VOL. 50, 1963 BIOCHEMISTRY: M. J. OSBORN 499 8 Davidson, J..N., and R. N. Smellie, Biochem. J., 52, 594 (1952). 9 Leslie, I., in The Nucleic Acids, ed. E. Chargaff and J. N. Davidson (New York: Academic Press, 1955), vol. 2, chap. 16. 10Jacobson, K. B., and S. Nishimura, Biochim. Biophys. Acta, 68, 490-493 (1963). 11Jacobson, K. B., Science, 138, 515 (1962). 12 Hiatt, H. H., J. Mol. Biol., 5, 217 (1962). 13 Schneider, W. C., in Methods in Enzymology, ed. S. P. Colowick and N. 0. Kaplan (New York: Academic Press, 1957), vol. 3, p. 680. 14 Britten, R. J., and R. B. Roberts, Science, 131, 32 (1960). 15 Bollum, F. J., J. Biol. Chem., 234, 2733 (1959). 16 Kenney, F. T., J. Biol. Chem., 234, 2707 (1959). 17 Robinson, C. L., and G. D. Novelli, Arch. Biochem. Biophys., 96, 459 (1962). 18 Brunngraber, E. P., Biochem. Biophys. Res. Comm., 8, 1 (1962). 19 Mans, R. J., and G. D. Novelli, Arch. Biochemn. Biophys., 94, 48 (1961). B Hoagland, M. B., and B. A. Askonas, these PROCEEDINGS, 49, 130 (1963). 21 Elson, D., L. W. Trent, and E. Chargaff, Biochim. Biophys. Acta, 17, 362 (1955). 22 Jacob, F., and J. Monod, J. Mol. Biol., 3, 318 (1961). STUDIES ON THE GRAM-NEGATIVE CELL WALL, I. EVIDENCE FOR THE ROLE OF 2-KETO-3-DEOXYOCTONATE IN THE LIPOPOLYSACCHARIDE OF SALMONELLA TYPHIMURIUM* BY M. J. OSBORNt DEPARTMENT OF MICROBIOLOGY, NEW YORK UNIVERSITY SCHOOL OF MEDiCINE Communicated by B. L. Horecker, July 24, 1963 The cell wall lipopolysaccharides which determine the somatic (o) antigen specificities of Salmonella typhimurium and E. coli consist of highly branched, com- plex polysaccharides linked to a glucosamine-containing lipid.' The polysaccharide moiety may contain as many as 6 neutral sugars; for example, L-glycero-D-man- noheptose, glucose, galactose, mannose, rhamnose, and abequose (3,6-dideoxy-D- galactose) have been identified2' I as components of the polysaccharide of S. typhimurium. The polysaccharides are thought'- to consist of an internal core structure, possibly similar in all enteric bacteria, to which are attached complex side chains bearing group or species specific antigenic determinants. New insight into both structure and mechanism of biosynthesis of these polysaccharides has recently been obtained3' 5 through the use of mutant organisms deficient in the syn- thesis of specific nucleotide sugars. It was first shown by Nikaido5' 6 that incom- plete polysaccharides are formed by mutants which are unable to synthesize UDP- galactose as a result of loss of the enzyme, UDP-galactose4-epimerase. The polysaccharides formed by these mutants characteristically lack not only galactose, but also certain other sugars present in the normal polymers, and appear to represent the innermost core region of the wild-type polysaccharide. Previous studies in our laboratory3 on a UDP-galactose-4-epimeraseless strain of S. typhimurium have shown that the incomplete "core" polysaccharide formed by this mutant contains glucose, L-glycero-D-mannoheptose, and phosphate. It has now been found that this polysaccharide also contains 2-keto-3-deoxyoctonate (KDO),7 recently identified by Heath and Ghalambor8 as a component of the lipopolysaccharide of 500 BIOCHEMISTRY: M. J. OSBORN PROC. N. A. S. E. coli 0111. In agreement with the results of these workers, the bulk of the KDO present in the lipopolysaccharide of the S. typhimurium mutant appears to be in glycosidic linkage at nonreducing terminal positions. In addition, however, ap- proximately 25 per cent of the total KDO occurs in a different position; this frac- tion is recovered in the purified, lipid-free polysaccharide, and occupies the reduc- ing-terminal position of glucose-heptose-phosphate chains. Most, if not all, of the polysaccharide chains appear to contain KDO at the reducing end, suggesting that the sugar acid may be involved in the linkage of the core polysaccharide to the lipid moiety of the intact lipopolysaccharide. Materials and Methods.-The previously described3 mutant of S. typhimurium lacking UDP- galactose-4-epimerase was grown commercially by the Grain Processing Corp. of Muscatine, Iowa, in a galactose-free peptone medium. Lipopolysaccharide was routinely prepared by phenol ex- traction of cell walls followed by precipitation with Mg++ as described earlier.3 Free KDO was isolated by chromatography on DEAE cellulose as described in the legend for Figure 2 after hydrolysis of lipopolysaccharide at pH 3.4, and was purified either by adsorption and elution from charcoal according to Heath and Ghalambor,'8 or by paper chromatography on Whatman no. 40 paper. Solvent systems employed for identification of KDO were: 2-butanone, acetic acid, H20 (8:1:1); ethyl acetate, acetic acid, H20 (3:1:3); n-butanol, pyridine, 0.1 N HCl (5:3:2); ethanol, acetic acid, H20 (80:1:19). 2-Keto-3-deoxyheptonolactone was a gift from Dr. D. B. Sprinson. KDO was determined by the thiobarbituric acid method of Weissbach and Hurwitz9 with minor modifications. For determination of KDO in the presence of a large excess of polysaccharide, the amounts of HI04 and NaAsO2 were increased to 10 umoles and 100 ,moles, respectively. A heat- ing time of 20 min was required to give maximal color development with polysaccharide-bound KDO. Under the assay conditions, 1.0 Mtmole of KDO gave an absorbancy of 19.0 at 548 mju in the Beckman DU spectrophotometer. Total lipopolysaccharide-bound KDO was determined after hydrolysis of lipopolysaccharide in 0.02 N H2S04 for 20 min at 1000. Heptose was determined by the cysteine-H2S04 reaction,'0 modified as follows: 2.25 ml of H2S04 (6 vol concentrated H2S04: 1 vol H20) were slowly added to duplicate samples (0.5 ml) in an ice-H20 bath, and mixed by shaking in the cold. After 3 min, the tubes were transferred to a 200 bath for 3 min, and then heated in a vigorously boiling H20 bath for exactly 10 min. The 10 min heating time minimized interference by other components of the polysaccharide. After cooling, 0.05 ml of 3% cysteine-HCl was added to one sample, the other serving as a blank. Absorbancy at 505 mjA and 545 mu was determined exactly 2 hr after addition of cysteine, and corrected for nonspecific absorbance in the blank minus cysteine. Under these conditions, 1.0 jmole of L-glycero-D-mannoheptose gave a value of A5ob - A, = 1.07. Glucose was determined with glucose oxidase (Worthington Glucostat reagents) after hy- drolysis of the polysaccharide in 1 N HCl for 5 hr at 100° in sealed, evacuated tubes. Internal standards were included, and the values reported have been corrected for 10-15% loss of added glucose during hydrolysis. Total carbohydrate was determined by the phenol-H2SO4 method," reducing sugar by the Nelson method'2 and phosphate by the procedure of Berenblum and Chain13 as modified by Ennor and Stocken."4 Results.-Identification of KDO in the lipopolysaccharide of the epimeraseless mutant: The presence of an unknown component was first suggested by the observation that the spectrum of the purified lipopolysaccharide in the cysteine- H2SO4 reaction contained a peak at 385-390 mu which could not be assigned to any of the known constituents. The material responsible for this reaction could be released from lipopolysaccharide by mild acid hydrolysis and purified as described in Materials and Methods. The purified compound showed a distinctive absorption spectrum in the cysteine-H2SO4 reaction (Xmax = 383 mu before cysteine, 390 m/u after cysteine addition, with diffuse absorption from 450-650 mu, and a secondary peak at 590 m, which appeared at 24-48 hr), and gave a reaction in the thiobar- VOL. 50, 1963 BIOCHEMISTRY: M. J. OSBORN 501 bituric acid test characteristic of 2-keto-3-deoxyaldonic acids (X max 549 my). The presence of an a-keto acid was confirmed by formation of the semicarbazone derivative'5 (Xmax = 250 mu). The isolated material was free of nitrogen and phos- phorus, and was chromatographically identical in 4 solvent systems with authentic KDO, kindly supplied by Dr. E. C. Heath."6 Identification of the S. typhimurium component as KDO was confirmed by periodate oxidation, which yielded HCHO, HCOOH, and formylpyruvate in the expected ratio of 1:3: 1. The present results support the conclusion of Heath and Ghalambor8 that KDO exists in glycosidic linkage as an integral component of the lipopolysaccharide. By two methods of lipopolysaccharide purification (phenol-Mg++ precipitation and Pronase digestion), KDO was recovered exclusively in the lipopolysaccharide frac- tion at each stage of purification. Both methods yielded lipopolysaccharide having a KDO: heptose ratio of approximately 0.3. The properties of lipopolysaccharide- bound KDO are consistent with glycosidic linkage to the polymer. The carbonyl group of bound KDO is resistant to borohydride reduction. No significant destruc- tion of KDO (measured by the thiobarbituric acid reaction after hydrolysis of the lipopolysaccharide) was observed after treatment of the intact lipopolysaccharide with NaBH4; exposure to borohydride after release of KDO from lipopolysaccharide by mild acid hydrolysis resulted in complete loss of reactivity toward thiobarbituric acid. The linkage is resistant to alkali, but extremely labile to acid hydrolysis, as judged 100- by appearance of reactivity in the thiobarbi- turic acid test and by liberation of free KDO, identified chromatographically. Figure 1 r2 8LX illustrates the time course of KDO release x2 during acid hydrolysis. At 1000, the half- m times of hydrolysis at pH 2.0, 3.0, and 3.4 ' pH 3 were approximately 6, 9, and 12 min, respec- z tively; at pH 4.5, the corresponding value 0 ;, epH 45 was 45 min. Isolation of polysaccharide-bound KDO: 40 / Hydrolysis of the insoluble mutant lipo- polysaccharide at pH 3.4 and 1000 resultedi in quantitative release of polysaccharide / and of KDO into the soluble fraction.