Archives of
Microbiology
Arch. Microbiol. 118, 219-221 (1978)
9
by Springer-Verlag 1978
Short Communication
Protoplast-Like Structures Formation from Two Species of Enterobacteriaceae by Fosfomycin Treatment Maria-Rosario Rodicio, Manuel-Benjamin Manzanal, and Carlos Hardisson Departamento de Microbiologia, Universidad de Oviedo, Oviedo, spain
Abstract. A procedure for protoplasts formation from Escherichia coli and Serratia marcescens by treatment
with fosfomycin alone is described. This method gives high and low yields of stable protoplasts from E. coli and S. marcescens respectively. In the last case numerous spheroplasts were obtained. Electron micrographs of intact cells, protoplasts and spheroplasts are shown. Key words: Protoplasts -
coli-
Fosfomycin -
tial phase. For the obtention of protoplasts, the cells were harvested and transferred to the stabilization medium (Nutrient Broth with 2 g Mg SO4 97 H20 and 200 g sucrose per litre), containing 10 pg/ml of fosfomycin (kindly provided by CEPA, Aranjuez, Spain). The protoplasts were prefixed in 5 ~ glutaraldehyde for 2h at room temperature and fixed by the Ryter and Kellenberger method (1958), dehydrated with acetone and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and photographed in a Philips EM 300 electron microscope operating at 80 KV. Intact bacteria were prepared without prefixation.
Escherichia
Serratia marcescens. Results and Discussion
Fosfomycin is an antibiotic discovered by Hendlin et al. (1969) from cultures of Streptomyces fradiae. It is a bactericidal agent that blocks the initial step of the cell wall synthesis by irreversible inhibition of the activity of the enzyme phosphoenolpyruvate (PEP): uridine diphospho-N-acetyl-glucosamine (UDP-GlcNac) enolpyruvyl transferase, by acting as a structural analogue of phosphoenolpyruvate (Kahan et al., 1974). Formation of spheroplasts from cultures of Proteus vulgaris in the presence of the antibiotic (Stapley et al., 1970) and from Pseudomonas aeruginosa using EDTA plus fosfomycin (Perez Urefia et al., 1974) has been mentioned. We describe in this paper a reproducible procedure for obtaining high yields of protoplasts by using fosfomycin alone.
Materials and Methods Esche~ 1, hia coli ML 1 and Serratia marcescens isolated from clinical
samples, were grown in Nutrient Broth until the middle of exponen-
Escherichia coli ML I and Serratia marcescens (Fig. 1) exhibited a great sensitivity to the fosfomycin. The formation of protoplasts began after about 1 h of incubation and about 1 h later the majority of the cells were protoplasts. In a recent work, Weiss (1976) reported the obtention of true protoplasts from E. coli ML 30 after treatment with EDTA and lysozyme. We have obtained from E. coli ML1, using fosfomycin alone, a good yield of protoplasts which are surrounded only by the cytoplasmic membrane (Fig. 2). The low molecular weight of fosfomycin allows its passage through the outer wall layer of the cell making previous EDTA treatment unnecessary. From S. marcescens, the yield of these true protoplasts was considerably less and frequently the outer membrane, although it desintegrated, was still partially surrounding the cell (Fig. 3). The rigid layer has been removed and a lot of tubular-vesicular projections which originated from the outer membrane were observed. These projections, which are formed by a unit membrane seem to be continuous with the outer membrane (Fig. 4). Similar projections were described by Lounatmaa et al. (1976) when Salmonella was
0302-8933/78/0118/0219/$ 00.60
220
Arch. Microbiol., Vol. 118 0978)
Fig. 1. Longitudinal section of an intact cell of Serratia marcescens. Cytoplasmic membrane (CM), rigid layer (RL), and outer membrane (OM). x 76915 Fig.2. Electron micrograph of a protoplast of Escherichia coll. x 53840 Fig.3. Spheroplasts of S. mareescens. Note the tubular-vesicular projections, x 30216 Fig.4. A higher magnification of the tubular-vesicular projections on the surface of the outer membrane, x 86118
treated with polymyxin. I n this case the projections seem to be the result of the action of the a n t i b i o t i c o n the m e m b r a n e . The forms o b t a i n e d are stable a n d useful for further experiments o n the intact protoplasts a n d spheroplasts or for the isolation o f subcellular structures.
Acknowledgements. M.-R. R. was the recipient of a predoctoral
fellowship from the Ministery of Education of Spain.
References Hendlin, D., Stapley, E. O., Jackson, M., Wallick, H., Miller, A. K., Wolf, F. J., Miller, T. W., Chaiet, L., Kahan, F. M., Foltz, E. L.,
M.-R. Rodicio et al. : Protoplast Formation by Fosfomycin Woodruff, H. B., Mata, J. M., Hernfindez, S., Mochales, S.: Phosphonomycin, a new antibiotic produced by strains of Streptomyces. Science 166, 122-123 (1969) Kahan, F. M., Kahan, J. S., Cassidy, P. J., Kropp, H.: The mechanism of action of fosfomycin (phosphonomycin). Ann. N.Y. Acad. Sci. 235, 364-386 (1974) Lounatmaa, K., Makela, H. P., Sarvas, H. : Effect of polymyxin on the ultrastructure of the outer membrane of wild-type and polymyxin-resistant strains of Salmonella. J. Bacteriol. 127, 1400-1407 (i976) Perez-Urefia, M. T., Espinosa, M., Barasoain, I., Portoles, A.: Estudio microbiol6gico de esferoplastos de Pseudomonas pat6genos producidos por antibiosis a nivel de pared y membrana. Microbiol. Espafi. 27, 235-255 (1974)
221 Ryter, A., Kellenberger, E.: Etude au microscope 61ectronique de plasmas contenant de l'acide d@oxyribonucl6ique. Z. Naturforsch. 13b, 597-605 (1958) Stapley, E. O., Hendlin, D., Mata, J. M., Jackson, M., Wallick, H., Hernfindez, S., Mochales, S., Currie, S. A., Miller, R. M.: Phosphonomycin. I. Discovery and in vitro biological characterization. Antimicrob. Agents. Chemother. 1969, 284-290 (1970) Weiss, R. L. : Protoplast formation in Escherichia coli. J. Bacteriol. 128, 668- 670 (1976)
Received February 27, 1978
Microbiology
Arch. Microbiol. 118, 219-221 (1978)
9
by Springer-Verlag 1978
Short Communication
Protoplast-Like Structures Formation from Two Species of Enterobacteriaceae by Fosfomycin Treatment Maria-Rosario Rodicio, Manuel-Benjamin Manzanal, and Carlos Hardisson Departamento de Microbiologia, Universidad de Oviedo, Oviedo, spain
Abstract. A procedure for protoplasts formation from Escherichia coli and Serratia marcescens by treatment
with fosfomycin alone is described. This method gives high and low yields of stable protoplasts from E. coli and S. marcescens respectively. In the last case numerous spheroplasts were obtained. Electron micrographs of intact cells, protoplasts and spheroplasts are shown. Key words: Protoplasts -
coli-
Fosfomycin -
tial phase. For the obtention of protoplasts, the cells were harvested and transferred to the stabilization medium (Nutrient Broth with 2 g Mg SO4 97 H20 and 200 g sucrose per litre), containing 10 pg/ml of fosfomycin (kindly provided by CEPA, Aranjuez, Spain). The protoplasts were prefixed in 5 ~ glutaraldehyde for 2h at room temperature and fixed by the Ryter and Kellenberger method (1958), dehydrated with acetone and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and photographed in a Philips EM 300 electron microscope operating at 80 KV. Intact bacteria were prepared without prefixation.
Escherichia
Serratia marcescens. Results and Discussion
Fosfomycin is an antibiotic discovered by Hendlin et al. (1969) from cultures of Streptomyces fradiae. It is a bactericidal agent that blocks the initial step of the cell wall synthesis by irreversible inhibition of the activity of the enzyme phosphoenolpyruvate (PEP): uridine diphospho-N-acetyl-glucosamine (UDP-GlcNac) enolpyruvyl transferase, by acting as a structural analogue of phosphoenolpyruvate (Kahan et al., 1974). Formation of spheroplasts from cultures of Proteus vulgaris in the presence of the antibiotic (Stapley et al., 1970) and from Pseudomonas aeruginosa using EDTA plus fosfomycin (Perez Urefia et al., 1974) has been mentioned. We describe in this paper a reproducible procedure for obtaining high yields of protoplasts by using fosfomycin alone.
Materials and Methods Esche~ 1, hia coli ML 1 and Serratia marcescens isolated from clinical
samples, were grown in Nutrient Broth until the middle of exponen-
Escherichia coli ML I and Serratia marcescens (Fig. 1) exhibited a great sensitivity to the fosfomycin. The formation of protoplasts began after about 1 h of incubation and about 1 h later the majority of the cells were protoplasts. In a recent work, Weiss (1976) reported the obtention of true protoplasts from E. coli ML 30 after treatment with EDTA and lysozyme. We have obtained from E. coli ML1, using fosfomycin alone, a good yield of protoplasts which are surrounded only by the cytoplasmic membrane (Fig. 2). The low molecular weight of fosfomycin allows its passage through the outer wall layer of the cell making previous EDTA treatment unnecessary. From S. marcescens, the yield of these true protoplasts was considerably less and frequently the outer membrane, although it desintegrated, was still partially surrounding the cell (Fig. 3). The rigid layer has been removed and a lot of tubular-vesicular projections which originated from the outer membrane were observed. These projections, which are formed by a unit membrane seem to be continuous with the outer membrane (Fig. 4). Similar projections were described by Lounatmaa et al. (1976) when Salmonella was
0302-8933/78/0118/0219/$ 00.60
220
Arch. Microbiol., Vol. 118 0978)
Fig. 1. Longitudinal section of an intact cell of Serratia marcescens. Cytoplasmic membrane (CM), rigid layer (RL), and outer membrane (OM). x 76915 Fig.2. Electron micrograph of a protoplast of Escherichia coll. x 53840 Fig.3. Spheroplasts of S. mareescens. Note the tubular-vesicular projections, x 30216 Fig.4. A higher magnification of the tubular-vesicular projections on the surface of the outer membrane, x 86118
treated with polymyxin. I n this case the projections seem to be the result of the action of the a n t i b i o t i c o n the m e m b r a n e . The forms o b t a i n e d are stable a n d useful for further experiments o n the intact protoplasts a n d spheroplasts or for the isolation o f subcellular structures.
Acknowledgements. M.-R. R. was the recipient of a predoctoral
fellowship from the Ministery of Education of Spain.
References Hendlin, D., Stapley, E. O., Jackson, M., Wallick, H., Miller, A. K., Wolf, F. J., Miller, T. W., Chaiet, L., Kahan, F. M., Foltz, E. L.,
M.-R. Rodicio et al. : Protoplast Formation by Fosfomycin Woodruff, H. B., Mata, J. M., Hernfindez, S., Mochales, S.: Phosphonomycin, a new antibiotic produced by strains of Streptomyces. Science 166, 122-123 (1969) Kahan, F. M., Kahan, J. S., Cassidy, P. J., Kropp, H.: The mechanism of action of fosfomycin (phosphonomycin). Ann. N.Y. Acad. Sci. 235, 364-386 (1974) Lounatmaa, K., Makela, H. P., Sarvas, H. : Effect of polymyxin on the ultrastructure of the outer membrane of wild-type and polymyxin-resistant strains of Salmonella. J. Bacteriol. 127, 1400-1407 (i976) Perez-Urefia, M. T., Espinosa, M., Barasoain, I., Portoles, A.: Estudio microbiol6gico de esferoplastos de Pseudomonas pat6genos producidos por antibiosis a nivel de pared y membrana. Microbiol. Espafi. 27, 235-255 (1974)
221 Ryter, A., Kellenberger, E.: Etude au microscope 61ectronique de plasmas contenant de l'acide d@oxyribonucl6ique. Z. Naturforsch. 13b, 597-605 (1958) Stapley, E. O., Hendlin, D., Mata, J. M., Jackson, M., Wallick, H., Hernfindez, S., Mochales, S., Currie, S. A., Miller, R. M.: Phosphonomycin. I. Discovery and in vitro biological characterization. Antimicrob. Agents. Chemother. 1969, 284-290 (1970) Weiss, R. L. : Protoplast formation in Escherichia coli. J. Bacteriol. 128, 668- 670 (1976)
Received February 27, 1978
