Received for publication 6 August 1976. Polymyxin-caused projections on the cell surface ofSalmonella typhimurium were seen as depressions in the outer ...
Vol. 128, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Nov. 1976, P. 665-667 Copyright X) 1976 American Society for Microbiology
Effect of Polymyxin on the Outer Membrane of Salmonella typhimurium: Freeze-Fracture Studies K. LOUNATMAA'* AND N. NANNINGA Laboratory of Electron Microscopy, University of Amsterdam, Amsterdam, The Netherlands Received for publication 6 August 1976
Polymyxin-caused projections on the cell surface of Salmonella typhimurium were seen as depressions in the outer concave fracture face and as protrusions in the outer convex fracture face, indicating participation of both leaflets of the outer membrane in these projections. Polymyxin affects the ultrastructure of the outer membrane of Escherichia coli (1-3, 6, 8), Pseudomonas aeruginosa (3), and Salmonella typhimurium (4). When studied in thin sections, polymyxin-treated cells carry protrusions on the cell surface. These protrusions vary in structure, depending on whether the bacterial strain is smooth or rough and whether the strain is polymyxin sensitive or polymyxin resistant (4). The typical polymyxin effect on smooth and polymyxin-sensitive (wild-type) cells is the occurrence of numerous finger-like projections, which seemingly arise as protrusions of the outer membrane. In thin sections the outer leaflet of the outer membrane was seen to continue into the polymyxin-caused projections, whereas the inner leaflet was never seen to do so. It was inferred that polymyxin may be preferentially acting at the level of the outer leaflet. Freeze-fracturing can be expected to give more and fundamentally different information on membrane ultrastructure than does thin sectioning. Consequently, we have used freeze-fracturing to study the effect of polymyxin on the outer membrane of Salmonella. The rough (rfa-mutation) strain (SH5014) and smooth strain (SH4247) of polymyxin-sensitive S. typhimurium (4) were grown in L broth, harvested by centrifugation in the logarithmic phase, and suspended in 0.01 M tris(hydroxymethyl)aminomethane buffer (pH 7.2) containing 10 ,ug of polymyxin E (Colomycin, colistin sulfate B.P.; Pharmax Ltd., Bexley, Kent) per ml. These suspensions were incubated for 30 min at 37°C, after which glycerol was added to a final concentration of 30% (vol/vol). The pellet collected by centrifugation was frozen in liquid Freon 22 cooled by liquid nitrogen. Fracturing was performed at - 120°C in the appara-
tus of Balzers. Micrographs of freeze-fractured replicas were printed in reverse; the direction of platinum shadowing is indicated by an arrow in the figures. Figures 1 and 2 show the fracture faces of the outer membrane of untreated S. typhimurium SH5014 after freeze-fracturing. The typical concave fracture face (OM) is composed of closely packed particl, The corresponding convex fracture face (OM) appears smooth with shallow grooves and occasional particles. Both of these fracture faces appear identical with the corresponding faces described earlier in E. coli (7). Identical fracture faces are obtained from the smooth parent strain (SH4247; not shown). Figures 3 and 4 show the effect of 10 jug of polymyxin per ml on the outer membrane of SH4247 in freeze-fractured cells. Numerous pitlike depressions are visible in the outer concave fracture face (OM) of Fig. 3 as compared with the untreated preparation in Fig. 1. The size and distribution of the closely packed particles, 8 nm in diameter, typical of this fracture face are not altered. Figure 4 shows the corresponding outer convex fracture face (OM) with numerous small, conelike elevations or particles similar in density and size to the pits on the corresponding concave fracture face (Fig. 3). It is therefore clear that both leaflets of the outer membrane participate in the polymyxin-caused projections. No alterations were observed in the fracture faces of the plasma membrane. Schindler and Teuber (5) demonstrated projections on the surface of polymyxin-treated cells in etched freeze-fracture preparations. However, they did not study possible alterations in the fracture faces of the outer membrane. They, therefore, could not see the participation of both leaflets of the outer membrane in the formation of the projections.
I Present address: Department of Electron Microscopy, University of Helsinki, Helsinki, Finland.
We wish to thank J. Raphael-Snijer, F. de Vries, and J. H. Leutscher for excellent technical assistance and P. Hel-
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FIG. 1-4. S. typhimurium. Outer concave (OA) and conzvev (OM) fracture face; the plane of cleavage running through the outer membrane (OM). Inner concave (PM) and convex (P 1) fracture face; the plane of cleavage running through the cytoplasmic membrane; CC, cytoplasmic contents. Arrow indicates the direction of shadowing. Bar indicates 0.2 ,um. FIG. 1. Concave fracture faces of the cell envelope of S. typhimurium rough strain SH5014. FIG. 2. Convex fracture faces of strain SH5014. FIG. 3. Outer concave fracture face (O0) of smooth strain SH4247 treated with 10 pg ofpolymyxin per ml. FIG. 4. Corresponding outer convex fracture face (O6)). 666
VOL. 128, 1976 ena Makela and M. Sarvas for their help and critical reading of this paper. This research was supported (for K.L.) by The Ministry of Education and Sciences, The Netherlands.
LITERATURE CITED 1. Chapman, G. B. 1962. Cytological aspects of antimicrobial antibiosis. J. Bacteriol. 84:169-179. 2. Koike, M., and K. lida. 1971. Effect of polymyxin on the bacteriophage receptors of the cell wall of gram-negative bacteria. J. Bacteriol. 108:1402-1411. 3. Koike, M., K. Iida, and T. Matsuo. 1969. Electron microscopic studies on mrode of action of polymyxin. J. Bacteriol. 97:448-452. 4. Lounatmaa, K., P. H. Makela, and M. Sarvas. Effect of polymyxin on the ultrastructure of the outer mem-
NOTES
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6.
7. 8.
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brane of wild-type and polymyxin-resistant strains of Salmonella. J. Bacteriol. 127:1400-1407. Schindler, P. R. G., and M. Teuber. 1975. Action of polymyxin B on bacterial membranes: morphological changes in the cytoplasm and in the outer membrane of Salmonella typhimurium and Escherichia coli B. Antimicrob. Agents Chemother. 8:95-104. Suganuma, A., K. Hara, T. Kishida, K. Nakjima, and J. Kavamata. 1968. Cytological changes of Escherichia coli caused by polymyxin E. Biken J. 11:149-155. van Gool, A. P., and N. Nanninga. 1971. Fracture faces in the cell envelope of Escherichia coli. J. Bacteriol. 108:474-481. Wahn, K., G. Lutsch, T. Rockstroh, and K. Zapf. 1968. Morphological and physiological investigations on the action of polymyxin B on Escherichia coli. Arch. Microbiol. 63:103-116.
