococcus lysodeikticus using different renaturation conditions.
WB. LMG 17680. washed and resuspended in MES buffer implemented with: (M) 1Æ5 U per ml of mutanolysin. Likewise. acidilactici DSM 20238. The extent of autolysis were not linked to the species but appeared as a strain dependent character in some cases inﬂuenced by the buffered solution composition (P. 94. (C) Cells harvested as described above and resuspended in MES buffer. pentosaceus DSM 20283) were characterized by low level of autolysis. PPE1Æ0. pentosaceus PPE1Æ0 became about two and ﬁve times more sensitive to the mutanolysin activity while pediocin resistant strains (PAC1Æ0 and PAC750F) were not affected by pediocin in their mutanolysin sensitivity. In speciﬁc. pentosaceus DSM 20283. the experiment was carried out as previously described for the evaluation of the osmotic sensitivity but decreasing the amount of mutanolysin from 5 U ml)1 to 1Æ5 U ml)1 and introducing 50 AU ml)1 of pediocin AcH/PA-1. the lowest values of mutanolysin activity were detected for strains showing the lowest values of autolysis extent in MES buffer (P. DISCUSSION The autolytic phenotype of P. carried out in MES buffer. only three strains (P. acidilactici DSM 20238 (pediocin sensitive) and its derivative mutant pediocin resistant (pedR) MI20238R and on P. P. The level of spontaneous autolysis evaluated during the experiment ranged between 0Æ7 and 6Æ2% and was therefore considered negligible (Fig. (1997). For all the strains cells were harvested in exponential growth phase. The results obtained (Fig. ATCC 8081. pedS ¼ pediocin sensitive strain C P. WB). 5). pentosaceus PPE1Æ0 and its derivative pedR mutant MIPPE1Æ0R. acidilactici DSM 20238. The peptidoglycan hydrolases proﬁle. acidilactici PAC1·0 (pedR) MP M C P. 7) were according to previous data related to the effect of pediocin on mutanolysin activity. The mutanolysin activity test. acidilactici PAC1Æ0 (pediocin producing and pediocin resistant). highlighted differences in the cell wall and/or cell osmotic sensitivity among the analysed strains. all pediocin sensitive strains tested showed an increase of the rate of autolysis in the presence of pediocin while in pedR strains the bacteriocin triggering effect on cell lysis was not detected. pentosaceus strains isolated from different environments was evaluated. This result was expected because ª 2003 The Society for Applied Microbiology. The majority of the strains tested showed a high level of autolysis both in potassium phosphate and in MES buffer. Despite the peptidoglycan hydrolase proﬁle is known to be speciesspeciﬁc as reported by Lortal et al. LMG 17674). was surprisingly complex because it was composed by 6–10 lytic bands. In speciﬁc. PPE1Æ0). Nevertheless. pedR ¼ pediocin resistant strain. lactis strains. 6) showed a strong synergic effect of the pediocin AcH/PA-1 and mutanolysin on cell lysis. Pediocin sensitive P. all the strains tested showed almost the same proﬁle with some variation in the intensity of the lytic signals and no species-speciﬁc proﬁle were detected. (2002) who described a correlation between cell wall sensitivity to mutanolysin and autolytic activity in L. acidilactici DSM 20238 (pedS) M C MP P. These results were in accordance with the data reported by Meijer and coworkers (Meijer et al. The results obtained (Fig. The effect of pediocin on the rate of autolysis was carried out on P. acidilactici PAC1Æ0. pentosaceus PPE1·0 (pedS) M MP Fig. its plasmid-cured derivative PAC750F (pediocin resistant). pentosaceus DSM 20283. Journal of Applied Microbiology. acidilactici and P. by comparing the mutanolysin activity and the extent of autolysis among all the tested strains it was not possible to extrapolate a strict correlation between the two enzymatic activities. ATCC 12697 and P. DSM 20282. acidilactici DSM 20238 and P. 561–570 . the highest values of mutanolysin activity were detected in highly autolytic strains (P. detected by the SDS– PAGE renaturing method. P. 6 Effect of pediocin AcH-PA-1 on the mutanolysin sensitivity of Pediococcus acidilactici and P. (MP) 1Æ5 U per ml of mutanolysin and 50 AU per ml of pediocin AcH-PA-1. acidilactici PAC750F (pedR) 0 20 40 60 Extent of autolysis (%) MP M 80 100 pediocin and mutanolysin. 2001) and by Ouzari et al. pentosaceus strains.AUTOLYTIC ACTIVITY IN PEDIOCOCCUS 567 C P.
5 0. 1991). 94. P. acidilactici and P. (600 nm) P. P. peptidases for Pediococcus and N-acetyl muramidase for L. pentosaceus was characterized by a main lytic band of 116 kDa detectable after few hours of renaturation and by several additional bands detectable only after 12 h of renaturation. In this context. pentosaceus MIPPE1Æ0R is the pedR mutant derived from P.2 1.e. In speciﬁc. pentosaceus PPE1·0 (pedS) P. 3) suggesting that this molecular species could be ascribed to the same ª 2003 The Society for Applied Microbiology. The peptidoglycan hydrolases proﬁle of P. The peptidoglycan hydrolases proﬁle visualized by renaturing SDS–PAGE do not allow to determine the exact number of enzymes present in bacteria. pedS ¼ pediocin sensitive strain.9 0.7 0. 7 Effect of pediocin AcH/PA-1 on the rate of autolysis of Pediococcus acidilactici and P. acidilactici PAC750F (pedR) O. The differences in enzymatic activity after PIC treatment between Pediococcus and Lactococcus peptidoglycan hydrolases. M O R A ET AL.6 0. (2000). post-translational modiﬁcation and proteolytic degradation may produce fragments that retain enzymatic activity as observed in L. pentosaceus PPE1Æ0 pedS. ﬁrst because only enzymes able to renature after SDS treatment are susceptible to detection. Moreover these two species were closely related at phylogentic level showing 16S rDNA similarity value above 98% (Collins et al. 1⋅3 1⋅2 1⋅1 1 0⋅9 0⋅8 0⋅7 0⋅6 0⋅5 1⋅3 1⋅2 1⋅1 1 0⋅9 0⋅8 0⋅7 0⋅6 0⋅5 0⋅4 1.D. On the other hand. preliminary experiments carried out using PIC on growing cells of Pediococcus strains did not determine variation in the peptidoglycan hydrolases proﬁles while a strong reduction in enzymatic activity was observed when the standard renaturation buffer was implemented with PIC. acidilactici DSM 20238 (pedS) 1 2 3 4 5 6 7 8 0 P.8 0. lactis. For all the strains cells were harvested in exponential growth phase.1 1 0. acidilactici DSM 20238 pedS P. washed and resuspended in potassium phosphate buffer (r) and implemented with 128 AU per ml of pediocin AcH/PA-1 (u). lactis BMG6Æ02. (600 nm) P. i. pentosaceus strains. acidilactici MI20238R is the pedR mutant derived from P. (1995) and by Selvarani et al. 561–570 . the reduction of enzymatic activity by PIC treatment was less evident in the main lytic band of 116 kDa (Fig. acidilactici PAC1·0 (pedR) P. Interestingly. a signiﬁcant inhibition in the enzymatic activity of peptidoglycan hydrolases of Pediococcus was detected when PIC was added in the renaturation buffer while no reduction in activity was detected for AcmA of L. (600 nm) P.568 D . 1993). These results suggested that proteolytic degradation should not be involved in the generation of secondary bands. pentosaceus were extremely similar at phenotypic level and hardly differentiated by physiological tests in which only maltose utilization seems to be discriminating (Tanasupawat et al. acidilactici MI20238R (pedR) 1 2 3 4 5 6 7 8 Time (h) Fig. secondly. could be explained assuming that autolytic enzymes of the two genera belong to different classes of enzymes.D. Journal of Applied Microbiology.D.4 0 O. acidilactici and P. lactis by Buist et al. pedR ¼ pediocin resistant strain. the number of bands detected may not reﬂect the exact number of peptidoglycan hydrolases present in the cell. pentosaceus MIPPE1·0R (pedR) O.
L. Mengaud. monocytogenes. we should exclude a simple mechanic action of the pediocin on the cell membrane. Chapot-Chartier. REFERENCES Bhowmik. A.A.. European Journal of Clinical Microbiology and Infection Disease 19. It was suspected that while cell death results from the direct action of pediocin.. M.K. 230–235. Despite the pediocin mechanism of antibacterial action has been investigated in several study (Ray and Miller 2000)..J. peptidoglycan hydrolases activity was not detected when cells of Pediococcus strains were used as target cells in renaturing SDS– PAGE. Further experiments were performed to evaluate the effect of pediocin AcH/PA-1 on the level and the rate of autolysis.. produced by P... a Streptococcus thermophilus peptidoglycan ª 2003 The Society for Applied Microbiology. Considering that pediocin resistant strains and mutants retained their autolytic activity without accelerating the autolysis rate in presence of pediocin. Leenhouts. Riesterer R. In this context we could assume that cell lysis requires the action of the lytic enzymes of the strains while pediocin could have an indirect role in triggering lysis determining a rapid collapse of the membrane potential and increasing the osmotic fragility of the cell. A. (1997) Evolution of lactic acid microﬂora during Grana cheese-making and ripening. Heinz.. A.M. Lohbrunner.A. Surprisingly. Rodriguez. lactis. Kok. Listeria and Clostridium strains. M. M. Ash. A. 1554–1563. and Marth. Staphylococcus. and Haandrikman. and Chapot-Chartier. 862–869. 1997). F. Journal of Applied Microbiology 89... Moter. This unexpected result could be explained by the absence of peptidoglycan hydrolases renaturation in presence of Pediococcus strains because of unknown interaction between lytic enzymes and some molecular component of the cell envelope or by other factors that should be investigated. M. U... Microbiologie Aliments Nutrition 15. J.. 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