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Ticles. the 5,6-Dimethylbenzimidazolylcobamide Coenzyme Has Been Identified in the Azotobacter

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Ticles. the 5,6-Dimethylbenzimidazolylcobamide Coenzyme Has Been Identified in the Azotobacter 1542 MICROBIOLOGY: H. NIKAIDO PROC. N. A. S. supernatant fluid, whereas the cobamide coenzyme remains in the ribosomal par- ticles. The 5,6-dimethylbenzimidazolylcobamide coenzyme has been identified in the azotobacter. We are grateful to David Perlman, the Squibb Institute for Medical Research, New Jersey, for determining vitamin B12 in our preparations and for the gift of cobalt58-labeled hydroxocobala- min. * Sabbatical leave October 1961-1962 at the University of Wisconsin. Aided by grant E-1417 (C6) from the National Institutes of Health and grant C-2826 from the National Science Founda- tion. t Appeared on the program for the Annual Meeting and in the abstracts published in Science as "Metabolism of Inorganic Nitrogen and Its Compounds in Microorganisms" by D. J. D. Nicholas and P. W. Wilson. l Holm-Hansen, 0. G. C., H. Gerloff, and F. Skoog, Physiol. Plantanum, 7, 665 (1954). 2 Shaukat-Ahmed, and H. J. Evans, Biochem. Biophys. Res. Comm., 1, 271 (1959) and Soil Science, 90, 205 (1960). 3 Lowe, R. H., and H. J. Evans, J. Bacteriol., 83, 210 (1962). 4Reisenauer, H. M., Nature, 186, 375 (1960). 5 Hallsworth, E. G., S. B. Wilson, and E. A. N. Greenwood, Nature, 187, 79 (1960). 6 Delwiche, C. C., C. M. Johnson, and H. M. Reisenauer, Plant Physiol., 36, 73 (1961). 7Lowe, R. H., H. J. Evans, and Shaukat-Ahmed, Biochem. Biophys. Res. Comm., 3, 675 (1960). 8 Nicholas, D. J. D., Y. Maruyama, and D. J. Fisher, Biochim. Biophys. Acta, 56, 623 (1962). 9 Hu, A. S. L., R. Epstein, H. 0. Halvorson, and R. M. Bock, Arch. Biochem. Biophys., 91, 210 (1960). 10 Nicholas, D. J. D., and D. J. Fisher, J. Sci. Food Agric., 11, 603 (1960). 11 Robrish, A. R., and A. G. Marr, J. Bacteriol., 83, 158 (1962). 1 Hirozi, K. K., H. 0. Halvorson, and R. M. Bock, Biochim. Biophys. Acta, 49, 212 (1961). 1 Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 14 Mejbaum, W., Z. physiol. Chem., 258, 117 (1939). 16 Kliewer, M., and H. J. Evans, Nature, 194, 108 (1962). 16 Alexander, M., and P. W. Wilson, these PROCEEDNGS, 41, 843 (1955). STUDIES ON THE BIOSYNTHESIS OF CELL WALL POLYSACCHARIDE IN MUTANT STRAINS OF SALMONELLA, II BY HIROSHI NIKAIDO INSTITUTE FOR PROTEIN RESEARCH, OSAKA UNIVERSITY, JAPAN* Communicated by Herman M. Kalckar, June 26, 1962 In the preceding paper, ' we described the properties of cell wall lipopolysaccharide (LPS)t from the mutant strains of Salmonella enteritidis and S. typhimurium, which cannot synthesize galactose.' LPS from wild type strains of these species contains glucose, galactose, mannose, rhamnose, 3,6-dideoxyhexose, and probably heptose, while LPS from these mutant strains (M mutants) contains only glucose and probably heptose as neutral sugar.1-3 When, however, M mutant cells were grown in the presence of galactose for a short period, they synthesized LPS, the composition of which was very similar to that in the wild type strains.24 This study was initiated in order to investigate the mechanism underlying these Downloaded by guest on September 24, 2021 VOL. 48, 1962 MICROBIOLOGY: H. NIKAIDO 1543 phenomena. The properties of the strains used were already described in the pre- ceding article.' Two hypotheses were considered to explain these observations. In the first and the simpler hypothesis, we assume that several sugars which cannot be found in M mutant LPS-mannose, rhamnose, and 3,6-dideoxyhexose-are all synthesized from galactose in Salmonella. Thus M mutant cells cannot synthesize galactose, and consequently the other sugars; but if they are grown in the presence of galac- tose, the exogenous galactose is converted into uridine diphosphate galactose (UDP- Gal) (for pathway, see Fig. 1) and then into the other sugars, which are now in- corporated into the cell wall polysaccharide. exogenous galactose I galactokinase Gal-i-P Gal-i-P uridyl Glu transferase A Glu IV< Glu UDPGal UDPGal4- epimerase Gal-Gal-Glu Gal-Glu B UTP<--- UDPG Glu I Gal-Gal-Glu Glucose-i-phosphate Glu-Gal-Man-Rha-Glu-Gal-Man-Rha-Gal-Gal-Glu L I L Gal-Glu C Glu Tyv-Man-Rha-Glu-Gal-Man-Rha-Gal-Gal-Glu JI* FIG. l.-Suggested pathway for the incorporation of exogenous galactose into LPS of M mutants. M mutants have a genetic defect at UDPGal4-epimerase (X), and accordingly cannot syn- thesize UDPGal from UTP and glucose-l-phosphate. Therefore, the cell wall polysaccharide remains at stage A, namely, poly-glucose. When galactose is added exogenously, it is metabolized by galactokinase and Gal-1-P uridyl transferase, and is transformed into UDPGal. UDPGal transfers its galactose moiety to A, forming B, and other sugars are subsequently transferred, thus finally completing the wild type-like polysaccharide C. The structures of polysaccharides are hypothetical. Monosaccharide sequences covered with brackets in C represent the hypothetical "repeating units." UTP, uridine triphosphate; Glu, glucose; Gal, galactose; Man, mannose; Rha, rhamnose: Tyv, tyvelose. The other more complicated hypothesis$ explains the situation in the following way. It is assumed that LPS or the O-polysaccharide of Salmonella has a "core" or "skeleton" of poly-glucose, and that all the side chains from this "core" must start with galactose. It is also assumed that mannose, rhamnose, and 3,6-dideoxyhexoses Downloaded by guest on September 24, 2021 1544 MICROBIOLOGY: H. NIKAIDO PROC. N. A. S. are present only in the side chains. Thus, M mutants cannot initiate the side chain containing various sugars, because of the lack of galactose. In the presence of galactose, however, they can make UDPGal, and add galactose onto the poly-glu- cose "core," thus initiating the formation of side chains. Incorporation of C14-Galactose with Intact Cells.-If the first hypothesis is correct, exogenously given C '4-galactose must be converted into various other sugars in the LPS by the intact M mutant cells. Galactose-1-C14 was therefore added to a grow- ing culture of 11-1-M cells at the concentration of 0.005%, and after 20 minutes' incubation, the cells were harvested, the 90% ethanol-insoluble fraction, containing LPS, was hydrolyzed with 2 N H2S04, and the hydrolyzate was chromatographed on paper. Spots of glucose, galactose, mannose, rhamnose, tyvelose, and ribose (probably from RNA) were found, but only the spot of galactose was radioactive. This result completely rules out the first hypothesis. Accumulation of Nucleoside Diphosphate Sugar Compounds in the Cell.-If the second hypothesis is correct, various sugars other than glucose and galactose cannot attach to the "core" simply because of the lack of attachment sites. Thus it would be expected that these sugars would accumulate in the soluble fraction, possibly in the form of the direct precursors before their transfer to the core. In fact, when the ethanol-soluble fraction of M mutant cells was treated with dilute acid, and was then chromatographed on paper, all the missing sugars in M mutant polysacchar- ide-except galactose-were demonstrated. It appeared most probable that the transfer of sugars to the core was done from nucleoside diphosphate sugar deriva- tives. Therefore, we looked for these compounds in the 80% ethanol-soluble frac- tion of M mutants grown without the addition of galactose. The ethanol extract was applied to a column of Dowex-1 (Cl') resin, and the elution was made with a HCl-NaCl system.5 The nucleotides containing 3,6-dideoxyhexoses were detected by the thiobarbituric acid reaction,6 and that containing rhamnose was detected by the cysteine-sulfuric acid reaction.7 The nucleotides were adsorbed to and then eluted from charcoal, and subsequently purified by paper chromatography. As a result of this study, nucleotides containing 3,6-dideoxyhexoses were isolated and were identified as cytidine diphosphate tyvelose, and cytidine diphosphate abe- quose.8 These were obtained from M mutants of S. enteritidis and of S. typhi- murium, respectively; the wild type strains of these bacteria contained in their LPS tyvelose and abequose respectively. It is interesting to note that these were the first cytidine diphosphate sugar compounds to be found in nature, together with the similar compounds isolated from A. vinelandii by Suzuki's group.9 The sugars in the latter compounds appear tohave a rather unusualstructure, and to be definitely different from 3,6-dideoxyhexoses.9 The nucleotide containing rhamnose seemed to be identical with thymidine diphosphate rhamnose.10 The nucleotide content of various strains is shown in Table 1, and it is quite clear that M mutant cells ac- cumulate large amounts of these nucleotides. These results support, or at least are consistent with, the second hypothesis. Several other pieces of evidence can be found which support the second hypothe- sis. Westphal and his co-workers demonstrated that usually smooth forms of Salmonella contain many sugars in their LPS, but that when they mutate to rough forms, various sugars are all lost at the same time, leaving only glucose and galac- tose.11 12 Furthermore, they showed'2 that when colitose was stripped off by mild Downloaded by guest on September 24, 2021 VOL. 48, 1962 MICROBIOLOGY: H. NIKAIDO 1545 TABLE 1 THE ACCUMULATION OF SOME NUCLEOSIDE DIPHOSPHATE SUGARS IN VARIOUS STRAINS -Nucleotide Accumulated- (pmoles/10 g dry wt. cells) CDP- Exp. Strain Description dideoxyhexose TDP-rhamnose 1 No. 11 Wild type (0.1)* (0.2)* 2 11-1-M M mutant 8.0 3.2 3 11-1-M " 5.8 ... t 4 7-M-1 " 2.6 ... t 5 7-M-1 " 2.9 1.7 The cells were grown in nutrient broth (without the addition of galactose) at 370C with aeration by shak- ing, and were harvested close to the end of the exponential phase of growth.
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