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Identification of Classical Propionibacterium Species Using 16S rDNA — Restriction Fragment Length Polymorphisms
System. App!. Microbio!. 21 , 419-428 (1998) _©_G_us_ta_vF_is_ch_er_V_er_lag_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
SYSTEI'V14l1C AND APPLIED MICROBIOLOGY
Identification of Classical Propionibacterium Species Using 16S rONA - Restriction Fragment Length Polymorph isms K.-H.]. RIEDEL\ B. D. WINGFIELD l and T. J. BRITZ2 1 2
Department of Microbiology and Biochemistry, University of the Orange Free State, Bloemfontein, South Africa Department of Food Science, University of Stellenbosch, Stellenbosch, South Africa
Received June 18, 1998
Summary The phenotypic identification of the classical propionibacteria is essentially still problematic and alternative techniques for the identification of the various species are required. A rapid and sensitive technique for the routine identification of the classical propionibacteria, based on the amplification of the 16S rRNA genes using the polymerase chain reaction and the subsequent restriction endonuclease digestion of the PCR products, was previously described. Although this technique enabled differentiation between the various classical species examined it was only evaluated on a limited number of type and reference strains. During this study, the taxonomic relationship between 135 Propionibacterium strains from diverse ecological niches, representing four classical species was investigated using this PCRJRFLP technique. Visual differentiation between the classical Propionibacterium species was possible after restriction endonuclease digestion of the PCR products obtained using primers 16sPI-16sP4 and 16sP3-16sP4 with the restriction endonucleases HaeIlI, AluI and HpaII, respectively. With the exception of strains independently identified as "P. rubrum" and "P. sanguineum", the results of this study confirm the consolidation of the "old" species into the various classical species as they currently exist. It was therefore concluded that the PCR/RFLP protocol is suitable for the rapid and routine identification of the classical propionibacteria. Key words: Classical Propionibacterium - PCR - RFLP - Taxonomy -16S ribosomal RNA
Introduction The classical propionibacteria, especially Propionibacterium freudenreichii are used extensively in the dairy industry as starter cultures for the manufacture of Swisstype cheese. The propionibacteria are also important in the food industry, where they are responsible for the production of organic acids, biomass, vitamin B12 and other specific metabolites (MARCOUX et al., 1992). Although the classical propionibacteria were extensively used for the commercial production of vitamin B 12 , this vitamin is currently being produced using a faster growing Pseudomonas strain (GLATZ, 1992). The production of propionic and other minor organic acids, CO 2, bacteriocins, vitamins and the antimutagenic properties displayed by propionibacteria (VOROBJEVA et al., 1996) may be of importance in the application of these organisms as probiotic agents in foods. The propionibacteria are also receiving increasing attention due to the fact that they rank amongst the most potent immunomodulatory stimulating cell populations involved in non-specific resistance (ROSZKOWSKI et al., 1990). Twenty three forms of the genus Propionibacterium were isolated and described during the period 1906 and
1944 (BRITZ and STEYN, 1980). Of these only 11 have been regarded as species, primarily as a result of the studies of VAN NIEL (1928). Other isolates which were described as new species (SAKAGUCHI et al., 1941; JANOSCHEK, 1944) were considered synonyms of known species and rejected. Since this period, there are several references to Propionibacterium isolates which could not be correctly identified, and that do not correspond to the species as described by BREED et al. (1957) (MALIK et al., 1968; SEYFRIED, 1968). Based primarily on the results of JOHNSON and CUMMINS (1972), MOORE and HOLDEMAN (1974) consolidated the 11 previously described Propionibacterium spp. into four species. They also transferred the anaerobic propionic acid-producing corynebacteria to the genus Propionibacterium to form the cutaneous species. Although this improved the taxonomy of the cutaneous species, it did not facilitate the identification of the classical species and several reports can be found in the literature (BRITZ and STEYN, 1980; BRITZ and RIEDEL, 1991, 1994, 1995; RIEDEL and BRITZ, 1992; 1993; DUPUIS et al., 1993) indicating problems encountered with the identification of classical strains. Due to
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K.-H. J. RIEDEL et al.
the close relationship between the various species, differentiation is difficult, as separation is currently based on five biochemical characteristics: fermentation of sucrose and maltose; reduction of nitrate; ~-hemolysis; colour of pigment; and isomer of diaminopimelic acid in the cell wall (CUMMINS and JOHNSON, 1986). Species differentiation is, however, not always reproducible due to variation in these specific phenotypic characteristics (BRITZ and STEYN, 1980; BRITZ and RIEDEL, 1991, 1994, 1995; RIEDEL and BRITZ, 1993). Alternative classification techniques, therefore, need to be investigated. Essentially due to the limitations of the phenotypic identification schemes, RIEDEL et al. (1994) developed a rapid and sensitive technique for the routine identification of the classical propionibacteria based on the amplification of the 16S rRNA genes using the polymerase chain reaction and the subsequent restriction endonuclease digestion of the PCR products. Although this technique enabled differentiation between the various classical species examined, it was only evaluated on a limited number of type and reference strains, all of dairy origin. The aim of this study was, therefore, to evaluate the suitability of the PCR/RFLP technique as previously described by RIEDEL et al. (1994) on classical Propionibacterium species from diverse ecological niches. The value of restriction endonuclease digestion of the amplified rDNA fragments, in the taxonomy of the classical Propionibacterium, was also determined and the results obtained were compared with those of various phenotypic and numerical analyses.
DNA isolation and purification: All strains were grown anaerobically at 32°C for four days in the medium of CHARFREITAG et al. (1988), with the addition of a 4% (v/v) mineral salts solution (HOLDEMAN et al., 1977). Genomic DNA was isolated, as described by SAMBROOK et al. (1989) from 0.5 g (wet weight) biomass of each strain. The methods used for agarose and polyacrylamide gel electrophoresis are as described by PERBAL (1988). Polymerase chain reaction: The primer design, reaction mixture composition and optimum conditions for amplification of the 16S rDNA are as described by RIEDEL et al. (1992, 1994). Restriction endonuclease digestion and gel electrophoresis: The resulting PCR products were extracted from the PCR reaction mixture using chloroform, purified using Quiagen columns (Diagen, Dusseldorf), precipitated with iso-propanol and redissolved in TE buffer (10 mM Tris-base, 1 mM EDTA, pH 8.0). The PCR products, obtained using the primer sets 16sPI-16sP4 and 16sP3-16sP4, were digested using the restriction endonucleases AZul, HaeIII and Hpall (Boehringer Mannheim, GmbH, Germany), respectively (RIEDEL et al., 1992, 1994). Restriction endonuclease digestions were performed on approximately 0.5 pg of amplified DNA using 5 U of restriction enzyme for 5 h at 37°C in the incubation buffers supplied by Boehringer Mannheim (GmbH, Germany). Restriction fragments were electrophoresed on 10% polyacrylamide gels (Merck) in TBE buffer (0.089 M Tris-base, 0.089 M Boric acid, 0.002 M EDTA) using a Hoefer SE 600 vertical slab gel unit at a constant voltage of 240 V. DNA molecular-weight marker V (0.25 pg) and DNA molecular-weight marker VIII (0.25 pg) were used as standards (Boehringer Mannheim, GmbH, Germany). The gels were stained using the silver staining technique described by PERBAL (1988). Restriction endonuclease fragment sizes were determined by linear regression analysis, using the molecular weight markers as standards.
Material and Methods
Results
Bacterial strains and culture conditions: The classical Propionibacterium strains used during this study, including various
A large portion (1110 bp) of the 165 rDNA region of the various classical Propionibacterium strains was successfully amplified using the polymerase chain reaction. Products of the predicted sizes, using primers 16sPI-16sP4 (approximately 1110-bp) and primers 16sP3-16sP4 (approximately 250-bp), were obtained and subjected to restriction endonuclease digestion. Comparison between the restriction endonuclease data of the amplified fragment of the various type and reference strains examined previously (RIEDEL et al., 1994) and that of the various strains included in this study was used to distinguish between the various species. Visual differentiation of the various species was possible using the chosen restriction endonucleases. A comparison between strains labelled as originally received, as identified using the classification system of CUMMINS and JOHNSON (1986) and results obtained from the PCR/RFLP as found during this and a previous study (RIEDEL et al., 1994) are given in Table 1.
type and reference strains as well as isolates from a variety of different sources, are given in Table 1. All cultures were preserved by Iyophilisation (JOUBERT and BRITZ, 1987) and stored at -20°C. Working cultures were obtained by inoculation of yeast extract lactate broth (YEL). The YEL-medium consisted of (gil): yeast extract 5.0; sodium lactate (50% v/v) 20.0; peptone 2.0; KH 2P0 4 10.0; hemin 10.0 ml and Tween 80 1.0 ml (pH 7.0) which was prepared according to the method of HOLDEMAN et al. (1977). All strains were cultured anaerobically at 32°C for four days in a Forma Scientific (Malinckrodt, Inc., Ohio, USA) anaerobic cabinet, using oxygen-free nitrogen as gas phase. Culture purity was checked by means of microscopical examination of Gram stains, by comparison of morphological characteristics and by gas chromatographical determination of the major metabolites. The phenotypic identification of the various strains was previously determined (BRITZ and RIEDEL, 1991, 1994; RIEDEL and BRITZ, 1992, 1993). Fermentation products: The major metabolites produced in YEL-medium were determined gas chromatographically using a Hewlett-Packard (Avondale, USA) gas chromatograph equipped with a flame ionisation detector and a 30 m x 0.53 mm i.d. Nukol (Supelco, Inc., Avondale, USA) capillary column. The chromatograph was programmed at an initial temperature of 120°C, then increased at a rate of 6°C/min and finally held at 165°C. The detector and inlet temperatures were 250 °C and 135°C, respectively. Nitrogen, at a flow rate of 5 mllmin, was used as the carrier gas.
Identification of strains based on the restriction endonuclease profile of the PCR products obtained using primers 16sPl-16sP4 The results obtained from the restriction endonuclease analysis (HaeIII, AZul) of the PCR products (16sP1-
Identification of the Classical Propionibacterium Species
421
Table 1. Propionibacterium strains used in this study. Culture Received as No Species
Isolated from Source<
Strain
Identified as Identification key#
PCRJRFLP
P. P. P. P. P. P. P. P. P.
P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici
Propionibacterium acidipropionici group 76 78 90 91 93 95 120 258 263 273 285 300 310 311 346 349 350 352 358 413 421 424 430 432 433 446 451 456
"P. peterssonii" "P. arabinosum" P. isolate P. isolate P. isolate P. iso/ate P. isolate P. isolate P. isolate P. iso/ate P. isolate P. iso/ate P. isolate P. isolate P. isolate P. iso/ate P. isolate P. iso/ate P. isolate "P. pentosaceum " "P. pentosaceum" P. acidipropionici "P. arabinosum" "P. arabinosum" "P. pentosaceum" "P. pentosaceum "P. arabinosum" "P. arabinosum"
6 6 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 10 4 1 4 7 7 6 1 6
P 35 P 50
1101 NCFB 570 ATCC 25562" NCFB 563 B 3569 B 3568 P9 ATCC4965 P78
Swiss cheese Buttermilk Gouda Emmentaler Gouda Gruyere Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Leerdammer Leerdammer Leerdammer Leerdammer Leerdammer Emmentaler Swiss cheese
acidipropionici acidipropionici acidipropionici acidipropionici acidipropionici acidipropionici acidipropionici acidipropionici acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. jensenii P. acidipropionici P. acidipropionici P. acidipropionici
Propionibacterium freudenreichii group 70 73 88 131 296 304 308 309 344 347 348 423 434 435 453 454
P. sp. P. freudenreichii P. isolate
P. freudenreichii P. iso/ate P. isolate P. iso/ate P. isolate P. isolate P. iso/ate P. iso/ate P. freudenreichii "P. shermanii" P. freudenreichii "P. shermanii; P. freudenreichii
6 6 8 9 6 8 8 8 8 8 8
P45 P 57
1
ATCC 620T' B3524 B 3523 P 67 P 73
7 7 6 6
Gouda Emmentaler Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Leerdammer Leerdammer Swiss cheese
P. P. P. P. P. P. P. P. P. P.
P. P. P. P. P.
P.
freud ss. shermanii freud ss. freudenreichii freud ss. shermanii freud ss. globosum freud. ss. freudenreichii freud ss. shermanii freud. ss. shermanii freud. ss. shermanii freud 55. shermanii freud. ss. shermanii freud. ss. freudenreichii freud. ss. freudenreichii freud. ss. shermanii freud. ss. freudenreichii freud. ss. shermanii freud. ss. freudenreichii
P. freudenreichii P. freudenreichii
P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreich ii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreich ii
Propionibacterium jensenii group 74 75 77 79 80 85 86
P. sp.
P. sp. "P. raffinosaceum" "P. peterssonii" P. jensenii P. isolate P. isolate
8 8 6 3 2 8 8
74 75 P 41 VPI5169 DSM 20535"
Emmentaler Emmentaler Emmentaler Buttermilk Gruyere Gouda
P. jensenii P. jensenii P. thoenii P. jensenii P. jensenii P. jensenii P. jensenii
P. acidipropionici jensenii acidipropionici jensenii jensenii P. jensenii P. jensenii
P. P. P. P.
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K.-H. J. RIEDEL et al.
Table 1. (Continued). Culture Received as No Species 87 89 96 98 99 100 121 122 257 259 261 262 264 265 266 267 268 269 270 271 272 274 275 277 278 279 280 281 282 283 284 287 289 290 293 295 299 301 303 313 314 345 351 353 354 355 356 357 359 420 422 427 429 431 449 450 452 455 457
P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate p. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. jensenii "P. technicum" "P. pituitosum" "P. raffinosaceum" "P. peterssonii" "P. raffinosaceum" "P. zeae" P. jensenii "P. technicum" "P. zeae"
Isolated from Source 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 4 4 4 4 4 6 6 6 6 6
Strain
NCFB 572 NCFB 567 NCFB 1077 NCFB 1078 NCFB 565 P P46 P 52 P 74 P 87
Gouda Gouda Emmentaler Gouda Gouda Gouda Gouda Emmentaler Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Leerdammer Leerdammer Leerdammer Leerdammer Leerdammer Leerdammer Leerdammer Buttermilk Edam
Emmentaler
Gruyere
Identified as Identification key#
PCRlRFLP
P. jensenii P. jensenii P. jensenii p. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. thoenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii
P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. acidipropionici P. jensenii P. acidipropionici P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. acidipropionici P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. acidipropionici P. thoenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. acidipropionici
Identification of the Classical Propionibacterium Species
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Table 1. (Continued). Culture Received as No Species
Isolated from Source
Identified as
Strain
Identification kel
PCRJRFLP
P 10 P4
P. jensenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. jensenii P. thoenii P. thoenii P. jensenii P. thoenii P. jensenii P. thoenii P. jensenii
P. jensenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. jensenii P. thoenii P. thoenii P. jensenii P. thoenii P. jensenii P. thoenii P. jensenii
Propionibacterium thoenii group 71 72 92 97 260 276 286 288 291 292 294 297 298 302 306 307 312 130 132 419 425 426 428 447 448
"P. rubrum"
P. thoenii P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate "P. intermedium" "P. wentii" P. thoenii .. P. sanguineum" "P. wentii" "P. ruburm" P. thoenii "P. rubrum"
6 6 8 8 8 8 8 8
Gruyere Emmentaler Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester
8 8 8 8 8 8 8 8 8 5 5 4 4 4 4 6 6
NCIB 8728 NCIB 8905 NCFB 568" NCFB 1080 NCFB 1083 NCFB 571 P 15 P 21
Emmentaler
Buttermilk
" Type strain Identification key of CUMMINS and JOHNSON (1986) 1- ATCC, American Type Culture Collection, Rockville, Maryland, USA 2 - DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany 3 - VPI, Virginia Polythechnic Institute, USA 4 - NCFB, National Collection of Food Bacteria, Shinfield, Reading, UK 5 - NCIB, National Collection of Industrial bacteria, UK 6 - G.W. Reinbold, Iowa State University, Iowa, USA 7 - U.S. Dept. of Agriculture, USA 8 - Environmental Bacteriology Culture Collection, University of the Orange Free State, South Africa 9 - Hansens Laboratory, Denmark 10 - National Research Council of Canada, Canada
#
16sP4) of the four type strains (P. acidipropionici ATCC 25562, P. freudenreichii subsp. freudenreichii ATCC 6207, P. jensenii - DSM 20535 and P. thoenii NCFB 568) are shown in Fig. 1.
• Propionibacterium acidipropionici group The restriction endonuclease patterns obtained for strains originally received labelled as either "P. pentosaceum" or "P. arabinosum" and now classified as P. acidipropionici (CUMMINS and JOHNSON, 1986), as well as isolates that were phenotypically identified as P. acidipropionici, were identical (results not shown) to the profile obtained for the P. acidipropionici type strain (ATCC 25562) (Fig. 1, lanes 3 and 4). Although originally received as "P. peterssonii" (UOFS 76), this strain gave
a restriction endonuclease profile identical to that of the P. acidipropionici species, confirming its phenotypic identity. "Propionibacterium raffinosaceum" - UOFS 77, which was phenotypically identified as a P. thoenii strain gave a restriction endonuclease profile identical to that of the P. acidipropionici species.
• Propionibacterium freudenreichii group Similarly, it was found that the restriction endonuclease pattern obtained for all strains originally received labelled as either P. freudenreichii or "P. shermanii" as well as isolates that were phenotypically identified as either P. freudenreichii subsp. freudenreichii, P. freudenreichii subsp. globosum or P. freudenreichii subsp. shermanii were identical (results not shown) to that obtained for
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1
2 3
4
5
6
7 8
9
o
504 04 -
2 2184 24 -
Fig. 1. Silver-stained 10% polyacrylamide gel displaying fragments obtained after restriction endonuclease digestion (HaeIIl and AZul) of the amplification products (1100 bp) obtained using primers 16sPl-16sP4. Lanes: 1 - DNA molecular weight marker V; 2DNA molecular weight marker VIII; 3 - P. acidipropionici ATCC 25562 - HaeIII; 4 - P. acidipropionici ATCC 25562 - AZul; 5 - P. freudenreichii subsp. freudenreichii ATCC 6207 - HaeIII; 6 - P. freudenreichii subsp. freudenreichii ATCC 6207 - AZul; 7 - P. jensenii DSM 20535 - HaeIII; 8 - P. jensenii DSM 20535 - AZul; 9 - P. thoenii NCFB 568 - HaeIII; 10 - P. thoenii NCFB 568 - AZuL
the P. freudenreichii subsp. freudenreichii type strain (ATCC 6207) (Fig. 1, lanes 5 and 6). • Propionibacterium jensenii group With a few exceptions, most of the strains originally received labelled or isolated and phenotypically identified as either P. jensenii, "P. peterssonii", "P. pituitosum", "P. raffinosaceum", "P. technicum" or "P. zeae" all gave restriction endonuclease profiles identical (results not shown) to the P. jensenii type strain (DSM 20535) (Fig. 1, lanes 7 and 8). One "P. technicum" strain, however, ("P. technicum" - UOFS 422) gave a restriction endonuclease profile similar to that of the P. thoenii species. In contrast, the other "P. technicum" strain included in this study ("P. technicum" - UOFS 455), was found to have a restriction endonuclease profile identical to that of the P. jensenii type (DSM 20535) and reference strains. Although most of the strains which were phenotypically identified as P. jensenii using the identification system of CUMMINS and JOHNSON (1986) (BRITZ and RIEDEL, 1991, 1994; RIEDEL and BRITZ, 1991,
1993) gave a restriction endonuclease pattern identical to that of the P. jensenii type (DSM 20535) (Fig. 1, lanes 7 and 8) and reference strains, a number of reference strains and isolates displayed a restriction endonuclease pattern identical to that of the P. acidipropionici type (ATCC 25562) (Fig. 1, lanes 3 and 4) and other P. acidipropionici strains. All the "P. rubrum" and "P. sanguineum" strains included in this study, displayed a restriction endonuclease pattern identical to that of the P. jensenii type (DSM 20535) and other P. jensenii strains. • Propionibacterium thoenii group All the isolates and reference strains which were phenotypically identified as P. thoenii displayed a restriction endonuclease pattern identical to that of the P. thoenii type strain (NCFB 568) (Fig. 1, lanes 9 and 10). • "Propionibacterium wentii" and "P. intermedium" Both the "P. wendii" strains included in this study gave a restriction endonuclease profile identical to that of the P. thoenii type (NCFB 568) (Fig. 1, lanes 9 and 10)
Identification of the Classical Propionibacterium Species
and other P. thoenii strains. The "P. intermedium" UOFS 130 strain included in this study, gave a restriction endonuclease profile identical to that of the P. jensenii type and other P. jensenii strains.
Identification of strains based on the restriction endonuclease profile of the peR product obtained using primers 16sP3-16sP4
Propionibacterium acidipropionici, P. jensen ii, P. freudenreichii and P. thoenii. The results obtained from the restriction endonuclease analysis using the restriction endonuclease HpaII, on the PCR products obtained using the primer set 16sP3-16sP4, of the type strains of the various classical Propionibacterium species are shown in Fig. 2. Due to identical restriction endonuclease profiles, visual differentiation between the P. acidipropionici, P. freudenreichii subspecies freudenreichii
and the P. thoenii type strains, were not possible. Similar results (not shown) were also observed for strains originally received labelled as either "P. pentosaceum" or "~Po arabinosum ", now classified as P. acidipropionici as well as all the strains and isolates phenotypically identified as P. acidipropionici using the classification system of CUMMINS and JOHNSON (1986). Differentiation between strains phenotypically identified as either P. thoenii, strains originally received labelled as "P. wentii" or any of the subspecies of P. freudenreichii was also not possible, confirming the results obtained for the type strains of the different species. However, differentiation of the P. jensenii type strain (DSM 20535) (Fig. 2, lane 5) and strains originally received labelled as either "P. rubrum", "P. sanguineum" or "P. intermedium", from the other classical species was possible using the restriction endonuclease Hpall. Strains originally received labelled as either P. jensenii, "P. peterssonii", "~Po pituitosum", "P.
2 3 4
242 184 24 Fig. 2. Silver-stained 10% polyacrylamide gel displaying fragments obtained after restriction endonuclease digestion (HpaII) of the amplification products (250 bp) obtained using primers 16sP3-16sP4. Lanes: 1 DNA molecular weight marker V; 2 DNA molecular weight marker VIII; 3 - P. acidipropionici ATCC 25562 (Partially digested fragment also visible); 4 - P. freudenreichii subsp. freudenreichii ATCC 6207; 5 - P. jensenii DSM 20535; 6 - P. thoenii NCFB 568.
89 -
425
426
K.-H. J. RIEDEL et al.
raffinosaceum", "P. technicum" or "P. zeae", which are now classified as P. jensenii, as well as "P. intermedium", all gave restriction endonuclease profiles (results not shown) identical to that of the P. jensenii type strain (DSM 20535) (Fig. 2, lane 5). Those strains which were phenotypically identified as P. jensen ii, but gave a restriction endonuclease profile similar to that of the P. acidipropionici species using the restriction endonucleases HaeIII and AluI on the larger PCR fragment (16sPl-16sP4), once again gave a endonuclease restriction profile similar to that of the P. acidipropionicilP. freudenreichiilP. thoenii group, confirming the incorrect phenotypic identification of these strains as P. jensenii.
Discussion In this study, the PCR and RFLP procedure, as optimised and described by RIEDEL et ai. (1994), was used to distinguish between 135 Propionibacterium strains representing the four classical species. With the exception of only the strains originally received labelled as "P. rubrum" of "P. sanguineum", the results of this study justify the classical species concept within the genus Propionibacterium, as it currently exists. The taxonomical position of strains originally received labelled as either "P. wentii" and "P. intermedium", which were excluded from the original analyses of JOHNSON and CUMMINS (1972) and MOORE and HOLDEMAN (1974), and thus not consolidated into any of the current species, was also determined. The results of this study confirm the consolidation of the strains originally received labelled as "P. arabinosum" and "P. pentosaceum" into the species P. acidipropionici (MOORE and HOLDEMAN, 1974). These results subsequently verify previous phenotypic (BRITZ and RIEDEL, 1991, 1994; RIEDEL and BRITZ, 1993) protein profile (RIEDEL and BRITZ, 1992) and PCRlRFLP studies (RIEDEL et aI., 1994). According to the current classification system of CUMMINS and JOHNSON (1986), MOORE and HOLDEMAN (1974) consolidated "P. peterssonii" into the P. jensenii species. This specific strain was, however, identified as and clustered in the P. acidipropionici group based on both the phenotypic and protein profile analyses (BRITZ and RIEDEL, 1991; RIEDEL and BRITZ, 1992). It can thus be assumed that this strain was probably mislabelled since the other "P. peterssonii" strains included in this study, were identified as P. jensen ii, as expected. In contrast to the phenotypic results (BRITZ and RIEDEL, 1991, 1994; RIEDEL and BRITZ, 1993), differentiation between the various subspecies of P. freudenreichii was not possible. These results conformed the protein profile results of BAER (1987) as well as RIEDEL and BRITZ (1992), the PCRlRFLP results of RIEDEL et ai. (1994) as well as the ribotyping results of RIEDEL and BRITZ (1996). DE CARVALHO et ai. (1994) could, however, differentiate between P. freudenreichii subspecies freudenreichii and P. freudenreichii subspecies shermanii
using ribosomal ribonucleic acid gene restriction patterns (ribotyping), confirming the results of BAER and RYBA (1992), who were able to differentiate the two subspecies based on their serological analysis. Since a high degree of DNA-DNA similarity has been observed between the three subspecies (JOHNSON and CUMMINS, 1972) and, as verified in this study, a low degree of polymorphism exists in the 16S rDNA, there seems to be no justification for the separation of this group into the various subspecies. The consolidation of the "P. peterssonii", "P. pituitosum", "P. raffinosaceum", "P. technicum" and "P. zeae" strains as members of the P. jensenii species (MOORE and HOLDEMAN, 1974) were also confirmed in this study. The similarity observed between the brown/red pigmented "P. rubrum", "P. sanguineum", "P. intermedium" and the P. jensenii strains verify previous phenotypic (MALIK et aI., 1968; SEYFRIED, 1968; BRITZ and RIEDEL, 1991, 1994, 1995; RIEDEL and BRITZ, 1993) electrophoretic protein profile (RIEDEL and BRITZ, 1992) and PCRlRFLP studies (RIEDEL et aI., 1994). These results clearly indicate that the "P. rubrum" and "P. sanguineum" strains should be grouped as members of the P. jensenii species, rather than in the P. thoenii species, as reported by MOORE and HOLDEMAN (1974) and CUMMINS and JOHNSON (1986). The P. thoenii and "P. rubrum" strains were consolidated into the species P. thoenii by MOORE and HOLDEMAN in 1974, based on their DNA-DNA similarity (JOHNSON and CUMMINS, 1972), similar pigment production and the production of ~-hemolysis on blood agar. They also considered "P. sanguineum" a synonym of the newly described P. thoenii species. Most of the phenotypic characteristics of "P. rubrum" and "P. sanguineum" which are used to differentiate between P. jensenii and P. thoenii were similar to that of the P. jensenii species (RIEDEL et aI., 1994), confirming the identification of these strains as P. jensenii. These results were subsequently confirmed by DE CARVALHO et ai. (1995), who, based on 16S ribosomal RNA sequence comparison, biochemical characteristics and DNA hybridisation, proposed the reclassification of "P. rubrum" as a ~-hemolytic biovar of P. jensenii. Both the National Collection of Industrial and Marine Bacteria (NCIMB) (Anon., 1986) and the American Type Culture Collection Catalogue (ATCC) (Anon., 1985) list a "P. intermedium" species, which was isolated by Demeter, but no further information is given. No reference to this strain or into which species it should be consolidated into was given by MOORE and HOLDEMAN (1974) or CUMMINS and JOHNSON (1986). The "P. intermedium" strain included in this study was phenotypically identified as P. jensenii (BRITZ and RIEDEL, 1991), which was subsequently confirmed by the PCRlRFLP results of this study. In terms of both the phenetic and PCRlRFLP results, the P. jensenii species should therefore have to be redefined to include the phenotypic description of the "P. rubrum", "P. sanguineum" and the "P. intermedium" strains. "Propionibacterium wentii", which is considered by the National Collection of Food Bacteria (NCFB) (Anon.,
Identification of the Classical Propionibacterium Species
1986) and the National Collection of Industrial and Marine Bacteria (NCIMB) (Anon., 1986) to be an invalid species, and listed only as a Propionibacterium species, was phenotypically identified as P. thoenii (BRITZ and RIEDEL, 1991). This stains phenotypic identity was subsequently confirmed by the results of the restriction endonuclease analysis of the 16S rDNA. Neither the NCFB nor the NCIMB give any further details possibly due to uncertainty as to which species this strain currently belongs to. The "P. wentii" strains were not included in the analyses of JOHNSON and CUMMINS (1972) and were subsequently not consolidated into any of the four classical species. In terms of both the phenetic and PCRlRFLP resuits, the P. thoenii species should therefore also have to be redefined to exclude the phenotypic description of the "P. rubrum" and "P. sanguineum" strains, but to include the phenotypic description of the "P. wentii" strains. In conclusion, the application of the polymerase chain reaction to amplify the 16S rDNA and subsequent restriction endonuclease digestion of this region permits both the specificity and sensitivity necessary for the rapid identification of members of the classical Propionibacterium. The results of this study confirm the previous report of RIEDEL et al. (1994). With the exception of strains originally received labelled as "P. rubrum" and "P. sanguineum", the results of this study confirm the consolidation of the various classical species by MOORE and HOLDEMAN (1974).lt is also evident from this study, that strains originally received labelled as "P. intermedium " and "P. wentii", which were not examined by MOORE and HOLDEMAN (1974), should be consolidated into the species P. jensenii and P. thoenii, respectively. Based on the results of this study it is apparent that the classical species concept within the genus Propionibacterium as it currently exists, is justified.
References Anon.: American Type Culture Collection Catalogue of Bacteria, Phages and rDNA vectors, pp. 128-129. (R. GHEMA, W. NIERMAN, P. PIENTA, eds.) 16th ed., Rockville, ATCC 1985 . Anon.: National collection of Food Bacteria Catalogue of Strains, p. 161. 3rd ed., Reading, university of Reading 1986. Anon.: National Collection of Industrial and Marine Bacteria Catalogue of Strains, pp. 106-107. (T. R. DANDO, J. E. P. YOUNG, eds.) 5th ed., Aberdeen, BPCC AUP Ltd. 1986. BAER, A. : Identification and differentiation of propionibacteria by electrophoresis of their proteins. Milchwiss. 42, 431-433 (1987). BAER, A., RYBA, I.: Serological identification of propionibacteria in milk and cheese samples. Int. Dairy J. 2, 299-310 (1992). BREED, R. S., MURRAY, E. G. D., SMITH, N. R.: Genus I. Propionibacterium Oral-Jensen 1909, pp. 569-577. In: Bergey's Manual of Determinative Bacteriology (R. S. BREED, E. G. D. MURRAY, N.-R. SMITH, eds.) 7th ed., BaiIW:re, London, Tindall and Cox 1957. BRITZ, T. J., RIEDEL, K.-H. J.: A numerical taxonomic study of Propionibacterium strains from dairy sources. J. Appl. Bacteriol. 71 , 407-416 (1991). BRITZ, T. J., RIEDEL, K.-H. J.: Propionibacterium species diversity in Leerdammer cheese. Int. J. Food Microbiol. 22, 257-267 (1994).
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BRITZ, T. J., RIEDEL, K.-H. J.: Numerical evaluation of the species groupings among the classical propionibacteria. Lait 75,309-314 (1995). BRITZ, T. J., STEYN, P. L.: Comparative studies on propionic acid bacteria. Phytophylactica 12,89-103 (1980). CHARFREITAG, 0., COLLINS, M. D., STACKEBRANDT, E. : Reclassification of Arachnia propionica as Propionibacterium propionicus comb. nov. Int. J. Syst Bacteriol. 38, 354-357 (1988). CUMMINS, C. S., JOHNSON, J. L.: Genus I. Propionibacterium Orla-Jensen 1909, pp. 1346-1353. In: Bergey's Manual of Systematic Bacteriology (P. H. A. SNEATH, N. S. MAIR, M. E. SHARPE, J. G. HOLT, eds.) vol. 2. Baltimore, Williams and Wilkins 1986. DE CARVALHO, A. F., GAUTIER, M., GRIMONT, F.: Identification of dairy Propionibacterium species by rRNA gene restriction patterns. Res. Microbiol. 145,667-676 (1994). DE CARVALHO, A. F., GUEZENEC, S., GAUTIER, M., GRIMONT, P. A. D.: Reclassification of "Propionibacterium rubrum" as P. jensenii. Res. Microbiol. 146,51-58 (1995). DUPUIS, c., CORRE, c., BOYAVAL, P.: Lipae and esterase activities of Propionibacterium freudenreichii subspecies freudenreichii. Appl. Environ. Microbial. 59,4004-4009 (1993). GLATZ, B. A.: The classical propionibacteria: their past, present, and future as industrial organisms. ASM News 58, 197-201 (1992). HOLDEMAN, L. V., CATO, E. P., MOORE, W. E. c.: Arachnua and Propionibacterium, pp. 56-65. In: Anaerobe Laboratory Manual, 4th ed., Virginia Polytechnic Institute and State University, Blacksburg, Virginia, Southern Printing Co. 1977. HOLT, J. G., KRIEG, N. R., SNEATH, P. H., STALEY, J. T., WILLIAMS, S. T.: Genus Propionibacterium, p. 580. In: Bergey's Manual of Determinative Bacteriology (J. G. HOLT, N. R. KRIEG, P. H., SNEATH, J. T. STATELY, S. T. WILLIAMS, eds. ) 9th ed., Baltimore, Williams and Wilkins 1994. JANOSCHEK, A.: Zur Systematik der Propionsaiirebakterien. Zentrlbl. Bakteriol. Parasitenk. Infect. Hyg. Abt. II 106, 321-337 (1944). JOHNSON, J. L., CUMMINS, C. S.: Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical propionibacteria, and strains of Arachnia propionica. J. Bacteriol. 109, 1047-1066 (1972). JOUBERT, W. A., BRITZ, T. J.: A simple and inexpensive method for the long term preservation of microbial cultures. J. Microbiol. Methods 7, 73-76 (1987). KASPAR, H. F.: Nitrite reduction to nitrous oxide by propionibacteria: detoxification mechanism. Arch. Microbiol. 133, 126-130 (1982). MALIK, A. c., REINHOLD, G. W., VEDAMUTHU, E. R.: Evaluation of the taxonomy of the Propionibacterium. Can. J. Microbi01. 14, 1185-1195 (1968). MARCOUX, Y., BEAULIEU, Y., CHAMPAGNE, C. P., GOULET, J.: Production of Propionibacterium freudenreichii subsp. shermanii in whey-based media. J. Ferment. Bioeng. 74, 95-99 (1992). MOORE, W. E. c., HOLDEMAN, L. Y.: Genus I. Propionibacterium Orla-Jensen 1909, pp. 633-641. In: Bergey's Manual of Determinative Bacteriology (R. E. BUCHANAN, N. E. GIBBONS, eds.) 8th ed., Baltimore, Williams and Wilkins 1974. PERBAL, B.: A practical guide to molecular cloning, 2nd ed. New York, John Wiley and Sons 1988. RIEDEL, K.-H. J., BRITZ, T. J.: Differentiation of "classical" Propionibacterium species by numerical analysis of electrophoretic protein profiles. System. Appl. Microbiol. 15, 567-572 (1992). RIEDEL, K.-H. ]., BRITZ, T. J.: Propionibacterium species diversity in anaerobic digesters. Biodiv. Conserv. 2, 400-411 (1993).
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RIEDEL, K.-H. J., BRITZ, T. J.: Justification of the "classical" Propionibacterium species concept by ribotyping. System. Appl. Microbiol. 19,370-380 (1996). RIEDEL, K.-H. J., WINGFIELD, B. D., BRITZ, T. J.: Combined influence of magnesium concentration and polymerase chain reaction specificity enhances. FEMS Microbiol. Lett. 92, 69-72 (1992). RIEDEL, K.-H. J., WINGFIELD, B. D., BRITZ, T. J.: Justification of the "classical" Propionibacterium species concept by restriction analysis of the 16S ribosomal RNA genes. System. Appl. Microbiol. 17,536-542 (1994). ROSZKOWSKI, W., ROSZKOWSKI, K., Ko., H. L., BEUTH, J., JELJASZEWICZ, J.: Immunomodulation by propionibacteria. Zbl. Bakt. 274, 289-298 (1990). SAKAGUCHI, K., IWASAKI, M., YAMADA, S.: A study on propionic acid fermentation. J. Agric. Chern. Soc. Jap. 17, 127 (1941). SAMBROOK, J., FRITSCH, E. F., MANIATIS, T.: Molecular cloning: A laboratory Manual, 2nd ed. New York, Cold Spring Harbour Laboratory, Cold Spring Harbour 1989.
SEYFRIED, P. L.: An approach to the classification of Lactobacilli using computer-aided numerical analysis. Can. J. Microbial. 14,313-318 (1968). VAN NIEL, C. B.: The propionic acid bacteria. Thesis. Haarlem, Delft, J. W. Boissevain & Co. 1928. VOROBJEVA, L. I., KHODJAEV, E. Yu., CHERDINCEVY, T. A.: The study of induced antimutagenesis of propionic acid bacteria. J. Microbiol. Methods 24, 249-258 (1996).
Corresponding author: K.-H. J. RIEDEL, Department of Microbiology and Biochemistry, University of the Orange Free State, Bloemfontein, 9300, South Africa. Tel: 027-51-4012585; Fax: 027-51-4482004
SYSTEI'V14l1C AND APPLIED MICROBIOLOGY
Identification of Classical Propionibacterium Species Using 16S rONA - Restriction Fragment Length Polymorph isms K.-H.]. RIEDEL\ B. D. WINGFIELD l and T. J. BRITZ2 1 2
Department of Microbiology and Biochemistry, University of the Orange Free State, Bloemfontein, South Africa Department of Food Science, University of Stellenbosch, Stellenbosch, South Africa
Received June 18, 1998
Summary The phenotypic identification of the classical propionibacteria is essentially still problematic and alternative techniques for the identification of the various species are required. A rapid and sensitive technique for the routine identification of the classical propionibacteria, based on the amplification of the 16S rRNA genes using the polymerase chain reaction and the subsequent restriction endonuclease digestion of the PCR products, was previously described. Although this technique enabled differentiation between the various classical species examined it was only evaluated on a limited number of type and reference strains. During this study, the taxonomic relationship between 135 Propionibacterium strains from diverse ecological niches, representing four classical species was investigated using this PCRJRFLP technique. Visual differentiation between the classical Propionibacterium species was possible after restriction endonuclease digestion of the PCR products obtained using primers 16sPI-16sP4 and 16sP3-16sP4 with the restriction endonucleases HaeIlI, AluI and HpaII, respectively. With the exception of strains independently identified as "P. rubrum" and "P. sanguineum", the results of this study confirm the consolidation of the "old" species into the various classical species as they currently exist. It was therefore concluded that the PCR/RFLP protocol is suitable for the rapid and routine identification of the classical propionibacteria. Key words: Classical Propionibacterium - PCR - RFLP - Taxonomy -16S ribosomal RNA
Introduction The classical propionibacteria, especially Propionibacterium freudenreichii are used extensively in the dairy industry as starter cultures for the manufacture of Swisstype cheese. The propionibacteria are also important in the food industry, where they are responsible for the production of organic acids, biomass, vitamin B12 and other specific metabolites (MARCOUX et al., 1992). Although the classical propionibacteria were extensively used for the commercial production of vitamin B 12 , this vitamin is currently being produced using a faster growing Pseudomonas strain (GLATZ, 1992). The production of propionic and other minor organic acids, CO 2, bacteriocins, vitamins and the antimutagenic properties displayed by propionibacteria (VOROBJEVA et al., 1996) may be of importance in the application of these organisms as probiotic agents in foods. The propionibacteria are also receiving increasing attention due to the fact that they rank amongst the most potent immunomodulatory stimulating cell populations involved in non-specific resistance (ROSZKOWSKI et al., 1990). Twenty three forms of the genus Propionibacterium were isolated and described during the period 1906 and
1944 (BRITZ and STEYN, 1980). Of these only 11 have been regarded as species, primarily as a result of the studies of VAN NIEL (1928). Other isolates which were described as new species (SAKAGUCHI et al., 1941; JANOSCHEK, 1944) were considered synonyms of known species and rejected. Since this period, there are several references to Propionibacterium isolates which could not be correctly identified, and that do not correspond to the species as described by BREED et al. (1957) (MALIK et al., 1968; SEYFRIED, 1968). Based primarily on the results of JOHNSON and CUMMINS (1972), MOORE and HOLDEMAN (1974) consolidated the 11 previously described Propionibacterium spp. into four species. They also transferred the anaerobic propionic acid-producing corynebacteria to the genus Propionibacterium to form the cutaneous species. Although this improved the taxonomy of the cutaneous species, it did not facilitate the identification of the classical species and several reports can be found in the literature (BRITZ and STEYN, 1980; BRITZ and RIEDEL, 1991, 1994, 1995; RIEDEL and BRITZ, 1992; 1993; DUPUIS et al., 1993) indicating problems encountered with the identification of classical strains. Due to
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the close relationship between the various species, differentiation is difficult, as separation is currently based on five biochemical characteristics: fermentation of sucrose and maltose; reduction of nitrate; ~-hemolysis; colour of pigment; and isomer of diaminopimelic acid in the cell wall (CUMMINS and JOHNSON, 1986). Species differentiation is, however, not always reproducible due to variation in these specific phenotypic characteristics (BRITZ and STEYN, 1980; BRITZ and RIEDEL, 1991, 1994, 1995; RIEDEL and BRITZ, 1993). Alternative classification techniques, therefore, need to be investigated. Essentially due to the limitations of the phenotypic identification schemes, RIEDEL et al. (1994) developed a rapid and sensitive technique for the routine identification of the classical propionibacteria based on the amplification of the 16S rRNA genes using the polymerase chain reaction and the subsequent restriction endonuclease digestion of the PCR products. Although this technique enabled differentiation between the various classical species examined, it was only evaluated on a limited number of type and reference strains, all of dairy origin. The aim of this study was, therefore, to evaluate the suitability of the PCR/RFLP technique as previously described by RIEDEL et al. (1994) on classical Propionibacterium species from diverse ecological niches. The value of restriction endonuclease digestion of the amplified rDNA fragments, in the taxonomy of the classical Propionibacterium, was also determined and the results obtained were compared with those of various phenotypic and numerical analyses.
DNA isolation and purification: All strains were grown anaerobically at 32°C for four days in the medium of CHARFREITAG et al. (1988), with the addition of a 4% (v/v) mineral salts solution (HOLDEMAN et al., 1977). Genomic DNA was isolated, as described by SAMBROOK et al. (1989) from 0.5 g (wet weight) biomass of each strain. The methods used for agarose and polyacrylamide gel electrophoresis are as described by PERBAL (1988). Polymerase chain reaction: The primer design, reaction mixture composition and optimum conditions for amplification of the 16S rDNA are as described by RIEDEL et al. (1992, 1994). Restriction endonuclease digestion and gel electrophoresis: The resulting PCR products were extracted from the PCR reaction mixture using chloroform, purified using Quiagen columns (Diagen, Dusseldorf), precipitated with iso-propanol and redissolved in TE buffer (10 mM Tris-base, 1 mM EDTA, pH 8.0). The PCR products, obtained using the primer sets 16sPI-16sP4 and 16sP3-16sP4, were digested using the restriction endonucleases AZul, HaeIII and Hpall (Boehringer Mannheim, GmbH, Germany), respectively (RIEDEL et al., 1992, 1994). Restriction endonuclease digestions were performed on approximately 0.5 pg of amplified DNA using 5 U of restriction enzyme for 5 h at 37°C in the incubation buffers supplied by Boehringer Mannheim (GmbH, Germany). Restriction fragments were electrophoresed on 10% polyacrylamide gels (Merck) in TBE buffer (0.089 M Tris-base, 0.089 M Boric acid, 0.002 M EDTA) using a Hoefer SE 600 vertical slab gel unit at a constant voltage of 240 V. DNA molecular-weight marker V (0.25 pg) and DNA molecular-weight marker VIII (0.25 pg) were used as standards (Boehringer Mannheim, GmbH, Germany). The gels were stained using the silver staining technique described by PERBAL (1988). Restriction endonuclease fragment sizes were determined by linear regression analysis, using the molecular weight markers as standards.
Material and Methods
Results
Bacterial strains and culture conditions: The classical Propionibacterium strains used during this study, including various
A large portion (1110 bp) of the 165 rDNA region of the various classical Propionibacterium strains was successfully amplified using the polymerase chain reaction. Products of the predicted sizes, using primers 16sPI-16sP4 (approximately 1110-bp) and primers 16sP3-16sP4 (approximately 250-bp), were obtained and subjected to restriction endonuclease digestion. Comparison between the restriction endonuclease data of the amplified fragment of the various type and reference strains examined previously (RIEDEL et al., 1994) and that of the various strains included in this study was used to distinguish between the various species. Visual differentiation of the various species was possible using the chosen restriction endonucleases. A comparison between strains labelled as originally received, as identified using the classification system of CUMMINS and JOHNSON (1986) and results obtained from the PCR/RFLP as found during this and a previous study (RIEDEL et al., 1994) are given in Table 1.
type and reference strains as well as isolates from a variety of different sources, are given in Table 1. All cultures were preserved by Iyophilisation (JOUBERT and BRITZ, 1987) and stored at -20°C. Working cultures were obtained by inoculation of yeast extract lactate broth (YEL). The YEL-medium consisted of (gil): yeast extract 5.0; sodium lactate (50% v/v) 20.0; peptone 2.0; KH 2P0 4 10.0; hemin 10.0 ml and Tween 80 1.0 ml (pH 7.0) which was prepared according to the method of HOLDEMAN et al. (1977). All strains were cultured anaerobically at 32°C for four days in a Forma Scientific (Malinckrodt, Inc., Ohio, USA) anaerobic cabinet, using oxygen-free nitrogen as gas phase. Culture purity was checked by means of microscopical examination of Gram stains, by comparison of morphological characteristics and by gas chromatographical determination of the major metabolites. The phenotypic identification of the various strains was previously determined (BRITZ and RIEDEL, 1991, 1994; RIEDEL and BRITZ, 1992, 1993). Fermentation products: The major metabolites produced in YEL-medium were determined gas chromatographically using a Hewlett-Packard (Avondale, USA) gas chromatograph equipped with a flame ionisation detector and a 30 m x 0.53 mm i.d. Nukol (Supelco, Inc., Avondale, USA) capillary column. The chromatograph was programmed at an initial temperature of 120°C, then increased at a rate of 6°C/min and finally held at 165°C. The detector and inlet temperatures were 250 °C and 135°C, respectively. Nitrogen, at a flow rate of 5 mllmin, was used as the carrier gas.
Identification of strains based on the restriction endonuclease profile of the PCR products obtained using primers 16sPl-16sP4 The results obtained from the restriction endonuclease analysis (HaeIII, AZul) of the PCR products (16sP1-
Identification of the Classical Propionibacterium Species
421
Table 1. Propionibacterium strains used in this study. Culture Received as No Species
Isolated from Source<
Strain
Identified as Identification key#
PCRJRFLP
P. P. P. P. P. P. P. P. P.
P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici
Propionibacterium acidipropionici group 76 78 90 91 93 95 120 258 263 273 285 300 310 311 346 349 350 352 358 413 421 424 430 432 433 446 451 456
"P. peterssonii" "P. arabinosum" P. isolate P. isolate P. isolate P. iso/ate P. isolate P. isolate P. isolate P. iso/ate P. isolate P. iso/ate P. isolate P. isolate P. isolate P. iso/ate P. isolate P. iso/ate P. isolate "P. pentosaceum " "P. pentosaceum" P. acidipropionici "P. arabinosum" "P. arabinosum" "P. pentosaceum" "P. pentosaceum "P. arabinosum" "P. arabinosum"
6 6 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 10 4 1 4 7 7 6 1 6
P 35 P 50
1101 NCFB 570 ATCC 25562" NCFB 563 B 3569 B 3568 P9 ATCC4965 P78
Swiss cheese Buttermilk Gouda Emmentaler Gouda Gruyere Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Leerdammer Leerdammer Leerdammer Leerdammer Leerdammer Emmentaler Swiss cheese
acidipropionici acidipropionici acidipropionici acidipropionici acidipropionici acidipropionici acidipropionici acidipropionici acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. jensenii P. acidipropionici P. acidipropionici P. acidipropionici
Propionibacterium freudenreichii group 70 73 88 131 296 304 308 309 344 347 348 423 434 435 453 454
P. sp. P. freudenreichii P. isolate
P. freudenreichii P. iso/ate P. isolate P. iso/ate P. isolate P. isolate P. iso/ate P. iso/ate P. freudenreichii "P. shermanii" P. freudenreichii "P. shermanii; P. freudenreichii
6 6 8 9 6 8 8 8 8 8 8
P45 P 57
1
ATCC 620T' B3524 B 3523 P 67 P 73
7 7 6 6
Gouda Emmentaler Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Leerdammer Leerdammer Swiss cheese
P. P. P. P. P. P. P. P. P. P.
P. P. P. P. P.
P.
freud ss. shermanii freud ss. freudenreichii freud ss. shermanii freud ss. globosum freud. ss. freudenreichii freud ss. shermanii freud. ss. shermanii freud. ss. shermanii freud 55. shermanii freud. ss. shermanii freud. ss. freudenreichii freud. ss. freudenreichii freud. ss. shermanii freud. ss. freudenreichii freud. ss. shermanii freud. ss. freudenreichii
P. freudenreichii P. freudenreichii
P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreich ii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreichii P. freudenreich ii
Propionibacterium jensenii group 74 75 77 79 80 85 86
P. sp.
P. sp. "P. raffinosaceum" "P. peterssonii" P. jensenii P. isolate P. isolate
8 8 6 3 2 8 8
74 75 P 41 VPI5169 DSM 20535"
Emmentaler Emmentaler Emmentaler Buttermilk Gruyere Gouda
P. jensenii P. jensenii P. thoenii P. jensenii P. jensenii P. jensenii P. jensenii
P. acidipropionici jensenii acidipropionici jensenii jensenii P. jensenii P. jensenii
P. P. P. P.
422
K.-H. J. RIEDEL et al.
Table 1. (Continued). Culture Received as No Species 87 89 96 98 99 100 121 122 257 259 261 262 264 265 266 267 268 269 270 271 272 274 275 277 278 279 280 281 282 283 284 287 289 290 293 295 299 301 303 313 314 345 351 353 354 355 356 357 359 420 422 427 429 431 449 450 452 455 457
P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate p. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. jensenii "P. technicum" "P. pituitosum" "P. raffinosaceum" "P. peterssonii" "P. raffinosaceum" "P. zeae" P. jensenii "P. technicum" "P. zeae"
Isolated from Source 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 4 4 4 4 4 6 6 6 6 6
Strain
NCFB 572 NCFB 567 NCFB 1077 NCFB 1078 NCFB 565 P P46 P 52 P 74 P 87
Gouda Gouda Emmentaler Gouda Gouda Gouda Gouda Emmentaler Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Leerdammer Leerdammer Leerdammer Leerdammer Leerdammer Leerdammer Leerdammer Buttermilk Edam
Emmentaler
Gruyere
Identified as Identification key#
PCRlRFLP
P. jensenii P. jensenii P. jensenii p. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. thoenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii
P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. acidipropionici P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. acidipropionici P. jensenii P. acidipropionici P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. acidipropionici P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. acidipropionici P. thoenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. jensenii P. acidipropionici
Identification of the Classical Propionibacterium Species
423
Table 1. (Continued). Culture Received as No Species
Isolated from Source
Identified as
Strain
Identification kel
PCRJRFLP
P 10 P4
P. jensenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. jensenii P. thoenii P. thoenii P. jensenii P. thoenii P. jensenii P. thoenii P. jensenii
P. jensenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. thoenii P. jensenii P. thoenii P. thoenii P. jensenii P. thoenii P. jensenii P. thoenii P. jensenii
Propionibacterium thoenii group 71 72 92 97 260 276 286 288 291 292 294 297 298 302 306 307 312 130 132 419 425 426 428 447 448
"P. rubrum"
P. thoenii P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate P. isolate "P. intermedium" "P. wentii" P. thoenii .. P. sanguineum" "P. wentii" "P. ruburm" P. thoenii "P. rubrum"
6 6 8 8 8 8 8 8
Gruyere Emmentaler Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester Anaerobic digester
8 8 8 8 8 8 8 8 8 5 5 4 4 4 4 6 6
NCIB 8728 NCIB 8905 NCFB 568" NCFB 1080 NCFB 1083 NCFB 571 P 15 P 21
Emmentaler
Buttermilk
" Type strain Identification key of CUMMINS and JOHNSON (1986) 1- ATCC, American Type Culture Collection, Rockville, Maryland, USA 2 - DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany 3 - VPI, Virginia Polythechnic Institute, USA 4 - NCFB, National Collection of Food Bacteria, Shinfield, Reading, UK 5 - NCIB, National Collection of Industrial bacteria, UK 6 - G.W. Reinbold, Iowa State University, Iowa, USA 7 - U.S. Dept. of Agriculture, USA 8 - Environmental Bacteriology Culture Collection, University of the Orange Free State, South Africa 9 - Hansens Laboratory, Denmark 10 - National Research Council of Canada, Canada
#
16sP4) of the four type strains (P. acidipropionici ATCC 25562, P. freudenreichii subsp. freudenreichii ATCC 6207, P. jensenii - DSM 20535 and P. thoenii NCFB 568) are shown in Fig. 1.
• Propionibacterium acidipropionici group The restriction endonuclease patterns obtained for strains originally received labelled as either "P. pentosaceum" or "P. arabinosum" and now classified as P. acidipropionici (CUMMINS and JOHNSON, 1986), as well as isolates that were phenotypically identified as P. acidipropionici, were identical (results not shown) to the profile obtained for the P. acidipropionici type strain (ATCC 25562) (Fig. 1, lanes 3 and 4). Although originally received as "P. peterssonii" (UOFS 76), this strain gave
a restriction endonuclease profile identical to that of the P. acidipropionici species, confirming its phenotypic identity. "Propionibacterium raffinosaceum" - UOFS 77, which was phenotypically identified as a P. thoenii strain gave a restriction endonuclease profile identical to that of the P. acidipropionici species.
• Propionibacterium freudenreichii group Similarly, it was found that the restriction endonuclease pattern obtained for all strains originally received labelled as either P. freudenreichii or "P. shermanii" as well as isolates that were phenotypically identified as either P. freudenreichii subsp. freudenreichii, P. freudenreichii subsp. globosum or P. freudenreichii subsp. shermanii were identical (results not shown) to that obtained for
424
K.-H. J. RIEDEL et al.
1
2 3
4
5
6
7 8
9
o
504 04 -
2 2184 24 -
Fig. 1. Silver-stained 10% polyacrylamide gel displaying fragments obtained after restriction endonuclease digestion (HaeIIl and AZul) of the amplification products (1100 bp) obtained using primers 16sPl-16sP4. Lanes: 1 - DNA molecular weight marker V; 2DNA molecular weight marker VIII; 3 - P. acidipropionici ATCC 25562 - HaeIII; 4 - P. acidipropionici ATCC 25562 - AZul; 5 - P. freudenreichii subsp. freudenreichii ATCC 6207 - HaeIII; 6 - P. freudenreichii subsp. freudenreichii ATCC 6207 - AZul; 7 - P. jensenii DSM 20535 - HaeIII; 8 - P. jensenii DSM 20535 - AZul; 9 - P. thoenii NCFB 568 - HaeIII; 10 - P. thoenii NCFB 568 - AZuL
the P. freudenreichii subsp. freudenreichii type strain (ATCC 6207) (Fig. 1, lanes 5 and 6). • Propionibacterium jensenii group With a few exceptions, most of the strains originally received labelled or isolated and phenotypically identified as either P. jensenii, "P. peterssonii", "P. pituitosum", "P. raffinosaceum", "P. technicum" or "P. zeae" all gave restriction endonuclease profiles identical (results not shown) to the P. jensenii type strain (DSM 20535) (Fig. 1, lanes 7 and 8). One "P. technicum" strain, however, ("P. technicum" - UOFS 422) gave a restriction endonuclease profile similar to that of the P. thoenii species. In contrast, the other "P. technicum" strain included in this study ("P. technicum" - UOFS 455), was found to have a restriction endonuclease profile identical to that of the P. jensenii type (DSM 20535) and reference strains. Although most of the strains which were phenotypically identified as P. jensenii using the identification system of CUMMINS and JOHNSON (1986) (BRITZ and RIEDEL, 1991, 1994; RIEDEL and BRITZ, 1991,
1993) gave a restriction endonuclease pattern identical to that of the P. jensenii type (DSM 20535) (Fig. 1, lanes 7 and 8) and reference strains, a number of reference strains and isolates displayed a restriction endonuclease pattern identical to that of the P. acidipropionici type (ATCC 25562) (Fig. 1, lanes 3 and 4) and other P. acidipropionici strains. All the "P. rubrum" and "P. sanguineum" strains included in this study, displayed a restriction endonuclease pattern identical to that of the P. jensenii type (DSM 20535) and other P. jensenii strains. • Propionibacterium thoenii group All the isolates and reference strains which were phenotypically identified as P. thoenii displayed a restriction endonuclease pattern identical to that of the P. thoenii type strain (NCFB 568) (Fig. 1, lanes 9 and 10). • "Propionibacterium wentii" and "P. intermedium" Both the "P. wendii" strains included in this study gave a restriction endonuclease profile identical to that of the P. thoenii type (NCFB 568) (Fig. 1, lanes 9 and 10)
Identification of the Classical Propionibacterium Species
and other P. thoenii strains. The "P. intermedium" UOFS 130 strain included in this study, gave a restriction endonuclease profile identical to that of the P. jensenii type and other P. jensenii strains.
Identification of strains based on the restriction endonuclease profile of the peR product obtained using primers 16sP3-16sP4
Propionibacterium acidipropionici, P. jensen ii, P. freudenreichii and P. thoenii. The results obtained from the restriction endonuclease analysis using the restriction endonuclease HpaII, on the PCR products obtained using the primer set 16sP3-16sP4, of the type strains of the various classical Propionibacterium species are shown in Fig. 2. Due to identical restriction endonuclease profiles, visual differentiation between the P. acidipropionici, P. freudenreichii subspecies freudenreichii
and the P. thoenii type strains, were not possible. Similar results (not shown) were also observed for strains originally received labelled as either "P. pentosaceum" or "~Po arabinosum ", now classified as P. acidipropionici as well as all the strains and isolates phenotypically identified as P. acidipropionici using the classification system of CUMMINS and JOHNSON (1986). Differentiation between strains phenotypically identified as either P. thoenii, strains originally received labelled as "P. wentii" or any of the subspecies of P. freudenreichii was also not possible, confirming the results obtained for the type strains of the different species. However, differentiation of the P. jensenii type strain (DSM 20535) (Fig. 2, lane 5) and strains originally received labelled as either "P. rubrum", "P. sanguineum" or "P. intermedium", from the other classical species was possible using the restriction endonuclease Hpall. Strains originally received labelled as either P. jensenii, "P. peterssonii", "~Po pituitosum", "P.
2 3 4
242 184 24 Fig. 2. Silver-stained 10% polyacrylamide gel displaying fragments obtained after restriction endonuclease digestion (HpaII) of the amplification products (250 bp) obtained using primers 16sP3-16sP4. Lanes: 1 DNA molecular weight marker V; 2 DNA molecular weight marker VIII; 3 - P. acidipropionici ATCC 25562 (Partially digested fragment also visible); 4 - P. freudenreichii subsp. freudenreichii ATCC 6207; 5 - P. jensenii DSM 20535; 6 - P. thoenii NCFB 568.
89 -
425
426
K.-H. J. RIEDEL et al.
raffinosaceum", "P. technicum" or "P. zeae", which are now classified as P. jensenii, as well as "P. intermedium", all gave restriction endonuclease profiles (results not shown) identical to that of the P. jensenii type strain (DSM 20535) (Fig. 2, lane 5). Those strains which were phenotypically identified as P. jensen ii, but gave a restriction endonuclease profile similar to that of the P. acidipropionici species using the restriction endonucleases HaeIII and AluI on the larger PCR fragment (16sPl-16sP4), once again gave a endonuclease restriction profile similar to that of the P. acidipropionicilP. freudenreichiilP. thoenii group, confirming the incorrect phenotypic identification of these strains as P. jensenii.
Discussion In this study, the PCR and RFLP procedure, as optimised and described by RIEDEL et ai. (1994), was used to distinguish between 135 Propionibacterium strains representing the four classical species. With the exception of only the strains originally received labelled as "P. rubrum" of "P. sanguineum", the results of this study justify the classical species concept within the genus Propionibacterium, as it currently exists. The taxonomical position of strains originally received labelled as either "P. wentii" and "P. intermedium", which were excluded from the original analyses of JOHNSON and CUMMINS (1972) and MOORE and HOLDEMAN (1974), and thus not consolidated into any of the current species, was also determined. The results of this study confirm the consolidation of the strains originally received labelled as "P. arabinosum" and "P. pentosaceum" into the species P. acidipropionici (MOORE and HOLDEMAN, 1974). These results subsequently verify previous phenotypic (BRITZ and RIEDEL, 1991, 1994; RIEDEL and BRITZ, 1993) protein profile (RIEDEL and BRITZ, 1992) and PCRlRFLP studies (RIEDEL et aI., 1994). According to the current classification system of CUMMINS and JOHNSON (1986), MOORE and HOLDEMAN (1974) consolidated "P. peterssonii" into the P. jensenii species. This specific strain was, however, identified as and clustered in the P. acidipropionici group based on both the phenotypic and protein profile analyses (BRITZ and RIEDEL, 1991; RIEDEL and BRITZ, 1992). It can thus be assumed that this strain was probably mislabelled since the other "P. peterssonii" strains included in this study, were identified as P. jensen ii, as expected. In contrast to the phenotypic results (BRITZ and RIEDEL, 1991, 1994; RIEDEL and BRITZ, 1993), differentiation between the various subspecies of P. freudenreichii was not possible. These results conformed the protein profile results of BAER (1987) as well as RIEDEL and BRITZ (1992), the PCRlRFLP results of RIEDEL et ai. (1994) as well as the ribotyping results of RIEDEL and BRITZ (1996). DE CARVALHO et ai. (1994) could, however, differentiate between P. freudenreichii subspecies freudenreichii and P. freudenreichii subspecies shermanii
using ribosomal ribonucleic acid gene restriction patterns (ribotyping), confirming the results of BAER and RYBA (1992), who were able to differentiate the two subspecies based on their serological analysis. Since a high degree of DNA-DNA similarity has been observed between the three subspecies (JOHNSON and CUMMINS, 1972) and, as verified in this study, a low degree of polymorphism exists in the 16S rDNA, there seems to be no justification for the separation of this group into the various subspecies. The consolidation of the "P. peterssonii", "P. pituitosum", "P. raffinosaceum", "P. technicum" and "P. zeae" strains as members of the P. jensenii species (MOORE and HOLDEMAN, 1974) were also confirmed in this study. The similarity observed between the brown/red pigmented "P. rubrum", "P. sanguineum", "P. intermedium" and the P. jensenii strains verify previous phenotypic (MALIK et aI., 1968; SEYFRIED, 1968; BRITZ and RIEDEL, 1991, 1994, 1995; RIEDEL and BRITZ, 1993) electrophoretic protein profile (RIEDEL and BRITZ, 1992) and PCRlRFLP studies (RIEDEL et aI., 1994). These results clearly indicate that the "P. rubrum" and "P. sanguineum" strains should be grouped as members of the P. jensenii species, rather than in the P. thoenii species, as reported by MOORE and HOLDEMAN (1974) and CUMMINS and JOHNSON (1986). The P. thoenii and "P. rubrum" strains were consolidated into the species P. thoenii by MOORE and HOLDEMAN in 1974, based on their DNA-DNA similarity (JOHNSON and CUMMINS, 1972), similar pigment production and the production of ~-hemolysis on blood agar. They also considered "P. sanguineum" a synonym of the newly described P. thoenii species. Most of the phenotypic characteristics of "P. rubrum" and "P. sanguineum" which are used to differentiate between P. jensenii and P. thoenii were similar to that of the P. jensenii species (RIEDEL et aI., 1994), confirming the identification of these strains as P. jensenii. These results were subsequently confirmed by DE CARVALHO et ai. (1995), who, based on 16S ribosomal RNA sequence comparison, biochemical characteristics and DNA hybridisation, proposed the reclassification of "P. rubrum" as a ~-hemolytic biovar of P. jensenii. Both the National Collection of Industrial and Marine Bacteria (NCIMB) (Anon., 1986) and the American Type Culture Collection Catalogue (ATCC) (Anon., 1985) list a "P. intermedium" species, which was isolated by Demeter, but no further information is given. No reference to this strain or into which species it should be consolidated into was given by MOORE and HOLDEMAN (1974) or CUMMINS and JOHNSON (1986). The "P. intermedium" strain included in this study was phenotypically identified as P. jensenii (BRITZ and RIEDEL, 1991), which was subsequently confirmed by the PCRlRFLP results of this study. In terms of both the phenetic and PCRlRFLP results, the P. jensenii species should therefore have to be redefined to include the phenotypic description of the "P. rubrum", "P. sanguineum" and the "P. intermedium" strains. "Propionibacterium wentii", which is considered by the National Collection of Food Bacteria (NCFB) (Anon.,
Identification of the Classical Propionibacterium Species
1986) and the National Collection of Industrial and Marine Bacteria (NCIMB) (Anon., 1986) to be an invalid species, and listed only as a Propionibacterium species, was phenotypically identified as P. thoenii (BRITZ and RIEDEL, 1991). This stains phenotypic identity was subsequently confirmed by the results of the restriction endonuclease analysis of the 16S rDNA. Neither the NCFB nor the NCIMB give any further details possibly due to uncertainty as to which species this strain currently belongs to. The "P. wentii" strains were not included in the analyses of JOHNSON and CUMMINS (1972) and were subsequently not consolidated into any of the four classical species. In terms of both the phenetic and PCRlRFLP resuits, the P. thoenii species should therefore also have to be redefined to exclude the phenotypic description of the "P. rubrum" and "P. sanguineum" strains, but to include the phenotypic description of the "P. wentii" strains. In conclusion, the application of the polymerase chain reaction to amplify the 16S rDNA and subsequent restriction endonuclease digestion of this region permits both the specificity and sensitivity necessary for the rapid identification of members of the classical Propionibacterium. The results of this study confirm the previous report of RIEDEL et al. (1994). With the exception of strains originally received labelled as "P. rubrum" and "P. sanguineum", the results of this study confirm the consolidation of the various classical species by MOORE and HOLDEMAN (1974).lt is also evident from this study, that strains originally received labelled as "P. intermedium " and "P. wentii", which were not examined by MOORE and HOLDEMAN (1974), should be consolidated into the species P. jensenii and P. thoenii, respectively. Based on the results of this study it is apparent that the classical species concept within the genus Propionibacterium as it currently exists, is justified.
References Anon.: American Type Culture Collection Catalogue of Bacteria, Phages and rDNA vectors, pp. 128-129. (R. GHEMA, W. NIERMAN, P. PIENTA, eds.) 16th ed., Rockville, ATCC 1985 . Anon.: National collection of Food Bacteria Catalogue of Strains, p. 161. 3rd ed., Reading, university of Reading 1986. Anon.: National Collection of Industrial and Marine Bacteria Catalogue of Strains, pp. 106-107. (T. R. DANDO, J. E. P. YOUNG, eds.) 5th ed., Aberdeen, BPCC AUP Ltd. 1986. BAER, A. : Identification and differentiation of propionibacteria by electrophoresis of their proteins. Milchwiss. 42, 431-433 (1987). BAER, A., RYBA, I.: Serological identification of propionibacteria in milk and cheese samples. Int. Dairy J. 2, 299-310 (1992). BREED, R. S., MURRAY, E. G. D., SMITH, N. R.: Genus I. Propionibacterium Oral-Jensen 1909, pp. 569-577. In: Bergey's Manual of Determinative Bacteriology (R. S. BREED, E. G. D. MURRAY, N.-R. SMITH, eds.) 7th ed., BaiIW:re, London, Tindall and Cox 1957. BRITZ, T. J., RIEDEL, K.-H. J.: A numerical taxonomic study of Propionibacterium strains from dairy sources. J. Appl. Bacteriol. 71 , 407-416 (1991). BRITZ, T. J., RIEDEL, K.-H. J.: Propionibacterium species diversity in Leerdammer cheese. Int. J. Food Microbiol. 22, 257-267 (1994).
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SEYFRIED, P. L.: An approach to the classification of Lactobacilli using computer-aided numerical analysis. Can. J. Microbial. 14,313-318 (1968). VAN NIEL, C. B.: The propionic acid bacteria. Thesis. Haarlem, Delft, J. W. Boissevain & Co. 1928. VOROBJEVA, L. I., KHODJAEV, E. Yu., CHERDINCEVY, T. A.: The study of induced antimutagenesis of propionic acid bacteria. J. Microbiol. Methods 24, 249-258 (1996).
Corresponding author: K.-H. J. RIEDEL, Department of Microbiology and Biochemistry, University of the Orange Free State, Bloemfontein, 9300, South Africa. Tel: 027-51-4012585; Fax: 027-51-4482004