Formation and Regeneration Protoplasts and Spheroplasts of … · Lactobacillus strain Protoplast...
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Vol. 54, No. 6 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1988, p. 1615-1618 0099-2240/88/061615-04$02.00/0 Copyright © 1988, American Society for Microbiology Formation and Regeneration of Protoplasts and Spheroplasts of Gastrointestinal Strains of Lactobacilli HUGH CONNELL, JOAN LEMMON, AND GERALD W. TANNOCK* Department of Microbiology, University of Otago, Dunedin, New Zealand Received 30 November 1987/Accepted 3 March 1988 Methods were developed for the formation of protoplasts and spheroplasts of gastrointestinal strains of Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus salivarius. Attempts to regenerate vegetative cells from protoplasts were not successful, but spheroplasts could be regenerated consistently for five of six strains. The microecology of lactobacilli inhabiting the gastroin- testinal tracts of mice, rats, fowl, and pigs is of particular interest because of the ability of some lactobacillus strains to colonize stratified, squamous epithelial surfaces in proximal regions of the digestive tract (5). Only certain strains of lactobacilli are able to adhere to and colonize these epithelial habitats, and such strains show specificities for particular animal hosts (5). Progress in understanding the molecular mechanisms involved in colonization requires the use of isogenic strains of lactobacilli that differ in a single charac- teristic (4). Methods for genetically manipulating gastroin- testinal isolates of lactobacilli are therefore required for this type of research to proceed. Conjugation in Lactobacillus reuteri has been described previously (4), but as has been demonstrated with other bacterial genera, transformation of lactobacillus cells by use of plasmid vectors may provide a more satisfactory means of genetically modifying a variety of gastrointestinal species of lactobacilli (2). We developed methods by which protoplasts and spheroplasts of L. reuteri, L. gasseri, and L. salivarius can be formed and by which vegetative cells can be reliably regenerated from spheroplasts. Our methods were derived following extensive studies in which enzyme concentrations, bacterial cell concentration, bacterial growth phase, osmot- ically protective media and buffers, and incubation times and conditions were evaluated to obtain optimal conditions for use in experiments with gastrointestinal lactobacilli. The optimal methods that we describe provide a first step in the development of a general method for the polyethylene glycol- or electroporation-mediated transformation of os- motically sensitive forms of lactobacilli indigenous to the gastrointestinal tract with plasmid DNA. We define lactoba- cillus protoplasts as osmotically fragile, spherical forms obtained after treatment of the bacteria with cell wall- degrading enzymes. Spheroplasts, in contrast, are osmoti- cally fragile forms which remain bacillus shaped after treat- ment with cell wall-degrading enzymes. Protoplasts of lactobacillus cells were formed by the following procedure. Lactobacilli MRS medium (10 ml; Difco Laboratories, Detroit, Mich.) was inoculated with an appropriate lactobacillus strain and incubated at 30°C for 16 h under anaerobic conditions. The bacterial cells were harvested by centrifugation, washed, and diluted in proto- plast buffer (3) to give a suspension with an optical density of 0.8 at a wavelength of 600 nm. Lysozyme (final concentra- tion, 1 mg/ml; Sigma Chemical Co., St. Louis, Mo.) and mutanolysin (final concentration, 25 jig/ml; Sigma) were * Corresponding author. added before the preparation was incubated at 37°C for 1 to 2 h (the minimum incubation time producing appreciable numbers of protoplasts, as judged by microscopic observa- tion), depending on the lactobacillus strain. The proportion of osmotically fragile forms was determined by diluting lysozyme-mutanolysin-treated and nontreated preparations in protoplast buffer and spread-plating portions of the dilu- tions on LCM agar plates (3). Protoplast formation was estimated by observation of spherical forms in the prepara- tion by phase-contrast microscopy (Fig. 1). Protoplast re- generation was attempted by spread-plating dilutions on plates of protoplast regeneration medium (3). Agar plate cultures were incubated under anaerobic conditions for up to 5 days at 37°C. The regeneration efficiency was calculated from the following equation: [(colony count from regenera- tion medium - colony count from LCM)/colony count from nontreated preparation on LCM] x 100. L. casei ATCC 393, for which regeneration of protoplasts has been described previously (3), formed protoplasts under these conditions (Table 1). A significant degree of protoplast regeneration (>5%) was observed with this strain in four of eight replicate experiments. The majority of cells in cultures of L. reuteri, L. gasseri, and L. salivarius also formed protoplasts by this method (Table 1). None of our gastrointestinal isolates, however, regenerated vegetative cells from protoplasts. Spheroplasts were prepared from lactobacillus cultures in 10-ml volumes of Lactobacilli MRS medium (Difco) incu- bated under anaerobic conditions for 3 h at 37°C. The optical FIG. 1. Phase-contrast micrographs of L. reuteri 100-63(3). (A) Bacillus shape of untreated cells or spheroplasts. (B) Spherical shape of protoplasts. Bars, 2 ,um. 1615 on January 9, 2021 by guest http://aem.asm.org/ Downloaded from
Transcript of Formation and Regeneration Protoplasts and Spheroplasts of … · Lactobacillus strain Protoplast...
Vol. 54, No. 6APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1988, p. 1615-16180099-2240/88/061615-04$02.00/0Copyright © 1988, American Society for Microbiology
Formation and Regeneration of Protoplasts and Spheroplasts ofGastrointestinal Strains of Lactobacilli
HUGH CONNELL, JOAN LEMMON, AND GERALD W. TANNOCK*
Department of Microbiology, University of Otago, Dunedin, New Zealand
Received 30 November 1987/Accepted 3 March 1988
Methods were developed for the formation of protoplasts and spheroplasts of gastrointestinal strains ofLactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus salivarius. Attempts to regenerate vegetative cellsfrom protoplasts were not successful, but spheroplasts could be regenerated consistently for five of six strains.
The microecology of lactobacilli inhabiting the gastroin-testinal tracts of mice, rats, fowl, and pigs is of particularinterest because of the ability of some lactobacillus strains tocolonize stratified, squamous epithelial surfaces in proximalregions of the digestive tract (5). Only certain strains oflactobacilli are able to adhere to and colonize these epithelialhabitats, and such strains show specificities for particularanimal hosts (5). Progress in understanding the molecularmechanisms involved in colonization requires the use ofisogenic strains of lactobacilli that differ in a single charac-teristic (4). Methods for genetically manipulating gastroin-testinal isolates of lactobacilli are therefore required for thistype of research to proceed.
Conjugation in Lactobacillus reuteri has been describedpreviously (4), but as has been demonstrated with otherbacterial genera, transformation of lactobacillus cells by useof plasmid vectors may provide a more satisfactory means ofgenetically modifying a variety of gastrointestinal species oflactobacilli (2). We developed methods by which protoplastsand spheroplasts of L. reuteri, L. gasseri, and L. salivariuscan be formed and by which vegetative cells can be reliablyregenerated from spheroplasts. Our methods were derivedfollowing extensive studies in which enzyme concentrations,bacterial cell concentration, bacterial growth phase, osmot-ically protective media and buffers, and incubation times andconditions were evaluated to obtain optimal conditions foruse in experiments with gastrointestinal lactobacilli. Theoptimal methods that we describe provide a first step in thedevelopment of a general method for the polyethyleneglycol- or electroporation-mediated transformation of os-motically sensitive forms of lactobacilli indigenous to thegastrointestinal tract with plasmid DNA. We define lactoba-cillus protoplasts as osmotically fragile, spherical formsobtained after treatment of the bacteria with cell wall-degrading enzymes. Spheroplasts, in contrast, are osmoti-cally fragile forms which remain bacillus shaped after treat-ment with cell wall-degrading enzymes.
Protoplasts of lactobacillus cells were formed by thefollowing procedure. Lactobacilli MRS medium (10 ml;Difco Laboratories, Detroit, Mich.) was inoculated with anappropriate lactobacillus strain and incubated at 30°C for 16h under anaerobic conditions. The bacterial cells wereharvested by centrifugation, washed, and diluted in proto-plast buffer (3) to give a suspension with an optical density of0.8 at a wavelength of 600 nm. Lysozyme (final concentra-tion, 1 mg/ml; Sigma Chemical Co., St. Louis, Mo.) andmutanolysin (final concentration, 25 jig/ml; Sigma) were
* Corresponding author.
added before the preparation was incubated at 37°C for 1 to2 h (the minimum incubation time producing appreciablenumbers of protoplasts, as judged by microscopic observa-tion), depending on the lactobacillus strain. The proportionof osmotically fragile forms was determined by dilutinglysozyme-mutanolysin-treated and nontreated preparationsin protoplast buffer and spread-plating portions of the dilu-tions on LCM agar plates (3). Protoplast formation wasestimated by observation of spherical forms in the prepara-tion by phase-contrast microscopy (Fig. 1). Protoplast re-generation was attempted by spread-plating dilutions onplates of protoplast regeneration medium (3). Agar platecultures were incubated under anaerobic conditions for up to5 days at 37°C. The regeneration efficiency was calculatedfrom the following equation: [(colony count from regenera-tion medium - colony count from LCM)/colony count fromnontreated preparation on LCM] x 100. L. casei ATCC 393,for which regeneration of protoplasts has been describedpreviously (3), formed protoplasts under these conditions(Table 1). A significant degree of protoplast regeneration(>5%) was observed with this strain in four of eight replicateexperiments. The majority of cells in cultures of L. reuteri,L. gasseri, and L. salivarius also formed protoplasts by thismethod (Table 1). None of our gastrointestinal isolates,however, regenerated vegetative cells from protoplasts.
Spheroplasts were prepared from lactobacillus cultures in10-ml volumes of Lactobacilli MRS medium (Difco) incu-bated under anaerobic conditions for 3 h at 37°C. The optical
FIG. 1. Phase-contrast micrographs of L. reuteri 100-63(3). (A)Bacillus shape of untreated cells or spheroplasts. (B) Sphericalshape of protoplasts. Bars, 2 ,um.
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TABLE 1. Protoplast formation from lactobacillus cells
Lactobacillus strain Protoplast No. of replica Incubationformation" expts time (min)
L. reuteri 100-63(3) 75 2 120L. reuteri 100-23 99 2 60L. reuteri D287 95 6 60L. gasseri 100-5 70 2 60L. salivarius 207 50 2 120L. salivarius RF59 80 2 60L. casei ATCC 393 99 8 120
" Proportion (percent) of lactobacillus cells seen as spherical forms inpreparations by phase-contrast microscopy.
TABLE 2. Spheroplast formation and regeneration oflactobacillus cells
Lactobacillus strain Spheroplast Regeneration No. of replicaformation" frequencyb expts
L. reuteri 100-63(3) 99 10-30 8L. reuteri 100-23 90 15-40 5L. reuteri D287 99 40-80 3L. gasseri 100-5 96 3-10 3L. salivarius 207 98 2-5 2L. salivarius RF59 91 0 2
" Proportion (percent) of osmotically fragile forms in the preparation;values are means of all experiments.
I Range of regeneration frequencies (percent) from experiments.
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FIG. 2. Transmission electron micrographs of sections of L. reuteri 100-63(3). (A) Untreated cells with the cell wall layer in closeassociation with the cell membrane. (B) Protoplasts with the cell wall absent. (C) Spheroplasts with the cell wall layer only loosely associatedwith th ba t ri l ll B r 0 2e c e a ce s. a s, . Ilm.
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FIG. 3. Transmission electron micrographs of sections of L. reuteri D287. (A) Untreated cells with the cell wall layer close to the cellmembrane. (B) Spheroplasts with the cell wall loosely associated with the bacterial cells. Bars, 0.2 ,um.
VOL. 54, 1988
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density of the cultures after the incubation period variedaccording to the lactobacillus strain used, but this did notaffect the efficiency of spheroplast formation. The vegetativecells, harvested by centrifugation, were washed in protoplastbuffer, suspended in protoplast buffer containing 25 ,ug oflysozyme per ml, and incubated at 37°C for 10 min. Bacillus-shaped cells were observed in these preparations whenobserved by phase-contrast microscopy (Fig. 1). Osmoticfragility (spheroplast formation) was determined by culturingdilutions of lysozyme-treated and nontreated preparations inprotoplast buffer on LCM agar plates. The cultures wereincubated under anaerobic conditions at 37°C for 2 days.Spheroplast formation was calculated from the followingequation: [(colony count from nontreated preparation -colony count from lysozyme-treated preparation)/colonycount from nontreated preparation] x 100. Regeneration ofvegetative cells from spheroplasts was determined by cul-turing protoplast buffer dilutions of preparations on sphero-plast regeneration agar (LCM medium supplemented with1.0% glucose, 0.5% bovine serum albumin, 25 mM magne-sium chloride, 25 mM calcium chloride, 2.5% gelatin, 0.5 Msucrose) incubated anaerobically at 37°C for 48 h. Regener-ation was calculated from [(colony count from regenerationmedium - colony count from LCM)/colony count fromnontreated preparation on LCM] x 100. Spheroplasts weregenerated from the cells of all lactobacillus strains tested,and regneration of vegetative cells was obtained consistentlyfor all but one strain (Table 2).To confirm that protoplasts or spheroplasts were formed
by the methods described above, preparations of treated ornontreated lactobacillus cells were prepared for transmis-sion electron microscopy by the method of Lee-Wicknerand Chassy (3). Preparations from lysozyme-mutanolysin-treated cultures contained spherical cells devoid of cell wallmaterial; lysozyme (25 jxg/ml)-treated cultures containedbacillus-shaped forms surrounded by a cell wall layer whichappeared detached from the cell protoplast (Fig. 2 and 3).We conclude that the cells of gastrointestinal strains of
lactobacilli treated with a combination of lysozyme-muta-nolysin can be converted into true protoplasts with a highefficiency. The protoplasts of our isolates, however, did notregenerate under conditions favorable for the regeneration ofL. casei ATCC 393. The combination of the two muralyticenzymes apparently damaged the cell walls of even nonpro-toplasted cells in the preparations to such a degree thatregeneration was impossible. Spheroplasts of gastrointesti-nal strains were easily produced by treatment of vegetativecells with a low concentration of lysozyme alone. Regener-ation of vegetative cells from spheroplasts occurred con-sistently for all but one strain. The use of spheroplasts intransformation experiments with polyethylene glycol or elec-troporation appears worthy of further study for the geneticmanipulation of gastrointestinal lactobacilli (1).
The support of Chr. Hansen's Biosystems, Copenhagen, Den-mark; The University Grants Committee, the University of OtagoResearch Committee; and Southland Frozen Meat Co. is gratefullyacknowledged.Thanks are also due to Roy Fuller, Dwayne Savage, Pierre
Raibaud, and Bruce Chassy for the provision of cultures.
LITERATURE CITED1. Chassy, B. M., and J. L. Flickinger. 1987. Transformation of
Lactobacillus casei by electroporation. FEMS Microbiol. Lett.44:173-177.
2. Gilmore, M. S. 1985. Molecular cloning of genes encodinggram-positive virulence factors. Curr. Top. Microbiol. Immunol.118:219-234.
3. Lee-Wickner, L.-J., and B. M. Chassy. 1984. Production andregeneration of Lactobacillus casei protoplasts. Appl. Environ.Microbiol. 48:994-1000.
4. Tannock, G. W. 1987. Conjugal transfer of plasmid pAMP1 inLactobacillus reuteri and between lactobacilli and Enterococcusfaecalis. Appl. Environ. Microbiol. 53:2693-2695.
5. Tannock, G. W., 0. Szylit, Y. Duval, and P. Raibaud. 1982.Colonization of tissue surfaces in the gastrointestinal tract ofgnotobiotic animals by lactobacillus strains. Can. J. Microbiol.28:1196-1198.
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