Vol. 133, No. 3

JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1300-1306 0021-9193/78/0133-1300$02.00/0 Copyright 0 1978 American Society for Microbiology

Printed in U.S.A.

Cell-Free Biosynthesis of the O-Acetylated NAcetylneuraminic Acid Capsular Polysaccharide of Group C Meningococci W. F. VANN, T.-Y. LIU, AND J. B. ROBBINS Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20014 Received for publication 3 October 1977

A cell-free system was established to study the biosynthesis of group C an a-2 -- 9-linked N-acetylneuraminic acid (NeuAc) homopolymer containing O-acetyl groups at either C7 or C8. Sialyltransferase activity, isolated from group C meningococcus strain C-li, catalyzed incorporation of [14C]NeuAc from CMP (CMP-["4C]NeuAc) into polymeric form. This sialyltransferase was stimulated by addition of meningococcus group C and Escherichia coli K92 capsular polysaccharides, the latter being an a-2 -.8- and a-2 -- 9-linked NeuAc heteropolymer. Group C meningococcal sialyltransferase did not require divalent ions but was stimulated by Mn2 . Attempts to demonstrate a lipid-soluble intermediate in the biosynthesis of this NeuAc polymer were unsuccessful. Meningococcal group C sialyltransferase incorporated NeuAc into a membrane-associated product. The polysaccharide can be extracted from the membrane-bound fraction with Triton X-100. The newly synthesized polysaccharide coprecipitates with authentic group C antigen in meningococcal group C antiserum and is degraded by sodium metaperiodate, indicating that the NeuAc polymer synthesized by the cell-free system consits of a-2 -. 9 linkage. Meningococcal group C spheroplast membranes contain an 0acetylase that can catalyze the transfer of acetyl groups from acetyl coenzyme A to the in vitro-synthesized polysaccharide.

meningococcal capsular polysaccharide,

N-Acetylneuraminic acid (NeuAc)-containing polymers have been reported as extracellular capsular polysaccharides of meningococcal groups B, C, X, and Y and Escherichia coli Kl and K92 antigens (3, 4, 9, 11-13). The groups B and C meningococcal polysaccharides are similar to the Ki (colominic acid) and K92 capsular polysaccharide antigens of E. coli, respectively (9, 13). All four polysaccharides are NeuAc polymers. The group C meningococcal polysaccha-

ride is an a-2 -- 9-linked homopolymer of NeuAc containing one 0-acetyl per residue located at either C7 or C8 (3, 12). A variant of group C meningococcus that produces a-2 -- 9-linked non-acetylated polysialic acid has been reported (1, 4). Both the group B meningococcal and E. coli Ki polysaccharides are a-2 -- 8-linked polysialic acid. The E. coli K92 polysaccharide contains both a-2 -. 8 and a-2 -- 9 linkages in its polysialic acid (6). These capsular polysaccharides are important in the virulence of pathogenic bacteria, and group C meningococcal polysaccharide has been successfully used as a va cine to prevent meningitis (2, 12). However, the mechanism of their biosynthesis is not clearly understood. Thus, an investigation of the metab-

olism of these surface polysaccharides is of interest. Colominic acid (E. coli Ki) has been reported to be synthesized from CMP-NeuAc by a siayltransferase involving a lipid-soluble intermediate (23). In this paper, we describe our studies designed to characterize the cell-fiee biosynthesis of 0-acetylated group C meningococcal polysaccharide by spheroplast membranes isolated from strain C-il. These group C meningococcal membranes had a sialyltransferase activity with different properties from that described for E. coli Kl. Further, we could not demonstrate organic solvent-extractable lipid intermediates reported to be obligatory forE. coli Ki biosynthesis (23, 26). MATERIALS AND METHODS Materials. Tryptic soy broth and yeast extract were purchased from Difco Laboratories. Morpholinopropane sulfonic acid (MOPS), CMP, and ethylenediaminetetraacetic acid were purchased from Calbiochem. NeuAc, acetyl coenzyme A (acetyl CoA), egg white lysozyme, ribonuclease, deoxyribonuclease, and N-ethyl maleimide were obtained from the Sigma Chemical Co. Sucrose and dithiothreitol were pur-

chased from Schwarz/Mann. The following radioactive compounds were ob1300

VOL. 133, 1978

tained from New England Nuclear Corp.: [4-14CJsialic CoA (specific activity, 0.96 mCi/mmol), [1-'4C]acetyl (specific activity, 54 mCi/mmol), and [3H]acetyl CoA (specific activity, 2.5 Ci/mmol). Sepharose 4B and Sephadex G-50 were purchased from Pharmacia Fine Chemicals, Inc., and Ultrogel 34 was obtained from LKB Instruments Inc. Bacterial polysaccharides from E. coli K92 strains (BOS 12 and N67) and Ki (strain K235) and group C (strain C-11) and group B (strain B-11) meningococci were prepared as described (10, 12; these bacterial strains may be obtained by writing to one of us). Group C meningococcal and Haemophi-

CELL-FREE GROUP C SYNTHESIS

1301

by multiple intravenous injections of Formalin-treated bacteria (10, 22). Preparation of spheroplast membranes. Encapsulated group C meningococci, strain C-11, were selected for capsular production by the antiserum agar technique (21) for growth in liquid medium. Bacteria, monitored for their concentration at 540 unm, were grown to mid-logarithmic phase on 3% tryptic soy broth (Difco) supplemented with 0.1% yeast extract dialysate. Dialyzed yeast extract was sterile-filtered outer fluid of a 48-h, 0.5-liter distilled-water dialysis at 4°C of 500 g of dry yeast extract (Difco). Spheroplast membranes were prepared by the method of either Osborn et al. (20) or Johnston and Gotschlich (13). After lysis and removal of unbroken cells at 12,000 x g, the membranes were collected by centrifugation at 100,000 x g for 1 h, stored in 0.01 M tris(hydroxymethyl)aminomethane (pH 7.5), and used without further fractionation. Sialyltransferase. Sialyltransferase was assayed for polymeric sialic acid synthesis in the reaction mixtures by using Whatman DE-81 ion-exchange paper in solvent system I (5). Polysialic acid remains at the origin, and CMP-NeuAc and NeuAc migrate near the solvent front (15, 23, 26). The reaction mixture consisted of 0.167 M MOPS (pH 7.6), 10 mM MnCl2, 0.67 mM CMP-NeuAc ([14C]NeuAc; specific activity, 1 mCi/mmol), and 0.1 mg of spheroplast membrane protein in a final volume of 60 1. The pH optima were studied between 6 through 9 in 0.167 M MOPS or 0.167 M tris(hydroxymethyl)aminomethane. The reactants were incubated at 35°C for 1 h, and the reaction was stopped by either (i) directly spotting 50 p1 on a paper chromatogram (2 by 28 cm) or (ii) prior mixing of the reactants with an equal volume of absolute ethanol and then spotting a 100-p1 final volume on the chromatogram. The paper chromatograms were developed for 90 min at room temperature and cut into 1.5-cm strips, and all segments were placed in Econofluor (New England Nuclear) for counting.

ammonium acetate, pH 8.0 (solvent I), and on Whatman no. 3 MM paper in butanol-pyridine-0.05 M Nethylmorpholium tetraborate (7:5:2; solvent II [5]). Transacetylase. Transacetylase activity was determined by miXing 0.25 M MOPS, 25 mM MgC12, 0.5 mM [1-'4C]acetyl CoA (specific activity, 0.5 mCi/mmol), and 0.3 mg of spheroplast membrane protein in a final volume of 0.2 ml. The mixture was incubated at 35°C for 30 min and applied to a Sepharose 4B column (1.1 by 30 cm) in 0.2 M ammonium acetate. The effluent was collected in 1.0-ml fractions, and 0.5 ml of each fraction was counted in Aquasol. Lipid extraction of the spheroplaat membrane. The methods of Folch et al. (9) using chloroformmethanol (2:1) and Fitzgerald-Chandler and Jann (8) using water-saturated butanol were employed. Triton X-100 extraction of spheroplast membrane. Spheroplast membrane preparations (0.1 to 1.0 mg in 0.5 ml) were mixed with 0.5 ml of MOPS, 50 p1 of 0.3 M MnCl2, and 0.5 ml of 2 mM CMP[14C]NeuAc. After incubation at 35°C for 1 h, the spheroplast membrane preparation was pelleted at 100,000 x g. The pellet of spheroplast membrane was homogenized for 20 min in 2% Triton X-100-0.25 M MOPS, pH 7.5, at 0°C. The suspension was centrifuged at 100,000 x g for 1 h, and the supernatant was applied to an Ultrogel 34 column (1.1 by 45 cm) and eluted with 0.2 M ammonium acetate (Fig. 1). The void volume fractions contained 4-14C-labeled NeuAc and were lyophFlized and redissolved in 1 ml of 10 mM tris(hydroxymethyl)aminomethane (pH 7.5) containing 50 ug of trypsin. The mixture was incubated at 350C for 6 h and deproteinized with 1 ml of 90% phenol in 10% saturated sodium acetate (pH 7.4) at 0°C. After washing the phenol phase with 1 ml of water, the aqueous phases were dialyzed exhaustively against water. Antibody binding. The trypsinized Triton X-100soluble fraction (2,000 cpm in 100 p1) was mixed with 200 p1 of either group C meningococcal or H. influenzae type b antisera. The polysaccharides were dissolved in 200 i1 of 0.02 M sodium phosphate in 0.15 M NaCl. The trypsinized soluble fraction, antisera, and polysaccharides were incubated for 1 h at 350C and 1 h at 40C and centrifuged at 5,000 x g for 20 min, and the precipitate was washed twice, suspended in 1 ml of 1.0% sodium dodecyl sulfate, and transferred to a scintillation vial containing 10 ml of Aquasol (New England Nuclear). The supernatant and washings were transferred to another scintilliation vial containing 10 ml of Aquasol. All samples were kept at 35°C overnight and transferred to the cold room before counting.

for amino acid analysis as previously described (16) and applied to a Beckman model 121M amino acid analyzer. Protein and NeuAc were determined by published methods (18, 27). Paper strips (1.5 by 2 cm) were placed in 10 ml of Econfluor, and column effluents were added to 10 ml of Aquasol (New England Nuclear) before counting in a Packard Tri-Carb 3375 liquid scintillation spectrometer. The counting efficiency of "4C on paper strips was 45% and 90 to 95% for the column effluents. Paper chromatography. Paper chromatograms were run on Whatman DE-81 equilibrated in 0.4 M

RESULTS Characteristics of group C meningococcal polysaccharide synthesized by cell-free transialidase. Application of the reaction mixture to Sepharose 4B resulted in spheroplast membranes and '4C-polysialic acid emerging at the void volume. The [14C]polysialic acid migrated with spheroplast membranes in sucrose density gradients. The [4-'4C]NeuAc-labeled polysialic acid was pelieted with the spheroplast membrane and sialyltransferase activities at

lus influenzae type b antisera were prepared in burros

Analytical procedures. Samples were hydrolyzed

1302

VANN, LIU, AND ROBBINS

J. BACTERIOL.

100,000 x g. The spheroplast membrane containing the "4C-labeled polysialic acid was extracted with Triton X-100 and passed through Sepharose 4B equilibrated with 0.1% Triton X100 in 0.2 M ammonium acetate. Both the void volume peak and a heterogeneous component contained ['4C]NeuAc. Chromatography of the Triton X-100-soluble fraction on Ultrogel 34 (Fig. 1) shows that the "4C-labeled polysialic acid was detected in the void volume. Thus, the extractable "4C-labeled product is a high-molecular-weight material. A similar elution profile was obtained when the Triton X-100-insoluble label was extracted with 1% sodium dodecyl sulfate at 100°C, precipitated with ethanol, treated with trypsin, and chromatographed on Ultrogel 34. Further, "C-labeled polymer, extracted from incubation mixtures with 45% hot phenol, emerges in the Sepharose 4B void volume. By these criteria, the newly synthesized polysaccharide appeared to be membrane associated. The Triton X-100-soluble product was reacted with trypsin and deproteinized with an equal volume of 90% saturated phenol at 100C and

then dialyzed against 0.2 M ammonium acetate in the cold room. This deproteinized fraction was studied by immune precipitation with group C meningococcal antiserum. All of the labeled polysaccharide coprecipitates with authentic group C meningococcal polysaccharide and group C meningococcal antiserum. In contrast, 50% of the ["4C]NeuAc remains in the supernatant of the control system (H. influenzae type b polysaccharide and antiserum; Table 1). This result suggests that the membrane-associated product contains antigenically active and, hence, polymeric NeuAc. Chemical analysis of the group C meningococcal polysaccharide has revealed that the NeuAc residues are a-(2-9) linked (17). The Triton X100-soluble fraction was subjected to mild acid hydrolysis (0.1 N H2SO4 at 800C for 1 h). The product was applied to paper chromatography in solvent system II and yielded only one radioactive component corresponding to authentic NeuAc. The 2 -- 9 linkage of the de-O-acetylated group C meningococcal polysaccharide is suggested by the degradation to NeuAc7 (5-ac-

etamido-3,5-dideoxy-L-arabiow-2-heptulosonic acid) after periodiate treatment (Fig. 2; 6, 25). In addition to polysialic acid, the membraneassociated product contains a peptide(s). Amino acid analysis reveals a limited number of amino acids. Asx, Ala, Glx, Thr, Ser, and Gly were

observed in the ratio of 3:3:2:1:1:1. The exact relationship between this peptide and the sialic acid polymer is currently under investigation. Properties of sialyltransferase. Figure 3 shows the dependence of this reaction on time and spheroplast membrane protein concentration. The amount of polymeric NeuAc formed is proportional to spheroplast membrane protein but is nonlinear versus time. The enzyme has a pH optimum between pH 7.0 and 8.0. Activity was 15% greater in MOPS than in tris(hydroxymethyl)aminomethane buffer at pH 7.5. Polysialic acid was formed between 13 and 420C. Optimum incorporation was observed between 35 and 42°C. Membranes were stored at -200C for 4 months without significant loss of sialyltransferase activity. As shown in Fig. 4, sialyltransferase is stimulated two- to threefold at MnCl2 concentrations between 10 and 20 mM. Slight inhibition was 20 30 50 10 observed at 50 mM MnCl2. It was activated to a EFFLUENT lesser extent by equivalent concentrations of FIG. 1. Gel filtration of the Triton X-100-soluble Ca2+, Co2+, and Mg2e. However, 10 mM ZnCl2 fraction on a column (1.1 by 45 cm) of Ultrogel 34. was inhibitory (Table 2). Manganese does not The Triton X-I00-soluble fraction was prepared from to be an appear absolute requirement because the particulate fraction of a sialyltransferase incubation mixture as described in the text and elated spheroplast membranes are prepared in 5 mM from the column in 1-mi fractions, and a 0.5-mi frac- ethylenediaminetetraacetic acid, and dialysis tion was counted and expressed as counts per minute against 25 mM ethylenedisminetetraacetic acid per mililiter. did not result in loss of enzyme activity. The 0

40

ml

60

CELL-FREE GROUP C SYNTHESIS

VOL. 133, 1978

TABLE 1. Immunoprecipitation ofpolysaccharides derived from biosythesis Anti-group C menin- Anti-Haemophillu Carrier pol- gococcal serum + pol- type b serum + polysaccharide ysaccharide ysaccharide added .e. Precipita- Soluble tation Soluble (pg) tion (cpm) (cpm) (cpm) t(aim)

10

2,170 2,160

17 10

494 492

807 1,237

100

1,834 2,093

81 13

650 628

1,126 1,187

0

30 20 TUBE NUMBER

10

40

90

FIG. 2. Gel filtration on a column (1.1 by 30 cm) of Sephadex G-50 of tysinized Triton X-100-soluble fraction, before (0) and after (0) periodate oxidation.

C meningococcal sialyltransferase differs from E. coli K235 enzyme with respect to its response to thiol reagents (23). Addition of dithiothreitol did not increase "4C-labeled NeuAc incorporation, and preincubation with 1 mM Nethylmaleimide for 10 min before addition of the substrate did not affect sialyltransferase activity. Sialyltransferase in the spheroplast membrane is stimulated by the addition of Triton X-100 with a mimal formation of polysialic acid at 0.1% detergent concentration. The addition of group C meningococcal polysaccharide or E. coli K92 polysaccharides [NeuAc polymers containing a-(2-9) liikages] increased polysialic acid formation (Table 2). This "primer" stimulation is specific, as E. coli Kl and meningococcal group B polysaccharides group

consisting of a-2

-*

8-sialic acid

no

stimulatory effect. Incasing the amount of K92 polysaccharide increased polysialic acid formation (Table 2). Monomeric NeuAc had no demonstrable effect.

1303

Lipid extraction. Because a lipid-extractable intermediate (undecaprenol phosphate) has been implicated in the synthesis of E. coli Kl polysaccharide (a-2 -. 8-linked polysialic acid), we were interested in whether this component is related to group C meningococcal polysaccharide formation (23). There was no detectable ['4C]NeuAc in the organic phases using two liquid extraction methods for 1.8 ml of reaction mixture (30-fold scale and up; 8, 9). Varying the conditions of the reaction mixture, such as the temperature, length of incubation, or increasing its volume did not yield lipid-extractable radioactivity. The controls, portions of the reaction mixture before lipid-extraction, demonstrated polysialic acid formation, indicating an active sialyltransferase. Bacitracin, which inhibits recycling of undecaprenol pyrophosphate, had no effect on sialyltransferase when included in the incubation mixture (Table 2). The addition of CMP to the incubation mixtures resulted in 70% inhibition. In contrast, similar concentrations of NeuAc were not inhibitory (Table 2). O-Acetylation of group C meningococcal polysaceharide strain C-li. The group C meningococcal polysaccharide of strain C-li is O-acetylated at positions 7 or 8 (3). Spheroplast membranes were assayed for their ability to incorporate [14C]acetyl groups into polysialic acid (24). The incorporation of acetyl groups into the membrane-associated polysaccharide was studied as described above. Incubation of spheroplast membranes with ['4C]acetyl CoA yielded a product that emerged with the void volume on Sepharose 4B (Fig. 5). This suggests that spheroplast membrane-bound polysialic acid can be acetylated in this cell-free system. The addition of purified group C polysaccharide from strain C-1l to the reaction mixture resulted in incorporation of ["4C]acetyl into soluble polysaccharide (Fig. 5). Fractions 17 through 37 contained both ["4C]acetyl and NeuAc. These fractions were treated with 2 N hydroxylamine in 2 N NaOH for 15 min and chromatographed on Sephadex G-50. All of the polysaccharide was excluded at the void volume, while the ['4C]acetyl was recovered as a single low-molecularweight peak. The alkali lability of this linkage is characteristic of O-acetyl groups (3, 6). Polysaccharide purified by Triton X-100 extractions of siayltransferase incubation mixtures (see above) were treated with [3H]acetyl CoA and spheroplast membranes. The ['4C]NeuAc of the labeled polysialic acid chromatographed with the [3H]acetyl (Fig. 6). The possibility that the membrane preparation might have covalently or noncovalently bound the [3H]acetyl CoA was eliminated by a blank experiment in which polysaccharide was not in-

1304

VANN, LIU, AND ROBBINS

J. BACTFJOI.

A.

En 0 z

2000

&

I0

cz 0 E

EQ54

B.

z 0

5 4000_

u, 1000 6

4( 0

,3

100 _

(A

0

20

40

60

80

1

1

20

40

100

1

I

IN I

60 80 100 120 140 mg PROTEIN

120

140

160

TIME (min.)

FIG. 3. Time course (A) and effect of membraneprotein concentration (B) on the sialyitransfera8e-catalyzed incorporation of "4C]NeuAc into chromatographically immobile product. The reaction conditions are given

in the text. woo-

cluded in the incubation mixture. Under such a

ooo__

condition, the incorporation of [3H]acetyl CoA wa insignificant. These data suggest that the spheroplast membranes contain an O-acetylase that will transfer acetyl group to in vitro-synthesized polysaccharide.

/

DISCUSSION /De novo synthesis of group C meningococcal S2 9 1 polysaccharide has been demonstrated by using 4W .ooo vspheroplast membranes incubated with CMP['4C]NeuAc. This biosynthetic property is comparable to sialyltransferase activities in other I? . systems (15, 23). The newly synthesized polymer was membrane associated and exhibited a molecular weight in excess of 200,000 as evidenced by its behavior on gel filtration on Ultrogel 34. It coprecipitated with an authentic sample of group C meningococcal polysaccharide with specific antibody against group C meningococci. With CMP-[14C]NeuAc as the donor, the siatransferred NeuAc to the C-polyo lyltransferase 10 2D SD saccharide that contains a-2 9 linkages and Mn++ (MM) the E. coli K92 polysaccharide that contains FIG. 4. Effect of Mn4+ on the incorporation Of alternating sequence of a-2 -* 9 and a-2 -. 8 114C]NeuAc into chromatographically immobile linkages. It did not utilize as an acceptor group B meningococcus NeuAc polymer consisting of product.

CELL-FREE GROUP C SYNTHESIS

VOL. 133, 1978

TABLE 2. Agents affecting group C meningococcal sialyltransferase activitya Concn Agent

% Activity 100 10 mM 29 10 mM 100 97 300 ,lg/ml 92 1mM 1 mM 112 10 mM 142 10 mM 143 10mM 148 MgCl2 10 mM 56 ZnCl2 292 100 Fg E. coli N-67 PSAb 140 100 izg E. coli Bos-12 PSA 100 100 tg E. coli K1 PSA a Control was the normal assay mixture as described in the text. The agents were added to the assay mixture, and the final volume was adjusted to 60 id. NEthylmaleimide (NEM) was added to the reaction mixture at 25°C for 10 min before the addition of the

None CMP NeuAc Bacitracin Nem Dithiothreitol CaCl2 CoCl2

substrate CMP-[14C]NeuAc. b SA, Polymeric sialic acid.

1305

a polyisoprenol-type lipid in the group C meningococcal polysaccharide synthesis because no, incorporation of ["4C]NeuAc could be demonstrated in the "lipid-extract" of sialyltransferase incubation mixtures with organic solvents. The lack of inhibition by bacitracin suggests sialyl-2pyrophosphoryl polyisoprenol is not an intermediate in the biosynthesis of group C meningococcal polysaccharide. Other bacterial polysaccharides may be synthesized without detectable undecaprenol intermediates. Thus, the biosynthesis of polyribitol phosphate by ribitol phosphate polymerase has an absolute requirement for lipoteichoic acid carrier but does not utilize an undecaprenol phosphate intermediate (7). Similarly, a lipid-extractable intermediate has not been demonstrated in the biosynthesis of rouxii chitin (19) and E. coli 09 lipopolysaccharide (14). It is possible that polyisoprenol phosphate is involved only in the first step, whereby a NeuAc

M00r

3000

I

2000

2000

CA, 0

E

I z

1000

UL

m

3

1000 TUBE NUMBER

FIG. 5. Incorporation of the [4C]acetyl group into group C meningococcal polysaccharide catalyzed by spheroplast membrane preparation. Polysaccharide was incubated with spheroplast, I'4Clacetyl CoA and applied to a column (1.1 by 30 cm) of Sepharose 4B. The effluent was measured for radioactivity (0) and assayed for sialic acid (0). The column was eluted with 0.1 M NH4HCO3, and fractions of 0.7 ml were collected.

a-2 -* 8 linkages. Interestingly, the former two polysialic acids cross-react immunologically but differ from the latter (10). Our results argue against the participation of

0

10

20

30

40

50

TUBE NUMBER

FIG. 6. Incorporation of [3H]acetyl (0) into

[14C]polysialic acid (0) catalyzed by spheroplast membrane. 4CJpolysialic acid was incubated with 3HJacetyl CoA and applied to a column (1.1 by 30 [

cm) ofSepharose 4B. The column was eluted with 0.1 M NH4HCO3, and fractions of 0. 7 ml were collected.

transferred from CMP-NeuAc to a molecule of isoprenol phosphate and that on this carrier the polysialic acid grows by direct tansfer of NeuAc from CMP-NeuAc. Such a mechanism has been proposed as one of the possible biosynthetic mechanisms for mannan of E. coli 09:K29 (14). It has been shown that nascent meningococcal C polysaccharide is membrane associated and contains a peptide(s) composed of Asx (3), Ala (3), Glx (2), Thr (1), Ser (1), and Gly (1). Whether such a peptide serves as a carrier for the synthesis of the group C polysaccharide is not known. The group C meningococcal polysaccharide derived from strain C-1l is O-acetylated at position 7 or 8. The spheroplast membrane used for the synthesis of polysialic acid contains an enzyme(s) capable of transferring the acetyl group from acetyl CoA to an exogenous polysaccharide. The ability of a polysialic acid to act as an acetyl acceptor led to the suggestion that the natural acceptor is probably the assembled polysialic acid rather than the CMP-NeuAc unit before it is transferred to the growing chains. The lipopolysaccharides of Mycobacterium phdei (24) and SabnoneUa anatum (21) appear to be O-acetylated after polymerization.

unit

J. BACTERIOL.

VANN, LIU, AND ROBBINS

1306 is

LrIERATURE CrIE 1. Apicella, IL A. 1972. Identification of a subgroup antigen on the Newieria meniWtidis Group C capsular polysaccharide. J. Infect Dis. 129:147-153. 2. Artenstein, IL S., R. Gold, J. G. Zimmerly, F. A. Whyle, H. Schneider, and C. HanIns. 1970. Prevention of meningococcal disease. N. Engl. J. Med. 282:417-420. 3. Bhattacharjee, A. K., H. J. Jenning, C. P. Kenny, A. Martin, and L C. P. Smith. 1975. Structural determination of the sialic acid polysaccharide of Neisseria meningitidis serogroup B and C with carbon 13 nuclear magnetic resonance. J. Bio. Chem. 260:1926-1932. 4. Bhattachrjee, A. K., H. J. Jennings, C. P. Kenny, A. Martin, and I. C. P. Smith. 1976. Structural determination ofthe polysaccharide antigens of Neisseria menigitidis serogroups Y, W-135, and BO. Can. J. Biochem 54I:1I-. 5. Carminatti, H., S. Passeron, IL Dankert, and E. Recondo. 1966. Separation of sugar nucleotides, phosphonc esters, and free sugars by paper chromatography with solvents containing borates of organic bases. J. Chromatogr. 18:342-348. 6. Egan, W., T.-Y. [iu, J. S. Cohen, D. Dorrow, E. C. Gotachllch, J. B. Robbins, and J. Robbins. 1977. Strutural studies on the sialic acid polysaccharide antigen of Escherichia coli strain Bos-12. Biochemistry 16:3687-3692. 7. Fiedler, F., and L Glaser. 1974. Purification of polyribitol phosphate polymerase and lipoteichoic acid carrier. J. Biol. Chem. 249:2684-2689. 8. Fitzgerald-Chandler, D. K., and K. Jann. 1971. Studies of the biosynthesis of the 09 antigen from Escherichia coli 09XK30(A):H12. Eur. J. Biochem. 24:222-231. 9. Folch, J., ML Lees, and G. H. Sloanes-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-9.

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