Release of alkaline phosphatase activity by aqueous-ether treatment of whole cells of Serratia marcescens.

The effect of aqueous-ether treatment according to the method of Ribi et al. (1961) on the release of alkaline phosphatase from cells of two strains of Serratia marcescens was studied. By this method, lipopolysaccharide-protein (endotoxin) complexes associated with alkaline phosphatase activities were released from both strain 08 and strain Bizio. SDS-polyacrylamide gel electrophoresis followed by enzymatic assay showed the presence of two active components in each strain. Fractions released from strain 08 contained alkaline phosphatase A (140,000 dalton) and alkaline phosphatase B (110,000) daltons) while those from strain Bizio contained alkaline phosphatase A' (190,000 daltons) and alkaline phosphatase B (110,000 daltons). Although it is known that saline plays a role in the release of alkaline phosphatase activities from cell envelope of Gram-negative bacteria the presence of saline in the extracting medium affects only slightly the chemical composition and not at all on the enzymatic nature of the released components. By comparing the enzymatic profiles of the materials released by other techniques, such as polymyxin B treatment and osmotic shock, it appears that alkaline phosphatase activities released by aqueous-ether treatment of whole cells of S. marcescens originate from the periplasmic space.

Relationship of structure to function in bacterial endotoxins. IX. Differences in the lipid moiety of endotoxic glycolipids.

Chemical, immunochemical, chromatographic, and endotoxic properties of five chromatographically pure glycolipids were compared. The preparations were extracted by chloroform-methanol from three Escherichia coli, one Salmonella minnesota, and one S. typhimurium Re heptoseless mutant strains. The local Shwartzman skin assay, the nonspecific resistance-enhancing effect, and the Limulus assays could not distinguish among the five glycolipids, all five being active in all three assays. Significant differences could be seen when the tumor resistance-enhancing effect of the glycolipids in mice was compared with the nonspecific TA3-Ha murine mammary adenocarcinoma growing in ascites form. Even greater variation was observed in the capacity of the preparations to enhance the nonspecific resistance of mice to virulent S. typhi 0901 infections. The data show that the five glycolipids are quite dissimilar in their biological effects. Similarly, thin-layer chromatography and molecular ratio determinations showed that differences exist in the chemical structure of the glycolipids. Accordingly, we claim that not only the polysaccharide but the lipid moiety as well may vary in various gram-negative endotoxin preparations.

Isolation and composition of oligosaccharide cores from endotoxins of two Serratia marcescens strains.

The oligosaccharide cores isolated from the acetic acid hydrolysates of endotoxins from Serratia marcescens 08 and Serratia marcescens Bizio were analyzed for their sugar composition. The intact oligosaccharide core from S. marcescens 08 consisted of 2-keto-3-deoxyoctonate, d-glycero-d-mannoheptose, l-glycero-d-mannoheptose, d-glucose, d-galactose, and d-glucosamine in a molar ratio of 2:1:5:3:1:3 and that from S. marcescens Bizio consisted of the same sugar components in a molar ratio of 2:1:5:5:1:2. This result indicates that endotoxins from S. marcescens genus may contain more than one structural type of oligosaccharide core. Both oligosaccharide cores also differ in their chemical compositions from cores of other Enterobacteriaceae.

Lipid content of antibiotic-resistant and -sensitive strains of Serratia marcescens.

The lipid content of antibiotic-resistant, nonpigmented strain (Bizio) and antibiotic-sensitive, pigmented strain (08) of Serratia marcescens was studied. The resistant strain contains at least three times more total extractable lipid and phospholipid than the sensitive strain. Lysophosphatidylethanolamine, phosphatidylserine, lecithin, phosphatidylglycerol, phosphatidylethanolamine, and polyglycerolphosphatide were identified in the phospholipid fractions of both strains.

Purification and partial characterization of a bacteriocin from Serratia marcescens.

Bacteriocin JF246 has been purified from mitomycin C-induced Serratia marcescens cells by salt extraction, ammonium sulfate fractionation, and chromatography on QAE-Sephadex and SP-Sephadex. The purified material is homogeneous on polyacrylamide gel electrophoresis in the presence of 2% sodium dodecyl sulfate or 6 m urea. In the absence of these agents, the bacteriocin associates into aggregates which can be dissociated with 0.4 m NaCl. The bacteriocin is probably composed of a single subunit with a molecular weight of 64,000 daltons. Analytical studies show the bacteriocin to be essentially protein in nature containing less than one residue of glucose or phosphorus per 64,000 daltons.