Biochemical conditions for the production of polysialic acid by Pasteurella haemolytica A2

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with (2–8) linkages. When the bacterium was grown at 37°C for 90 h in 250 ml shake flasks at 200 rpm in Brain heart infusion broth (BHIB), it accumulated, attaining a level of 60 g/ml. Release of this polymer was strictly regulated by the growth temperature, and above 40° no production was detected. The pathway for the biosynthesis of this sialic acid capsular polymer was also examined in P. haemolytica A2 and was seen to involve the sequential presence of three enzymatic activities: Neu5Ac lyase activity, which synthesizes Neu5Ac by condensation of N-acetyl-D-mannosamine and pyruvate with apparent Km values of 91 mM and 73 mM, respectively; a CMP-Neu5Ac synthetase, which catalyzes the production of CMP-Neu5Ac from Neu5Ac and CTP with apparent Km values of 2 mM and 0.5 mM, respectively, and finally a membrane-associated polysialyltransferase, which catalyzes the incorporation of sialic acid from CMP-Neu5Ac into polymeric products with an apparent CMP-Neu5Ac Km of 250 M.     

High production of polysialic acid [Neu5Ac alpha(2-8)-Neu5Ac alpha(2-9)]n by Escherichia coli K92 grown in a chemically defined medium. Regulation of temperature

The capsular polysaccharide of Escherichia coli K92 consists of a linear polymer of Neu5Ac with alternating alpha(2-8) and alpha(2-9) linkages. It accumulates when the bacterium is grown at 37 degrees C in a defined medium containing D-xylose and L-asparagine as carbon and nitrogen sources. Release of the capsular polymer into the medium was maximal (450 micrograms x ml-1) in the stationary phase of growth (76 h). This medium could be useful for obtaining sufficient polymer to develop effective vaccines. The enzyme, CMP-Neu5Ac synthetase, was not detected in cells grown at 20 degrees C. The lack of this enzyme explains the absence of polymer biosynthesis when the bacterium was grown at 20 degrees C.     

Characterization of cpsF and its product CMP-N-acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli

Group B Streptococcus (GBS) is the foremost cause of neonatal sepsis and meningitis in the United States. A major virulence factor for GBS is its capsular polysaccharide, a high molecular weight polymer of branched oligosaccharide subunits. N -acetylneuraminic acid (Neu5Ac or sialic acid), at the end of the polysaccharide side chains, is critical to the virulence function of the capsular polysaccharide. Neu5Ac must be activated by CMP-Neu5Ac synthetase before it is incorporated into the polymer. We showed previously that a transposon mutant of a serotype III GBS strain which had no detectable capsular Neu5Ac was deficient in CMP-Neu5Ac-synthetase activity (Wessels et al ., 1992). In this paper, we report the identification and characterization of cpsF , a gene interrupted by transposon insertion in the previously described Neu5Ac-deficient mutant. The predicted amino acid sequence of the cpsF gene product shares 57% similarity and 37% identity with CMP-Neu5Ac synthetase encoded by the Escherichia coli K1 gene, neuA . The enzymatic function of the protein encoded by cpsF was established by cloning the gene in E. coli under the control of the T7 polymerase/promoter. Lysates of E. coli in which the cpsF gene product was expressed, catalysed the condensation of CTP with Neu5Ac to form CMP-Neu5Ac. In addition, when a CMP-Neu5Ac synthetase-deficient mutant of E. coli K1 was transformed with cpsF , K1 antigen expression was restored. We conclude that cpsF encodes CMP-Neu5Ac synthetase in type III GBS, and that the GBS enzyme can function in the capsule-synthesis of a heterologous bacterial species.     

In vitro synthesis of colominic acid by membrane-bound sialyltransferase of Escherichia coli K-235. Kinetic properties of this enzyme and inhibition by CMP and other cytidine nucleotides

The membrane-bound sialyltransferase obtained from Escherichia coli K-235 grown in a chemically defined medium (ideal for colominic acid production) was studied. The in vivo half-life calculated for this enzyme was 20 h. Kinetic tests revealed (at 33 degrees C and pH 8.3) hyperbolic behaviour with respect to CMP-Neu5Ac (Km250 microM) and a transition temperature at 31.3 degrees C. The enzyme was inhibited by NH4+, some divalent cations and by several agents that react with thiol groups. Detergents and fatty acids also inhibited the sialyltransferase activity. In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of [14C]Neu5Ac into a protein-complex intermediate and therefore into free polymer. CDP and CTP also inhibited (91% and 84%) this enzyme activity whereas cytosine and cytidine had no effect. CMP inhibition corresponded to a competitive model the calculated Ki was 30 microM. Incubations of protein[14C]Neu5Ac with CMP, CDP and CTP led to de novo synthesis of CMP-[14C]Neu5Ac. The presence of colominic acid, which usually displaces the reaction equilibrium towards polymer synthesis, did not affect this de novo CMP-[14C]Neu5Ac formation. CMP also inhibited in vivo colominic acid biosynthesis.     

Characterization of CMP-N-acetylneuraminic acid synthetase of group B streptococci.

The capsular polysaccharide is a critical virulence factor for group B streptococci associated with human infections, yet little is known about capsule biosynthesis. We detected CMP-Neu5Ac synthetase, the enzyme which activates N-acetylneuraminic acid (Neu5Ac, or sialic acid) for transfer to the nascent capsular polysaccharide, in multiple group B streptococcus serotypes, all of which elaborate capsules containing Neu5Ac. CMP-Neu5Ac synthetase isolated from a high-producing type Ib strain was purified 87-fold. The enzyme had apparent Km values of 7.6 for Neu5Ac and 1.4 for CTP and a pH optimum of 8.3 to 9.4, required magnesium, and was stimulated by dithiothreitol. This is the first characterization of an enzyme involved in group B streptococcus capsular polysaccharide biosynthesis.     

Regulation of colominic acid biosynthesis by temperature: role of cytidine 5''-monophosphate N-acetylneuraminic acid synthetase

Synthesis of colominic acid in Escherichia coli K-235 is strictly regulated by temperature. Evidence for the role of cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase in this regulation was obtained by measuring its level in E. coli grown at 20 and 37°C. No activity was found in E. coli grown at 20°C. CMP-Neu5Ac started to be quickly synthesized when bacteria grown at 20°C were transferred to 37°C and was halted when cells grown at 37°C were transferred to 20°C. These findings suggest that temperature regulates the synthesis of this enzyme and therefore the concentration of CMP-Neu5Ac necessary for the biosynthesis of colominic acid.     

Induction of capsular polysaccharide synthesis by rho-fluorophenylalanine in Escherichia coli wild type and strains with altered phenylalanyl soluble ribonucleic acid synthetase

Escherichia coli K-12 strain AB259 can be induced to form capsular polysaccharide (mucoid clones) by dl-p-fluorophenylalanine (FPA; 5 x 10(-6)m on agar plates at 37 C or 8 x 10(-5)m in liquid medium at 30 C). The change was shown to be phenotypic. An increase in enzymes probably involved in capsular polysaccharide synthesis [phosphomannose isomerase (3.3-fold), uridine diphosphate-d-galactose-4-epimerase (2.5-fold), and guanine diphosphate-l-fucose synthetase] was demonstrated as a result of growth in FPA. These increases appear sufficient to account for the increased synthesis of capsular polysaccharide due to growth in FPA. FPA-resistant derivatives of strain AB259 were obtained by selecting mutants on FPA-containing agar or by transducing in an altered phenylalanyl soluble ribonucleic acid synthetase that activates FPA poorly. Mucoid clones were formed by these strains only in the presence of 30 to 1,000 times as much FPA. Among these strains, there was a close correlation between incorporation of FPA-C(14) and induction of capsular polysaccharide synthesis. The results are thus consistent with the following model: FPA is incorporated into the protein product of the R(1) gene (repressor) and alters it sufficiently to allow derepression of several enzymes.     

Evidence of free radical participation in N-glycolylneuraminic acid generation in liver of chicken treated with gallotannic acid

The occurrence of N-glycolylneuraminic acid (Neu5Gc) in cancerous tissue and inflammatory diseases, conditions associated with increased oxidative stress suggests the participation of reactive oxygen radicals in Neu5Gc generation, where an oxygen atom is transferred. To study this possibility, we treated two groups of domesticated birds and rabbits with different dosages of gallotannic acid (GTA), a compound known to cause generation of reactive oxygen species (ROS). The antioxidant status and leukocyte capacity, as well as amount and form of sialic acids were assessed in plasma and liver. Results showed that while lipid peroxides were increased, white blood cell (WBC) count was decreased significantly in all treated groups. The increased sialic acids and low protein contents were observed in plasma, possibly as a result of decreased sialic acid cycling crucial for formation of new glycoconjugates in tissues, caused by decreased protein synthesis due to microsomal degranulation. The activities of antioxidant enzymes were also decreased in treated groups, implying increased oxidative stress. The presence of Neu5Gc and apparent absence of Neu5Ac hydroxylase activity in liver of chicken treated with GTA indicate that free radicals might be involved in the non-enzymatic hydroxylation of N-acetylneuraminic acid (Neu5Ac) to Neu5Gc in liver, which normally does not express this sialic acid.     

Construction of a functional CMP-sialic acid biosynthesis pathway in Arabidopsis

Previous studies have reported that plants contain negligible amounts of free or protein-bound N-acetylneuraminic acid (Neu5Ac). This is a major disadvantage for the use of plants as a biopharmaceutical expression system, since N-glycans with terminal Neu5Ac residues are important for the biological activities and half-lives of recombinant therapeutic glycoproteins in humans. For the synthesis of Neu5Ac-containing N-glycans, plants have to acquire the ability to synthesize Neu5Ac and its nucleotide-activated derivative, cytidine monophospho-N-acetylneuraminic acid. In this study, we have generated transgenic Arabidopsis (Arabidopsis thaliana) plants expressing three key enzymes of the mammalian Neu5Ac biosynthesis pathway: UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminic acid phosphate synthase, and CMP-N-acetylneuraminic acid synthetase. Simultaneous expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase and N-acetylneuraminic acid phosphate synthase resulted in the generation of significant Neu5Ac amounts (1,275 nmol g(-1) fresh weight in leaves) in planta, which could be further converted to cytidine monophospho-N-acetylneuraminic acid (2.4 nmol g(-1) fresh weight in leaves) by coexpression of CMP-N-acetylneuraminic acid synthetase. These findings are a major step toward the production of Neu5Ac-containing glycoproteins in plants.     

Engineering of a sialic acid synthesis pathway in transgenic plants by expression of bacterial Neu5Ac-synthesizing enzymes

Plants are a low-cost and contamination-free factory for the production of recombinant pharmaceutical proteins. However, plant-made pharmaceuticals differ from their mammalian homologues by the structure of their N -linked glycans. For instance, most mammalian glycoproteins harbour terminal sialic acids that control their half-life in the bloodstream. The absence of the whole sialylation machinery in plants is of major concern as non-sialylated plant-made pharmaceuticals may not perform at their full potential in humans, because of their removal from the circulation through the involvement of hepatic cell receptors. In this context, we have investigated the synthesis of N -acetylneuraminic acid (Neu5Ac) in the cytosol of plants by either the re-routing of the endogenous 3-deoxy- d - manno -2-octulosonic acid (Kdo) biosynthetic pathway or the expression of microbial Neu5Ac-synthesizing enzymes. In this paper, we demonstrate that the plant Kdo-8P synthase is not able to use N -acetyl d -mannosamine as a substrate, and thus re-routing of the Kdo pathway for the synthesis of Neu5Ac is not possible. Consequently, we expressed genes encoding Neu5Ac lyase from Escherichia coli and Neu5Ac synthase ( neuB2 ) from Campylobacter jejuni in plants. These resulted in the production of functional enzymes in the cytosol, which in turn can catalyse the synthesis of Neu5Ac in vitro . Experiments were carried out on two models, Bright Yellow 2 (BY2) tobacco cells and Medicago sativa (alfalfa), the perennial legume crop.     

NeuA O-acetylesterase activity is specific for CMP-activated O-acetyl sialic acid in Streptococcus suis serotype 2

Several bacteria causing meningitis, such as Escherichia coli K1, Streptococcus suis, Neisseria meningitidis, and group B Streptococci (GBS), produce sialic acid (Neu5Ac)-containing capsular polysaccharide (CPS). Biosynthesis of the Neu5Ac-containing CPS requires CMP-Neu5Ac as substrate, which is synthesized by CMP-Neu5Ac synthetase from CTP and Neu5Ac. In E. coli or GBS, the NeuA protein encoded by the neuA gene has been known encoding a bifunctional enzyme that possesses both CMP-Neu5Ac synthetase and O-acetylesterase activity. In this report, we found that the S. suis NeuA (SsNeuA) was also a bifunctional CMP-Neu5Ac synthetase/O-acetylesterase. Biochemical analyses revealed that the SsNeuA strictly de-O-acetylated CMP-O-acetyl-Neu5Ac, whereas the E. coli NeuA (EcNeuA) preferentially de-O-acetylated CMP-O-acetyl-Neu5Ac. E. coli devoid of NeuA O-acetylesterase activity was unable to produce capsule and only CMP-Neu5Ac synthetase activity of the EcNeuA or SsNeuA could not restore its ability to produce capsule. These results suggest that the O-acetylesterase is essential for the synthesis of capsular Neu5Ac in E. coli, probably in S. suis and GBS as well. Our findings are key to understanding the biosynthesis of capsular Neu5Ac in E. coli, S. suis and GBS.     

Purification, characterization and immunological properties of the capsular polysaccharide of Pasteurella haemolytica serotype T15: its identity with the K62 (K2ab) capsular polysaccharide of Escherichia coli and the capsular polysaccharide of Neisseria meningitidis serogroup H

Capsular polysaccharide from two strains of Pasteurella haemolytica serotype T15 was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, proved to be very similar in structure to the capsular polysaccharide of P. haemolytica serotype T4 and identical to the previously described K62 (K2ab) capsular polysaccharide of Escherichia coli, and the capsular polysaccharide of Neisseria meningitidis serotype H, i.e. ----(2-glycerol-3)----(phosphate)----(4-alpha-D-galactopyranose -1)---- with partial O-acetylation on the galactose residues. Electron microscopy with Protein A-gold labelled antisera showed that the polysaccharide was peripherally located on the surface of all three organisms. Chemical removal of O-acetyl groups from the polysaccharide yielded a structure identical to that previously described for E. coli K2 (K2a). Both O-acetylated and de-O-acetylated P. haemolytica T15 polymers, when absorbed on to sheep erythrocytes in passive haemagglutination assays, yielded identical antibody titres with sera raised against P. haemolytica T15, E. coli K2 or N. meningitidis H whole cells. De-O-acetylation of the Pasteurella polysaccharide influenced its precipitability with immune sera, but this could not be related to the absence of O-acetyl groups because the non-acetylated E. coli K2 polymer readily precipitated with a line of 'identity' with the acetylated P. haemolytica T15 polymer.     

Influence of growth temperature on the development of Escherichia coli polysaccharide K antigens

The established seventy-one Escherichia coli polysaccharide capsular K antigenic test strains were examined for the development of the K antigen after growth at 37 degrees C and 18 degrees C. Twenty-eight K antigens were not detectable after growth of bacteria at 18 degrees C, while the remaining 43 antigens were developed at both temperatures. Most of the temperature-dependent antigens belonged to electrophoretically fast-moving K polysaccharides of rather low molecular weight, characteristically found among the common K antigens from extraintestinal disease isolates. Lipopolysaccharide O antigens were developed at both growth temperatures.     

Growth temperature-dependent variation in the bacteriophage-inactivating capacity and antigenicity of Yersinia enterocolitica lipopolysaccharide

Growth temperature affected the structure of Yersinia enterocolitica Ye 3827 lipopolysaccharide (LPS). Although Y. enterocolitica Ye 3827 synthesized smooth LPS when grown at a low temperature (25 degrees C), partial smooth-rough transition occurred when the bacteria were grown at the physiological temperature (37 degrees C). The structural alteration was detected by bacteriophage-inactivation assay and chemical and immunological analyses. LPS prepared from bacteria grown at 25 degrees C inactivated a number of bacteriophages that recognize the O-antigenic polysaccharide portion of LPS, whereas more than 3000 times the amount of LPS from bacteria grown at 37 degrees C was required for the same degree of inactivation. The antigenic determinant(s) responsible for the major reaction between 25 degrees C-LPS and anti-25 degree C-bacteria was located on the O-antigenic polysaccharide portion of LPS, but those responsible for the major reaction between 37 degrees C-LPS and anti-37 degrees C-bacteria were located on the R-core or inner portion of LPS.     

Engineering sialic acid synthetic ability into insect cells: identifying metabolic bottlenecks and devising strategies to overcome them

Previous studies have indicated negligible levels of both sialylation and the precursor N-acetylneuraminic acid (Neu5Ac) in a number of insect cell lines grown in serum-free medium. The overexpression of the human sialic acid 9-phosphate synthase (SAS) in combination with N-acetylmannosamine (ManNAc) feeding has been shown to overcome this limitation. In this study we evaluated the potential bottlenecks in the sialic acid synthesis pathway in a Spodoptera frugiperda (Sf9) insect cell line and devised strategies to overcome them by overexpression of the enzymatic pathway enzymes combined with appropriate substrate feeding. Coexpression of SAS and UDP-GlcNAc 2-epimerase/ManNAc kinase, the bifunctional enzyme initiating sialic acid biosynthesis in mammals, resulted in Neu5Ac synthesis without use of any external media supplementation to demonstrate that Neu5Ac could be generated intracellularly in Sf9 cells using natural metabolic precursors. N-Acetylglucosamine (GlcNAc) feeding in combination with this coexpression resulted in much higher levels of Neu5Ac compared to levels obtained with ManNAc feeding with SAS expression alone. The lower Neu5Ac levels obtained with ManNAc feeding suggested limitations in the transport and phosphorylation of ManNAc. The bottleneck in phosphorylation was likely due to utilization of GlcNAc kinase for phosphorylation of ManNAc in insect cells and was overcome by expression of ManNAc kinase. The transport limitation was addressed by the addition of tetra-O-acetylated ManNAc, which is easily taken up by the cells. An alternative sialic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), could also be generated in insect cells, suggesting the potential for controlling not only the production of sialic acids but also the type of sialic acid generated. The levels of KDN could be increased with virtually no Neu5Ac generation when Sf9 cells were fed excess GlcNAc. The results of these studies may be used to enhance the sialylation of target glycoproteins in insect and other eukaryotic expression systems.     

NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharide in group B Streptococcus

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) was enhanced by CTP and Mg(2+), the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac(2) followed by CMP activation of Neu5Ac or activation of Neu5,9Ac(2) followed by de-O-acetylation of CMP-Neu5,9Ac(2). Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.     

Biosynthesis and assembly of the polysialic acid capsule in Escherichia coli K1. Role of a low-density vesicle fraction in activation of the endogenous synthesis of sialyl polymers

Escherichia coli K1 synthesizes a polysialic acid capsule when grown at 37 but not 15 degrees C. The derangement in sialyl polymer synthesis appears to result from the inability of 15 degrees C membranes to synthesize or assemble a functional endogenous acceptor (Troy, F.A., and McCloskey, M.A. (1979) J. Biol. Chem. 254, 7377-7387). Membranes from cells grown at 15 degrees C spontaneously gained the ability to synthesize sialyl polymer after incubation at 33 degrees C for 2-4 h. The incubation-dependent activation of the endogenous synthesis of sialyl polymer in 15 degrees C membranes possessed two unusual features. First, the sialyltransferase was localized in a low density vesicle fraction (LDV; rho = 1.11 g/cm3). Second, this fraction catalyzed protein synthesis, and protein synthesis was required for activation. A study of the LDV fraction showed: 1) their light density resulted from a 5- to 8-fold enrichment in lipid phosphate to protein ratio and their sialyltransferase activity was enriched 40-fold compared with unfractionated total membranes; 2) they contained proteins characteristic of inner and outer membranes including leader peptidase and lipoprotein; 3) they constituted 8% of the mass of unfractionated total membranes yet contained all of the endogenous sialyltransferase activity in 15 degrees C membranes. In contrast, LDV from 37 degrees C grown cells accounted for 4.8% of the membrane mass and only 12.5% of the endogenous sialyltransferase activity; 4) they were multilamellar and averaged 0.7 mu in diameter. Based on these results, we believe the LDV fraction is of physiological importance in sialyl polymer synthesis. Growth at 15 degrees C allowed identification and study of the LDV fraction possibly because of the altered thermotropic properties of the membrane phospholipids that occur when E. coli is grown at low temperature.     

Physiological studies of a temperature-sensitive sporulation mutant of Bacillus cereus T.

Growth of temperature-sensitive mutant Bacillus cereus T JS22-C occurred normally at the restrictive temperature (37 degrees C), but sporulation was blocked at stage 0. The production of extracellular and intracellular proteases and of alkaline phosphatase occurred at 37 degrees C, but the expression of a functional tricarboxylic acid cycle did not. At the permissive temperature (26 degrees C), the mutant sporulated at a slightly lower frequency (60%) and at a lower rate than the parent strain. The oxidation of organic acids, which accumulate in the growth medium began at T0 in cultures of the parent strain but was delayed until about T3 in cultures of the mutant. Later events in sporulation were also delayed in the mutant by about 3 h. Experiments in which the temperature of growth was shifted from 37 to 26 degrees C or from 26 to 37 degrees C at various times showed that the temperature-sensitive event began approximately 1 h after the end of exponential growth and ended when the cells reached the end of stage II (septum formation). The absence of a functional tricarboxylic acid cycle in cells of the mutant grown at 37 degrees C or shifted from 26 to 37 degrees C before T1 did not appear to be due to a lesion in one of the structural genes of the tricarboxylic acid cycle but was more likely due to the inability of the cells to derepress the synthesis of some of the enzymes of that cycle.     

Protein synthesis is required for in vivo activation of polysialic acid capsule synthesis in Escherichia coli K1.

The kinetics of in vivo expression of the polysialosyl (K1) capsular antigen in Escherichia coli has been studied. Growth of E. coli K1 strains at 15 degrees C prevents K1 polysaccharide synthesis (F. A. Troy and M. A. McCloskey, J. Biol. Chem. 254:7377-7387, 1979). Synthesis is reactivated in cells grown at 15 degrees C after upshift to 37 degrees C. The early expression and resultant morphology of K1 capsular antigen was monitored in temperature upshift experiments by using electron microscopy. Morphological stabilization of the capsule was achieved by treatment of cells with an antiserum specific for the alpha, 2-8-linked polysialosyl antigen. The kinetics of K1 capsule expression in growing cells was measured by bacteriophage adsorption with phage K1F, which required the K1 capsule for binding. The results of temperature upshift experiments showed that capsule first appeared on the cell surface after 10 min. Subsequent bacteriophage binding increased linearly with time until a fully encapsulated state was reached 45 min after upshift. The initiation of K1 capsule appearance was dependent on protein synthesis and the addition of chloramphenicol before temperature upshift prevented any expression of the K1 antigen. Chloramphenicol reduced the rate of K1 synthesis when added after temperature upshift. We conclude from these results that protein synthesis is a prerequisite for activation of capsule expression in vivo, but not for subsequent elongation of polysialosyl chains.