Letters in Applied Microbiology 1997, 25, 345–348
Cell-wall protein profiles of dairy thermophilic lactobacilli M. Gatti, E. Fornasari and E. Neviani Istituto Sperimentale Lattiero Caseario, Lodi, Italy 1446/97: received 18 February 1997 and accepted 29 April 1997 M . G AT T I, E. F OR NA S AR I A N D E . N E VI AN I . 1997. SDS–PAGE fingerprinting of cell-wall proteins extracted from 119 strains belonging to different species of lactic acid bacteria have been compared. The method of extraction and electrophoretic separation utilized in this work was found to be a reliable and rapid way for characterizing thermophilic lactobacilli species and strains. A protein of approximately 50 kDa was found to be characteristic for all the Lact. helveticus strains, and two cell-wall proteins of about 20 and 30 kDa were typical of the species Lact. delbrueckii, but the discrimination between the subspecies lactis and bulgaricus was not possible by the electrophoretic technique used. The other thermophilic species studied in this work presented cell-wall protein patterns that permitted their differentiation from both Lact. helveticus and Lact. delbrueckii.
INTRODUCTION
The analysis of whole-cell protein profiles was successfully used to investigate the taxonomic status of different microorganisms (Pot et al. 1993; Klein et al. 1994; Dykes and von Holy 1994; Samelis et al. 1995; Torriani et al. 1996). Since 1953 (Houwink 1953) an increasing number of studies has brought to light the presence of protein surface layers on the bacterial cell envelope (Masuda and Kawata 1981; Masuda and Kawata 1983; Bhouwmik et al. 1985; Watanabe et al. 1987; Sleytr and Messner 1988; Lortal et al. 1991; Crow et al. 1995; Valence and Lortal 1995). In some cases, a divergence in biological properties of these protein components has been recognized (Oram 1971; Watanabe et al. 1987; Akcelik and Tunail 1992; Crow et al. 1995; Valence and Lortal 1995). The presence of surface proteins has been used to study and compare several strains of lactobacilli (Reniero et al. 1990; Masuda 1992; Yasui et al. 1995; Boot et al. 1996). Thermophilic lactobacilli are the most important microflora present in natural or selected starters used for several Italian dairy products and particularly for Parmesan cheese (Neviani and Carini 1994). The aim of this work was to investigate the cell-wall protein profile in several species of thermophilic lactobacilli. MATERIALS AND METHODS Strains
From the ISLC (Istituto Sperimentale Lattiero Caseario) collection, the following strains were used for this study: 38 Correspondence to: Dr Erasmo Neviani, Istituto Sperimentale Lattiero Caseario, Via A. Lombardo 11, 20075 Lodi, Italy (e-mail:
[email protected]). © 1997 The Society for Applied Bacteriology
Lactobacillus helveticus, 27 Lact. delbrueckii ssp. bulgaricus, 18 Lact. delbrueckii ssp. lactis, eight Lact. acidophilus, seven Lact. fermentum, eight Streptococcus thermophilus, four Enterococcus faecium and nine Ent. faecalis. Most of the strains were isolated from both natural whey starter and from cheeses. According to Bergey’s Manual, the strains had been classified using the following criteria: cell morphology, sugar fermentation, growth temperature range, good growth temperature, salt resistance, lactic acid isomer, NH3 from arginine, and CO2 production (Kandler and Weiss 1986). Results were confirmed by Api 50 CHL and APILAB Plus (BioMe´rieux, Marcy-l’Etoile, France). Additional tests for the identification of Lact. helveticus strains were performed by a specific oligonucleotide probe (Tailliez et al. 1994). The strains were grown at 42 °C in MRS or M17, and after overnight incubation the cultures were centrifuged (5000 g for 10 min).
Growth conditions and cell-wall protein extraction and analysis
The harvested cells were washed twice in distilled sterile water, resuspended in 1·5 ml distilled water (A600 nm 2) and centrifuged. Cell-wall proteins were extracted from final pellets with 0·5 ml of 0·01 mol l−1 Tris–HCl, 0·01 mol l−1 EDTA, 0·01 mol l−1 NaCl, 2% SDS, pH 8·0, at 100 °C for 5 min. After treatment, the suspensions were centrifuged (11 600 g for 10 min) and the supernatant fluids examined on SDS– PAGE by Phast System (Pharmacia, Uppsala, Sweden) and stained with Coomassie blue (Heukeshoven and Dernick 1988).
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The pelleted cells were treated with lysozyme (Samelis et al. 1995) to obtain the total free-cell protein extract.
RESULTS
The SDS–PAGE fingerprinting of cell-wall proteins extracted as described in the Methods proved to be a fast and reliable way to characterize thermophilic lactobacilli species and strains. The interference of intracellular proteins in the cell-wall protein extract was proved to be negligible by comparing the cell-wall and total free-cell protein electrophoretic patterns (data not shown). The protein profile patterns for 119 strains are summarized in Fig. 1. Similar patterns were observed for strains belonging to each species studied. A comparison of cell-wall protein profiles on SDS gel found results to be different and specific for each species. Lactobacillus helveticus strains were characterized by about 50 kDa of protein. This protein was not observed for the other species studied, but was specific for Lact. helveticus. For 21% of the strains of the species tested this protein was the only one detectable using the extraction condition described in the Methods (Fig. 1a, lane 1). A large proportion of strains presented an additional 66 kDa of protein (Fig. 1a, lane 2), while some other strains (13%) showed an additional protein of about 38 kDa (Fig. 1a,
lane 3). Those strains presenting the first protein never presented the second, and vice versa. In some cases, there was a partial overlapping of the band at about 50 kDa (data not shown); however, this phenomenon was not repeatable and was seen only for those strains with the 50 kDa band as the only cell-wall protein. In 20% of the strains, the cell-wall protein profile was more complex, and always showed the characteristic band of about 50 kDa as well as numerous other small bands of molecular weight varying between 100 kDa and 10 kDa (Fig. 1a, lane 4). The electrophoretic profile of 80% of the strains belonging to the Lact. delbrueckii ssp. bulgaricus species was characterized by only two proteins (31 kDa and 20 kDa), coloured in varying intensities (Fig. 1b, lanes 1, 2 and 3); for the remaining 20% of the strains, no band was evident (Fig. 1b, lane 4) with the extraction conditions and electrophoresis used in the study. All the strains of the Lact. delbrueckii ssp. lactis species showed a band of about 20 kDa, similar to that found in Lact. delbrueckii ssp. bulgaricus (Fig. 1c). A majority of the strains (55%) presented an additional six proteins of different molecular weights (of about 13, 15, 31, 45, 55 and 66 kDa) (Fig. 1c, lane 1); 5% showed only a 20 kDa band (Fig. 1c, lane 2), while 40% of the strains also had one at about 31 kDa, a pattern similar to that obtained on the wall of Lact. delbrueckii ssp. bulgaricus (Fig. 1c, lane 2).
Fig. 1 SDS–PAGE fingerprinting of cellwall proteins of 119 different strains belonging to different species of lactic acid bacteria. (a) Protein profile patterns of 38 Lactobacillus helveticus strains; lane 1, eight strains; lane 2, 18 strains; lane 3, five strains, lane 4, seven strains. (b) Protein profile patterns of 27 Lact. delbrueckii subspecies bulgaricus strains; lane 1, 13 strains; lane 2, seven strains; lane 3, two strains; lane 4, five strains. (c) Protein profile patterns of 18 Lact. delbrueckii subspecies lactis strains; lane 1, 10 strains; lane 2, one strain; lane 3, seven strains. (d) Protein patterns of eight Lact. acidophilus strains (lane A); seven Lact. fermentum strains (lane B); eight Streptococcus thermophilus strains (lane C) and 13 Enterococcus faecalis and Ent. faecium strains (lane D). Lane M, low molecular weight standards; rabbit muscle phosphorylase, 97·400; bovine serum albumin, 66·200; ovalbumin, 45·000; carbonic anhydrase, 31·000; soybean trypsin inhibitor, 21·500; hen egg white lysozyme, 14·000 © 1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 25, 345–348
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Lactobacillus acidophilus was characterized by three bands (20, 31 and 55 kDa) (Fig. 1d, lane A), and Lact. fermentum, by five (70, 30, 25, 23 and 20 kDa) (Fig. 1d, lane B). The electrophoretic profiles of the eight strains of the Strep. thermophilus species showed the presence of numerous bands (more than 10) of different molecular weight ranging from 10 to 70 kDa (Fig. 1d, lane C). With the extraction conditions and electrophoresis procedure used in the study, no cell-wall band was seen for Ent. faecium or Ent. faecalis (Fig. 1d, lane D).
DISCUSSION
The fact that thermophilic lactobacilli possess different protein surface layer profiles is of great interest in investigating the functional role of the cell surface occurring in different ecosystems and among strains; this information could prove useful in classifying thermophilic strains. These results emphasize that the patterns of cell-wall proteins can be used to study and compare several strains of thermophilic lactic acid bacteria. Different species present varying and typical protein patterns; even among strains belonging to the same species it is possible to identify different cell-wall protein profiles showing strain-specific patterns. The protein of about 50 kDa was found to be characteristic of all the Lact. helveticus strains identified by Lortal et al. (1991) in Lact. helveticus ATCC 12046 and by Valence and Lortal (1995) in Lact. helveticus ISLC5. This protein was shown to be specific for the species. Similarly, Lact. delbrueckii strains presented two different, typical and specific cell-wall proteins (20 and 30 kDa). However, while it may be possible to recognize the Lact. helveticus and Lact. delbrueckii species, it is nonetheless impossible to discriminate between Lact. delbrueckii ssp. bulgaricus and Lact. delbrueckii ssp. lactis because of the similarity in cell-wall protein profile of the greater part of their strains. The other thermophilic species studied in this work presented cell-wall protein patterns that permited their differentiation from both Lact. helveticus and Lact. delbrueckii. Furthermore, for most of the micro-organisms studied in this paper, it seemed to be possible to discern a relationship between the source of the strains and their cell-wall pattern. From the Lact. helveticus, strains, 90% of those characterized by the cell-wall protein pattern described in Fig. 1a lanes 2 and 3 had been isolated from Grana cheese, while 83% of those characterized by the cell-wall protein pattern described in Fig. 1a lanes 1 and 4 had been isolated from Provolone cheese. From the Lact. delbrueckii ssp. bulgaricus strains, 82% of those characterized by the cell-wall protein profile shown in Fig. 1b lane 1 had been isolated from yoghurt, while 93% of the strains belonging to the same species but characterized by the cell-wall protein profile
shown in lanes 2, 3 and 4 (Fig. 1b) had been isolated from natural starter. These data suggest that differences in cell-wall protein profiles of the strains might be related to their adaptation to different ‘ecological niches’ and cheese technology. REFERENCES Akcelik, M. and Tunail, N. (1992) A 30 kDa cell wall protein produced by plasmid DNA which encodes inhibition of phage adsorption in Lactococcus lactis supsp. lactis P25. Milchwissenchaft 47, 215–217. Bhouwmik, T., Johnson, M.C. and Ray, B. (1985) Isolation and partial characterization of the surface protein of Lactobacillus acidophilus strains. International Journal of Food Microbiology 2, 311–321. Boot, H.J., Kolen, C.P., Pot, B., Kersters, K. and Pouwels, P.H. (1996) The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus. Microbiology 142, 2375–2384. Crow, V.L., Gopal, P.K. and Wicken, A.L. (1995) Cell surface differences of lactococcal strains. International Dairy Journal 5, 45–68. Dykes, G.A. and von Holy, A. (1994) Strain typing in the genus Lactobacillus. Letters in Applied Microbiology 19, 63–66. Heukeshoven, J. and Dernik, R. (1988) Increased sensitivity for Coomassie staining of sodium dodecyl sulfate-polyacrylamide gels using PhastSystem development unit. Electrophoresis 9, 60–61. Houwink, A.L. (1953) A macromolecular monolayer in the cell wall of Spirillum spec. Biochimica et Biophysica Acta 10, 360–366. Kandler, O. and Weiss, N. (1986) Regular, nonsporing Gram-positive rods. In Bergey’s Manual of Systematic Bacteriology 2, pp. 1208–1260. Baltimore: William and Wilkins. Klein, G., Hack, B., Zimmerman, K. and Reuter, G. (1994) Protein fingerprinting as a method for strain-specific differentiation of technologically useful Lactobacillus strains from clinical isolates. Berl Munch Tierarztl Wonchenschr 107, 302–307. Lortal, S., Rousseau, M., Boyaval, P. and Van Heijenoort, J. (1991) Cell wall and autolytic system of Lactobacillus helveticus ATCC 12046. Journal of General Microbiology 137, 549–559. Masuda, K. (1992) Heterogeneity of S-layer proteins of Lactobacillus acidophilus strains. Microbiology Immunology 36, 297–301. Masuda, K. and Kawata, T. (1981) Characterization of regular array in the wall of Lactobacillus buchneri and its reattachment in the other wall components. Journal of General Microbiology 124, 81– 90. Masuda, K. and Kawata, T. (1983) Distribution and chemical characterization of regular arrays in the cell walls of strains of the genus Lactobacillus. FEMS Microbiology Letters 20, 145–150. Neviani, E. and Carini, S. (1994) Microbiology of Parmesan cheese. Microbiology – Aliments – Nutrition 12, 1–8. Oram, J.D. (1971) Isolation and properties of a phage receptor substance from the plasma membrane of Streptococcus lactis ML3. Journal of General Virology 13, 59–71. Pot, B., Hertel, C., Ludwing, W., Descheemaeker, P., Kersters, K. and Schleifer, K.H. (1993) Identification and classification of Lactobacillus acidophilus, Lact. gasseri and Lact. johnsonii strains
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