Formation and Regeneration of Protoplasts and Spheroplastsof ...

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,

GERALD W. TANNOCK* Department of Microbiology, University of Otago, Dunedin, New Zealand AND

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 gastrointestinal 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 characteristic (4). Methods for genetically manipulating gastrointestinal 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, osmotically 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 osmotically sensitive forms of lactobacilli indigenous to the gastrointestinal tract with plasmid DNA. We define lactobacillus protoplasts as osmotically fragile, spherical forms obtained after treatment of the bacteria with cell walldegrading enzymes. Spheroplasts, in contrast, are osmotically fragile forms which remain bacillus shaped after treatment 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 protoplast buffer (3) to give a suspension with an optical density of 0.8 at a wavelength of 600 nm. Lysozyme (final concentration, 1 mg/ml; Sigma Chemical Co., St. Louis, Mo.) and mutanolysin (final concentration, 25 jig/ml; Sigma) were *

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 observation), 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 dilutions on LCM agar plates (3). Protoplast formation was estimated by observation of spherical forms in the preparation by phase-contrast microscopy (Fig. 1). Protoplast regeneration 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 regeneration 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) incubated 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.

Corresponding author. 1615

1616

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APPL. ENVIRON. MICROBIOL.

TABLE 1. Protoplast formation from lactobacillus cells Lactobacillus strain

L. L. L. L. L. L. L.

reuteri 100-63(3) reuteri 100-23 reuteri D287 gasseri 100-5 salivarius 207 salivarius RF59 casei ATCC 393

Protoplast

No. of replica

75 99 95 70 50 80 99

expts 2 2 6 2 2 2 8

formation"

TABLE 2.

Spheroplast formation and regeneration of lactobacillus cells

Incubation time (min)

Lactobacillus strain

120 60 60 60 120 60 120

L. L. L. L. L. L.

" Proportion (percent) of lactobacillus cells seen as spherical forms in preparations by phase-contrast microscopy.

Spheroplast formation"

reuteri 100-63(3) reuteri 100-23 reuteri D287 gasseri 100-5 salivarius 207 salivarius RF59

99 90 99 96 98 91

Regeneration

frequencyb 10-30 15-40

40-80 3-10 2-5 0

No. of replica

expts

8 5 3 3 2 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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