Jun 27, 1972 - Lipid-linked glucose is formed from uridine diphosphate-glucose and fica- prenol phosphate only in strains ofShigella flexneri with glucosylated ...
Vol. 112, No. 3 Printed in USA.
JOURNAL OF BACrERIOLOGY, Dec. 1972, p. 1420-1421 Copyright 0 1972 American Society for Microbiology
Formation of Polyisoprenol Phosphate Glucose in Shigella flexneri W. JANKOWSKI, T. CHOJNACKI, AND EWA JANCZURA Institute of Biochemistry and Biophysics, Polish Academy of Sciences and State Institute of Hygiene, Warszawa, Poland
Received for publication 27 June 1972
Lipid-linked glucose is formed from uridine diphosphate-glucose and ficaprenol phosphate only in strains of Shigella flexneri with glucosylated 0 antigen.
The synthesis of 0 antigen in Salmonella involves the lipid intermediates that have the structure of polyisoprenol (C55) pyrophosphate sugar (9). It was demonstrated that also polyisoprenol monophosphate glucose is involved in the biosynthesis of glucosylated antigens in Salmonella (5, 8). In these two papers a high rate of formation of this lipid was characteristic only for the strains with glucosylated 0-side chain. It was therefore plausible to assume that the formation of a specific lipid-linked sugar represents the target of the mechanism of genetic determination of the structure of 0 antigen. The possibility of studying the reactions of lipid intermediates in Shigella flexneri 2a by employing the derivatives of plant ficaprenol (7) has already been demonstrated (3). Since the structure of 0 antigen in several serotypes and variants of S. flexneri has been established by Simmons (6), we have checked whether in this species the occurrence of glucosylated antigen is correlated with the ability of a given strain to form lipid-linked glucose. S. flexneri serotypes 2a, 3a, 4a, 5b, and 6 and variants X and Y were obtained from the Shigella Laboratory of the Department of Bacteriology, State Institute of Hygiene, Warsaw. They were grown as described previously (3) and were collected from cultures in the exponential phase. The cells were washed with and suspended in 0.145 M NaCl-0.02 M tris(hydroxymethyl)aminomethane (Tris) buffer, pH 7.5, to give identical optical density at 650 nm for each strain and were sonically disrupted in an MSE sonic oscillator. The treated suspension was centrifuged at 7,500 x g for 30 min at 4 C, and the supernatant fraction was centrifuged at 105,000 x g for 1 hr at 4 C to obtain a pellet (particulate fraction). This pellet was washed by resuspending in 0.145 M NaCl-0.02 M Tris buffer, pH 7.5, and recen-
trifuged at 105,000 x g as above. The pellet was suspended in an'appropriate volume of 0.145 M NaCl-0.02 M Tris buffer, pH 7.5, to obtain the same concentration of protein (0.46 mg/ml) for preparations from all strains. The suspension served as the enzyme preparation. It could be stored frozen for several weeks with no appreciable loss of enzymic activity. Uridine diphosphate (UDP)- "C-glucose (40 mCi/mmole) was prepared from glucose-U- 14C (Chemapol, Czechoslovakia) by the method of Wright and Robbins (10) and purified by chromatography on PEI-cellulose column (3). Ficaprenol phosphate was the same as in the previous paper (4). The formation of labeled lipids from UDP"4C-glucose was studied similarly as described by Behrens and Leloir (1). The reaction mixture (final volume 0.2 ml) contained 0.15 mm UDP-
1420
"IC-glucose, 0.01 M Mg.-ethylenediaminetetraacetic acid (EDTA), 0.1 M mercaptoethanol, 0.2 M glycylglycine buffer, pH 7.5, 0.6% Triton X-100, and particulate enzyme (0.046 mg of protein). In tests with ficaprenol phosphate, the lipid (20 nmoles) dissolved in ethanol was added first to the tube, followed by 20 ,liters of 0.1 M Mg,-EDTA, and the solvents were evaporated. After adding the other components, the reaction mixtures were incubated at 37 C for 30 min. The lipids were extracted and washed as described in a previous paper (3). The radioactivity of lipid extracts was measured in a Packard Tri-Carb scintillation spectrometer with Bray's scintillation fluid (2). The labeling of lipids (Table 1) in the absence of polyisoprenol phosphate was low, irrespective of the type of 0 antigen characteristic for a given strain. The structure of 0 antigens of the serotypes and variants presented in Table 1 was taken from the paper of Simmons (6). Upon addition of ficaprenol phosphate, the increase of the radioactivity in lipids was found only for
VOL 112, 1972
NOTES
I ABLE 1. Effect of ficaprenol phosphate on the formation of lipid-linked glucose in various serotypes and variants of Shigella flexneri
Lipid-linked glucose formed
SeyrpoeSerotype
~ ~ ~ ~ ~(nmoles)
Structure of 0 antigen
or
With fica-
varn-
Con- peo tro prenol trol phos-
ant
phate
Glc -GIcNAc
12 AcGlc
Rha -4 Rha-
2a
-
3a
4 13 114 1 - -GIcNAc - Rha - Rha- -
-
0.06
1.66
0.10
1.16
0.08
1.20
0.07
0.55
Glc 4a
4 -
1
13
1.42
-GIcNAc --+ Rha --4 Rha- -
Glc
Glc
5b -4-GlcNAc 3
6
Y
-
6
-
141
-+
1
Rha - + Rh- -
13
12
1
12
14
1
-GIcNAc --4 Rha --* Rha- -GIcNAc -_4 Rha -__ Rha- -
0.06 0.08
0.05 0.05
Glc X
'4 1 3 '14 - -GIcNAc ZRa-
1
Rha- -
0.08
1.25
the serotypes 2a, 3a, 4a, 5b and for variant X. All of these strains contain 0 antigen to which are attached a-glucosyl or 0-acetylated aglucosyl secondary side chains. In preparations from serotype 6 and from variant Y that do not contain glucosyl side chains, ficaprenol phosphate did not stimulate the formation of lipidlinked glucose.
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The data of Table 1 suggest that in S. flexneri a polyisoprenol monophosphate glucose might act as the glucose donor in the formation of the specific 0 antigen, similarly as in some Salmonella species with antigens specified by. glucosyl units (5, 8). Whereas in the latter the observed difference in the rate of formation of lipid-linked glucose between strains with glucosylated and nonglucosylated antigen might imply either the lack of the lipid acceptor or the lack of enzyme synthesizing polyisoprenol monophosphate glucose, the present experiments on S. flexneri indicate that the enzyme is missing in the strains with nonglucosylated 0 antigen. This work was supported by the Polish Academy of Sciences within the project 09.3.1.1.1.9. LITERATURE CITED 1. Behrens, N. H., and L. F. Leloir. 1970. Dolichol monophosphate glucose: an intermediate in glucose transfer in liver. Proc. Nat. Acad. Sci. U.S.A. 66:153-159. 2. Bray, G. A. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1:279-285. 3. Jankowski, W., and T. Chojnacki. 1972. Enzymic formation of polyisoprpnol phosphate sugars. Acta Biochim. Polon. 19:51-69. 4. Jankowski, W., and T. Chojnacki. 1972. Formation of lipid-linked sugars in rat liver and brain microsomes. Biochim. Biophys. Acta 260:93-97. 5. Nikaido, H., K. Nikaido, T. Nakae, and H. Makela. 1971. Glucosylation of lipopolysaccharide in Salmonella: biosynthesis of 0-antigen factor 12,; I. Over-all reaction. J. Biol. Chem. 246:3902-3911. 6. Simmons, D. A. R. 1971. Immunochemistry of Shigella flexneri 0-antigens: a study of structural and genetic aspects of the biosynthesis of cell-surface antigens. Bacteriol. Rev. 35:117-148. 7. Stone, K. J., A. R. Wellburn, F. W. Hemming, and J. F. Pennock. 1967. The characterization of ficaprenol-10, -11 and -12 from the leaves of Ficus elastica (decorative rubber plant). Biochem. J. 102:325-330. 8. Wright, A. 1971. Mechanism of conversion of the Salmonella 0 antigen by bacteriophage ('4. J. Bacteriol. 105:927-936. 9. Wright, A., M. Dankert, P. Fennessey, and P. W. Robbins. 1967. Characterization of polyisoprenoid compound functional in 0-antigen biosynthesis. Proc. Nat. Acad. Sci. U.S.A. 57:1798-1803. 10. Wright, A., and P. W. Robbins. 1965. The enzymatic synthesis of uridine diphosphate/"4C/glucose. Biochim. Biophys. Acta 104:594-596.
