Effect of ph on Turbidity and Ultrastructures of Endotoxins Extracted from Salmonella minnesota Wild Type and Re Mutant

Transcription

1 Microbiol. I mmunol. Vol. 29 (1), 75-80, 1985 Effect of ph on Turbidity and Ultrastructures of Endotoxins Extracted from Salmonella minnesota Wild Type and Re Mutant Ken-ichi AMANO, Toshinobu SATO, and Kazue FuKusHI* Department of Bacteriology, Hirosaki University School of Medicine, Hirosaki, Aomori 036 (Accepted for publication, October 26, 1984) It has been reported that the physical state of endotoxic lipopolysaccharide (LPS) is influenced by inorganic cations (Nat, K+, Mg2t, and Fe2+) and low molecular basic amines (putrescine, spermidine, spermine, and ethanolamine) (6, 8). On the other hand, Schramm et al (9) showed the ultrastructural differences between Escherichia coli LPS in ph 7 and 10 solutions. However, they did not observe these structures at acidic ph. In this paper, we observed the effect of ph on the turbidity and the ultrastructures of endotoxic LPS and glycolipid (ReG1) from Re mutant, and suggested that the ultrastructure and the turbidity of the endotoxins were under the influence of ph. S. minnesota 1114 (wild type) and R595 (Re mutant) were cultivated as described previously (1-3). The LPS and ReG1 were extracted with hot phenol-water (PW) according to the method of Westphal and Jann (10). The ReG1 was also extracted with phenol-chloroform-petroleum ether (PCP) according to the method of Galanos et al (7) and chloroform-methanol (CM) according to the method of Chen et al (4). The extracts (crude endotoxins) were purified by ultra-centrifugation (100,000 x g for 2 hr, repeated twice). Endotoxins and phosphatidylethanolamine (PEA, Sigma Chemical Co., St. Louis, MO, U.S.A.) (1 mg) were solubilized using an ultrasonic bath (Branson Cleaning Equipment, Shelton, CT, U.S.A.) in 2.2 ml each of the following 20 mm buffers; sodium citrate-na2hpo4 (ph 3.0, 5.0, and 6.7), sodium citrate-naoh (ph 3.0, 4.5, and 6.0), tris(hydroxymethyl)aminomethane-hc1 (ph 7.0 and 9.0), and glycine-naoh (ph 9.0 and 11.0), and incubated at 60 C for 20 min. After standing for 30 min at room temperature, the turbidity of these samples was measured with the absorbance at 500 nm. Electron microscopy was performed as described previously (1-3). We measured the turbidity in different ph solutions of the LPS and ReG1 to determine the effect of ph on the bacterial endotoxins in aqueous solution. As shown in Fig. 1, the wild-type LPS in the acidic buffers (ph 3.0) represented relatively high turbidity ( absorbance at 500 nm (Abs)/mg/2.2 ml), while the LPS in the buffers between ph 4.5 and 11.0 showed low turbidity (

2 76 K. AMANO ET AL Fig. 1. ph dependence of turbidity on wild-type LPS from Salmonella minnesota. The turbidity assays were as described in the text. The following buffers at the indicated ph values were used:, sodium citrate-na2hpo4;, sodium citrate-naoh; ~, Tris-HC1;, glycine-naoh; œ, distilled water. Abs/mg/2.2 ml). These data suggested that the LPS in the acidic buffers (ph 3.0) formed larger aggregates than the LPS in the buffers between ph 4.5 and On the other hand, the LPS dissolved in distilled water presented a turbidity similar to that of the LPS in the buffer at ph 3.0. Figure 2 shows that the absolute absorbance of ReGl-PW and ReG1-PCP is quite different (e.g., these turbidity at ph 3.0 was and Abs/ mg/2.2 ml, respectively), while the turbidity of these glycolipids has a similar tendency with respect to ph. Furthermore, these data indicated that the two ReG1 solutions at low ph represented higher turbidity than those at high ph in the same buffers. The values of turbidity of two ReGIs solubilized in distilled water corresponded to the absorbance of the original ReG1 solutions at around ph 3.0 (0.065 and 0.18 Abs/mg/2.2 ml, for ReG1-PCP and ReG1-PW, respectively). The turbidity of the ReGl-CM solution was relatively constant ( Abs/mg/2.2 ml) whenever this ReG1 was dissolved in any of the buffers or water, suggesting that the ReG1-CM aggregates were not under the influence of the ph of the buffers. To clarify whether or not the stable aggregates of ReGl-CM were due to the presence of phospholipids, we measured the turbidity of PEA, a major component in the phospholipids of Salmonella (data not shown). As shown in Fig. 3, the ph did not affect the turbidity of PEA. However, because the absorbance of PEA, dissolved in water, was eight times lower than that of ReGl-CM (0.004 and 0.32 Abs/mg/2.2 ml, respectively), it was suggested that the high turbidity of ReGl-CM is caused not only by the presence of PEA but also by the unknown character of the ReG1-CM. The endotoxic LPS and ReG1 were solubilized in distilled water, with the ph adjusted by HC1 or NaOH, and observed by electron microscopy after shadowing with platinum-palladium, to clarify the influence of ph on the ultrastructures of endotoxins and the relationship between the turbidity of endotoxin solutions and these ultrastructures. The LPS showed amorphous and spherical aggregates at

3 NOTES 77 A B C Fig. 2. ph dependence of turbidity on ReGls from S. minnesota. The turbidity assays were as described in the text. The buffers used were as indicated in the legend of Fig. 1. A, ReGI-PW; B, ReGI-PCP; C, ReGl-CM. Fig. 3. ph dependence of turbidity on phosphatidylethanolamine. The turbidity assays were as described in the text, The used buffers were indicated in the legend of Fig. 1.

4 78 K. AMANO ET AL Fig. 4. LPS with Electron micrographs in different ph platinum-palladium. 3.0 solution; b, in ph 4.5; d, in ph e, in ph ; of solutions a, wild-type shadowed 1.PS in ph c, in ph 5.4;

5 79 NOTES Fig. 5. Electron micrographs of wild-type LPS stained with I% sodium phosphotungstate, ph 3.0 (a), ph 3.5 (b), and ph 5.0 (c). ph 3.0, while this material formed a mixture of amorphous aggregates and filamentous forms at ph 4.5 (Fig. 4, a and b). At ph 5.4, all the LPS presented a filamentous form which had a width of nm on the average (Fig. 4c). Because the width of bilayers of wild-type LPS stained positively with uranyl formate was approximately nm (5), the LPS filaments at ph 5.4 might be a single bilayer. The ultrastructure of LPS at ph 7.0 and 10.0 showed a filamentous form over 20 nm wide (Fig. 4, d and e), suggesting that these filaments are in a bundle of the filaments seen at ph 5.4. Based on the comparison of the LPS ultrastructures, it appeared that the amorphous or spherical aggregation of LPS changed to the filamentous form between ph 4 and 5. On the other hand, we observed the ultrastructures of LPS stained negatively with sodium phosphotungstate (PTA) at different ph (Fig. 5). The LPS stained at ph 3.0 represented a spherical aggregation, but this endotoxin stained at ph 3.5 and 5.0 formed mixtures of spherical, amorphous, and filamentous forms. The transformation of the LPS stained with PTA occurred between ph 3.0 and 3.5.

6 80 K. AMANO ET AL Based on the results of the turbidity assay and the electron microscopy of LPS, it was suggested that the forms of LPS aggregates are transformed from spheres into filaments in proportion to the decrease in the turbidity of the LPS solution. As described in a previous paper (1), the shadowed ultrastructures of ReGl-PW, ReGl- PCP, and ReGl-CM showed spherical forms, small flat pieces, and wide sheets, respectively, regardless of ph. Furthermore, in this study, the ReGls did not show any changes in the ultrastructures when stained with PTA at ph 3.0 and 6.0 (data not shown). REFERENCES 1) Amano, K., and Fukushi, K Chemical and ultrastructural differences in endotoxic glycolipids from Salmonella minnesota Re mutant extracted with various solvent systems. Microbiol. Immunol. 28: ) Amano, K., and Fukushi, K Chemical and ultrastructural comparison of endotoxins extracted from Salmonella minnesota wild type and R mutants. Microbiol. Immunol. 28: ) Amano, K., and Fukushi, K Electron microscopic studies of endotoxins treated with alkaline and acid reagents. Microbiol. Immunol. 28: ) Chen, C.H., Johnson, A.G., Kasai, N., Key, B.A., Levin, J., and Nowotny, A Heterogeneity and biological activity of endotoxic glycolipid from Salmonella minnesota R595. J. Infect. Dis. 198: 543 S51. 5) Fukushi, K., Asano, H., and Sasaki, J The physical structure of endotoxin extracted from wild-type and R mutants of Salmonella. J. Electron Microsc. 26: ) Galanos, C., and Luderitz, O Electrodialysis of lipopolysaccharides and their conversion to uniform salt forms. Eur. J. Biochem. 54: ) Galanos, C., LUderitz, O., and Westphal, O A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 9: ) Nixdorf, K., Gmeiner, J., and Martin, H.H Interaction of lipopolysaccharide with detergents and its possible role in the detergent resistance of the outer membrane of gram-negative bacteria. Biochim. Biophys. Acta 510: ) Schramm, V.G., Westphal, O., and Liideritz, O Doer bakterielle Reizstoffe III. Mit: Physikalisch-chemisches Verhalten eines hochgereinigten Coli-Pyrogens. Z. Naturforschung. 7B: ) Westphal, O., and Jann, O Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure, p In Whister, R. L. (ed), Method in carbohydrate chemistry, Vol. 5, Academic Press, Inc., New York. (Received for publication, February 17, 1984)