Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene

The Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase gene is located on a 3.3-kilobase (kb) HindIII fragment of the plasmid pSR23 which contains the genes for K1 capsule production (Vann, W. F., Silver, R. P., Abeijon, C., Chang, K., Aaronson, W., Sutton, A., Finn, C. W., Lindner, W., and Kotsatos, M. (1987) J. Biol. Chem. 262, 17556-17562). The CMP-NeuAc synthetase gene expression was increased 10-30-fold by cloning of a 2.7-kb EcoRI-HindIII fragment onto the vector pKK223-3 containing the tac promoter. The complete nucleotide sequence of the gene encoding CMP-NeuAc synthetase was determined from progressive deletions generated by selective digestion of M13 clones containing the 2.7-kb fragment. CMP-NeuAc synthetase is located near the EcoRI site on this fragment as indicated by the detection of an open reading frame encoding a 49,000-dalton polypeptide. The amino- and carboxyl-terminal sequences of the encoded protein were confirmed by sequencing of peptides cleaved from both ends of the purified enzyme. The nucleotide deduced amino acid sequence was confirmed by sequencing several tryptic peptides of purified enzyme. The molecular weight is consistent with that determined from sodium dodecyl sulfate-gel electrophoresis. Gel filtration and ultracentrifugation experiments under nondenaturing conditions suggest that the enzyme is active as a 49,000-dalton monomer but may form aggregates.     

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.     

Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis.

The activity of the cytoplasmic CMP-2-keto-3-deoxyoctulosonic acid synthetase (CMP-KDO synthetase), which is low in Escherichia coli rough strains such as E. coli K-12 and in uncapsulated strains such as E. coli O111, was significantly elevated in encapsulated E. coli O10:K5 and O18:K5. This enzyme activity was even higher in an E. coli clone expressing the K5 capsule. This and the following findings suggest a correlation between elevated CMP-KDO synthetase activity and the biosynthesis of the capsular K5 polysaccharide. (i) Expression of the K5 polysaccharide and elevated CMP-KDO synthetase activity were observed with bacteria grown at 37 degrees C but not with cells grown at 20 degrees C or below. (ii) The recovery kinetics of capsule expression of intact bacteria, in vitro K5 polysaccharide-synthesizing activity of bacteria, and CMP-KDO synthetase activity of bacteria after temperature upshift from 18 to 37 degrees C were the same. (iii) Chemicals which inhibit capsule (polysaccharide) expression also inhibited the elevation of CMP-KDO synthetase activity. The chromosomal location of the gene responsible for the elevation of this enzyme activity was narrowed down to the distal segment of the transport region of the K5 expression genes.     

KfoE encodes a fructosyltransferase involved in capsular polysaccharide biosynthesis in Escherichia coli K4

Escherichia coli K4 synthesizes a capsular polysaccharide (CPS) consisting of a fructose-branched chondroitin (GalNAc-GlcA(fructose)n), which is a biosynthetic precursor of chondroitin sulfate. Here, the role of kfoE in the modification of the chondroitin backbone was investigated using knock-out and recombinant complementation experiments. kfoE disruption and complementation had no significant effect on cell growth. CPS production was increased by 15 % in the knock-out strain, and decreased by 21 % in the knock-out strain complemented with recombinant kfoE. CPS extracted from the knock-out strain was chondroitin, whereas CPS extracted from the complemented strain was a fructose-branched chondroitin. The results demonstrated that the kfoE gene product altered the fructose group at the C3 position of the GlcA residue during production of K4CPS.     

Neuraminidase associated with coliphage E that specifically depolymerizes the Escherichia coli K1 capsular polysaccharide.

Plaque morphology indicated that the five Escherichia coli K1-specific bacteriophages (A to E) described by Gross et al. (R. J. Gross, T. Cheasty, and B. Rowe, J. Clin. Microbiol. 6:548-550, 1977) encode K1 depolymerase activity that is present in both the bound and free forms. The free form of the enzyme from bacteriophage E was purified 238-fold to apparent homogeneity and in a high yield from ammonium sulfate precipitates of cell lysates by a combination of CsCl density gradient ultracentrifugation, gel filtration, and anion-exchange chromatography. The enzyme complex had an apparent molecular weight of 208,000, as judged from its behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was dissociated by sodium dodecyl sulfate at 100 degrees C to yield two polypeptides with apparent molecular weights of 74,000 and 38,500. Optimum hydrolytic activity was observed at pH 5.5, and activity was strongly inhibited by Ca2+; the Km was 7.41 X 10(-3) M. Rapid hydrolysis of both the O-acetylated and non-O-acetylated forms of the K1 antigen, an alpha 2----8-linked homopolymer of N-acetylneuraminic acid, and of the meningococcus B antigen was observed. Limited hydrolysis of the E. coli K92 antigen, an N-acetylneuraminic acid homopolymer containing alternating alpha 2----8 and alpha 2----9 linkages, occurred, but the enzyme failed to release alpha 2----3-, alpha 2----6-, or alpha 2----9-linked sialic residues from a variety of other substrates.     

CMP-N-acetylneuraminic acid synthetase from Escherichia coli K1 is a bifunctional enzyme: identification of minimal catalytic domain for synthetase activity and novel functional domain for platelet-activating factor acetylhydrolase activity

Escherichia coli CMP-NeuAc synthetase (EC 2.7.7.43) catalyzes the synthesis of CMP-NeuAc from CTP and NeuAc, which is essential for the formation of capsule polysialylate for strain K1. Alignment of the amino acid sequence of E. coli CMP-NeuAc synthetase with those from other bacterial species revealed that the conserved motifs were located in its N termini, whereas the C terminus appeared to be redundant. Based on this information, a series of deletions from the 3'-end of the CMPNeuAc synthetase coding region was constructed and expressed in E. coli. As a result, the catalytic domain required for CMP-NeuAc synthetase was found to be in the N-terminal half consisting of amino acids 1-229. Using the strategy of tertiary structure prediction based on the homologous search of the secondary structure, the C-terminal half was recognized as an alpha1-subunit of bovine brain platelet-activating factor acetylhydrolase isoform I. The biochemical analyses showed that the C-terminal half consisting of amino acids 228-418 exhibited platelet-activating factor acetylhydrolase activity. The enzyme properties and substrate specificity were similar to that of bovine brain alpha1-subunit. Although its physiological function is still unclear, it has been proposed that the alpha1-subunit-like domain of E. coli may be involved in the traversal of the blood-brain barrier.     

Structure of the Escherichia coli 0104 polysaccharide and its identity with the capsular K9 polysaccharide

Spontaneous mutants of Rhizobium leguminosarum biovar viciae strain C1204b were selected for their ability to tolerate 0.2 M NaCl, a growth-inhibiting level of salt for the parental strain. Transposon-mediated salt-sensitive mutants of strain C1204b were screened for their inability to grow in 0.08 M NaCl. Quantitation of the free-amino acid pools in the mutants grown in NaCl revealed a dramatic increase in glutamine, serine, glutamate and proline, and to a lesser extent alanine and glycine in the salt-tolerant mutants in comparison with the parental strain exposed to NaCl; but only glutamate and proline increased in the salt-sensitive mutants under NaCl stress. Extracellular polysaccharide levels were quantitated for the salt-tolerant mutants and determined to be approximately two-fold higher than for the parental strain. Although the mutations that occurred in the NaCl-tolerant and NaCl-sensitive strains did not interfere with nodule formation, no nitrogenase activity could be observed in the NaCl tolerant mutants as evaluated by acetylene reduction.     

Biosynthesis of the Escherichia coli K5 polysaccharide, a representative of group II capsular polysaccharides: polymerization in vitro and characterization of the product.

Biosynthesis of the capsular K5 polysaccharide of Escherichia coli, which has the structure 4)-beta GlcA-1,4-alpha GlcNAc-(1, was studied with membrane preparations from an E. coli K5 wild-type strain and from a recombinant K-12 strain expressing the K5 capsule. Polymerization occurs at the inner face of the cytoplasmic membrane without the participation of lipid-linked oligosaccharides. The serological K5 specificity of the in vitro product was determined with a K5-specific monoclonal antibody in an antigen-binding assay. The K5 polysaccharide, as obtained from the membranes after an in vitro incubation, has 2-keto-3-deoxyoctulosonic acid as the reducing sugar, which indicates that the polysaccharide grows by chain elongation at the nonreducing end.     

Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinant Escherichia coli

Abstract The genes directing the expression of group II capsules in Escherichia coli are organized into three regions. The central region 2 is type specific and thought to determine the synthesis of the respective polysaccharide, whilst the flanking regions 1 and 3 are common to all group II gene clusters and direct the surface expression of the capsular polysaccharide. In this communication we analyze the involvement of region 1 and 3 genes in the synthesis of the capsular KS polysaccharide. Recombinant E. coli strains harboring all KS specific region 2 genes and having various combinations of region 1 and 3 gene were studied using immunoelectron microscopy. Membranes from these bacteria were incubated with UDP[¹⁴C]GlcA and UDPG1cNAc in the absence or presence of KS polysaccharide as an exogenous acceptor. It was found that recombinant strains with only gene region 2 did not produce the K5 polysaccharide. Membranes of such strains did not synthesize the polymer and did not elongate K5 polysaccharide added as an exogenous acceptor. An involvement of genes from region 1 (notably kps C and kps S) and from region 3 (notably kps T) in the K5 polysaccharide synthesis was apparent and is discussed.     

CMP-N-acetylneuraminic acid synthetase of Escherichia coli: high level expression, purification and use in the enzymatic synthesis of CMP-N-acetylneuraminic acid and CMP-neuraminic acid derivatives.

The gene encoding CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) in Escherichia coli serotype O7 K1 was isolated and overexpressed in E.coli W3110. Maximum expression of 8-10% of the soluble E.coli protein was achieved by placing the gene with an engineered 5'-terminus and Shine-Dalgarno sequence into a pKK223 vector derivative behind the tac promoter. The overexpressed synthetase was purified to greater than 95% homogeneity in a single step by chromatography on high titre Orange A Matrex dye resin. Enzyme purified by this method was used directly for the synthesis of CMP-NeuAc and derivatives. The enzymatic synthesis of CMP-NeuAc was carried out on a multigram scale using equimolar CTP and N-acetylneuraminic acid as substrates. The resultant CMP-NeuAc, isolated as its disodium salt by ethanol precipitation, was prepared in an overall yield of 94% and was judged to be greater than 95% pure by 1H NMR analysis. N-Carbomethoxyneuraminic acid and N-carbobenzyloxyneuraminic acid were also found to be substrates of the enzyme; 5-azidoneuraminic acid was not a substrate of the enzyme. N-Carbomethoxyneuraminic acid was coupled to CMP at a rate similar to that observed with NeuAc, whereas N-carbobenzyloxyneuraminic acid was coupled greater than 100-fold more slowly. The high level of expression achieved with the E.coli synthetase, together with the high degree of purity readily obtainable from crude cell extracts, make the recombinant bacterial enzyme the preferred catalyst for the enzymatic synthesis of CMP-N-acetylneuraminic acid.     

An extraintestinal, pathogenic isolate of Escherichia coli (O4/K54/H5) can produce a group 1 capsule which is divergently regulated from its constitutively produced group 2, K54 capsular polysaccharide.

We are studying an O4/K54/H5 Escherichia coli bacteremic isolate (CP9) as a model pathogen for extraintestinal infection. Its group 2, K54 capsular polysaccharide is an important virulence determinant and confers serum resistance. In this study the effect of the group 1 capsule regulators, RcsA, RcsB, and Lon protease, on the regulation of CP9's capsular polysaccharides was assessed. It was established that in the presence of multicopy rcsA or with disruption of lon, CP9 can be induced to produce a group 1 capsule. RcsA, RcsB, and Lon are present in this K54 background and regulate group 1 capsule expression in a fashion similar to that described for K-12 strains. Two independent group 2 capsule gene protein fusions (cl1.29::TnphoA and cl1.137::TnphoA) were used to evaluate the effects of these regulators on group 2 K54 capsule production. Disruption of lon resulted in 1.9-fold (TR293 [cl1.29::TnphoA lon-146]) and 3.4-fold (TR1373 [cl1.137::TnphoA lon-146]) decreases in fusion activity at 28 degrees C, relative to the baseline level. However, decreases in fusion activity at 42 degrees C were only 1.2- and 1.4-fold, respectively. Inactivation of both lon and rcsA or lon and rcsB restored fusion activity to baseline levels at 28 degrees C, but only a partial restoration of activity was seen at higher temperatures. To assess whether these differences in fusion activity reflected a functional change in capsule production, the effects of 80% normal human serum (NHS) were tested against CP9 and TR93 (lon-146). Since the group 2 K54 capsule protects against the bactericidal activity of 80% NHS, a decrease in its production results in an increase in serum sensitivity. Viable counts of CP9 increased 10-fold in 80% NHS over 3 h at 28 degrees C, as expected. In contrast to CP9, TR93 (lon-146) incurred a 10-fold loss in viability under the same conditions. The levels of RcsA are increased in TR93 (lon 146) as consequence of lon disruption; therefore, these results in conjunction with the cl1::TnphoA protein fusion data establish RcsA as a negative regulator of the group 2 K54 capsular polysaccharide. Furthermore, these results also suggest existence of another Lon-sensitive negative regulator of group 2 K54 capsule production, which is active higher temperatures.     

Influence of metal ions and temperature on the conformation of Escherichia coli K1 capsular polysaccharide

Escherichia coli K1 secretes a homopolymer capsular polysaccharide (CPS) consisting of alpha 2,8 linked N-acetylneuraminic acid (poly 2,8NeuNAc). Typically poly 2,8NeuNAc is arranged in low and high order alpha helices with carboxyl and hydroxyl groups extending from the helices. Several properties of CPS such as antigenicity and metal binding can be influenced by its structural conformation. We examined the influences of metal ions and temperature on the secondary structure of polya2,8NeuNAc. Conformation alteration was detected by ultraviolet (UV) spectroscopy and circular dichroism (CD). The majority of metal ions tested had no detectable influence on poly a2,8NeuNAc structure. In contrast, Yb³⁺., Hg²⁺, and Cu²⁺ ions greatly altered the UV and CD spectra, which suggests that these ions had disrupted the alpha helical structure of poly 2,8NeuNAc. These changes were influenced by the metal ion concentration. When poly 2,8NeuNAc was incubated at temperatures ranging from 20 - 60°C, alterations in its UV absorption spectra were also seen. The most significant change occurred between 35 and 40°C. In summary, this study suggests that the higher order structure and function of bacterial CPS may be influenced by environmental factors     

Primary structure of the Escherichia coli serotype K42 capsular polysaccharide and its serological identity with the Klebsiella K63 polysaccharide.

The Escherichia coli K42 capsular polysaccharide consists of leads to 3)-alpha-D-Galp-(1 leads to 3)-alpha-D-GalUAp-(1 leads to 3)-alpha-L-Fucp-(1 leads to repeating units. The E. coli K42 and Klebsiella K63 antigens are serologically identical.     

Escherichia coli K1-specific bacteriophage CUS-3 distribution and function in phase-variable capsular polysialic acid O acetylation

Escherichia coli K1 is the leading cause of human neonatal sepsis and meningitis and is important in other clinical syndromes of both humans and domestic animals; in this strain the polysialic acid capsule (K1 antigen) functions by inhibiting innate immunity. Recent discovery of the phase-variable capsular O acetylation mechanism indicated that the O-acetyltransferase gene, neuO, is carried on a putative K1-specific prophage designated CUS-3 (E. L. Deszo, S. M. Steenbergen, D. I. Freedberg, and E. R. Vimr, Proc. Natl. Acad. Sci. USA 102:5564-5569, 2005). Here we describe the isolation and characterization of a CUS-3 derivative (CUS-3a), demonstrating its morphology, lysogenization of a sensitive host, and the distribution of CUS-3 among a collection of 111 different K1 strains. The 40,207-bp CUS-3 genome was annotated from the strain RS218 genomic DNA sequence, indicating that most of the 63 phage open reading frames have their closest homologues in one of seven different lambdoid phages. Translational fusion of a reporter lacZ fragment to the hypervariable poly-Psi domain facilitated measurement of phase variation frequencies, indicating no significant differences between switch rates or effects on rates of the methyl-directed mismatch repair system. PCR analysis of poly-Psi domain length indicated preferential loss or gain of single 5'-AAGACTC-3' nucleotide repeats. Analysis of a K1 strain previously reported as "locked on" indicated a poly-Psi region with the least number of heptad repeats compatible with in-frame neuO expression. The combined results establish CUS-3 as an active mobile contingency locus in E. coli K1, indicating its capacity to mediate population-wide capsule variation.     

Translocation of group 1 capsular polysaccharide in Escherichia coli serotype K30. Structural and functional analysis of the outer membrane lipoprotein Wza

The late steps in assembly of capsular polysaccharides (CPS) and their translocation to the bacterial cell surface are not well understood. The Wza protein was shown previously to be required for the formation of the prototype group 1 capsule structure on the surface of Escherichia coli serotype K30 (Drummelsmith, J., and Whitfield, C. (2000) EMBO J. 19, 57-66). Wza is a conserved outer membrane lipoprotein that forms multimers adopting a ringlike structure, and collective evidence suggests a role for these structures in the export of capsular polymer across the outer membrane. Wza was purified in the native form and with a C-terminal hexahistidine tag. WzaHis6 was acylated and functional in capsule assembly, although its efficiency was slightly reduced in comparison to the native Wza protein. Ordered two-dimensional crystals of WzaHis6 were obtained after reconstitution of purified multimers into lipids. Electron microscopy of negatively stained crystals and Fourier filtering revealed ringlike multimers with an average outer diameter of 8.84 nm and an average central cavity diameter of 2.28 nm. Single particle analysis yielded projection structures at an estimated resolution of 3 nm, favoring a structure for the WzaHis6 containing eight identical subunits. A derivative of Wza (Wza*) in which the original signal sequence was replaced with that from OmpF showed that the native acylated N terminus of Wza is critical for formation of normal multimeric structures and for their competence for CPS assembly, but not for targeting Wza to the outer membrane. In the presence of Wza*, CPS accumulated in the periplasm but was not detected on the cell surface. Chemical cross-linking of intact cells suggested formation of a transmembrane complex minimally containing Wza and the inner membrane tyrosine autokinase Wzc.     

Isolation from Klebsiella and characterization of two rcs genes that activate colanic acid capsular biosynthesis in Escherichia coli

Two genes, designated rcsA (regulation of capsule synthesis) and rcsB, that had been cloned from the chromosome of Klebsiella aerogenes (K. pneumoniae) capsular serotype K21 were capable of activating expression of colanic acid capsular polysaccharide in Escherichia coli K12. The Klebsiella rcsA gene encoded a polypeptide of 23 kDa that was required for the induction of a mucoid phenotype at less than or equal to 30 degrees C but not at greater than or equal to 37 C. The Klebsiella rcsB locus encoded no apparent polypeptides and was not capable by itself of causing the overproduction of colanic acid. However, when present in the same cell with rcsA, either in cis or in trans, rcsB caused expression of mucoidy in E. coli at all growth temperatures. These findings are best explained if the Klebsiella rcsA gene product acts as a positive regulator of colanic acid biosynthesis in E. coli and that activity of this protein is in turn subject to regulation by Lon protease. The Klebsiella rcsB locus may exert its effect by preferentially binding a negative regulator of capsular biosynthesis, possibly Lon itself. DNA sequences homologous to the Klebsiella K21b rcsA and rcsB genes were found in the genomes of all other capsular serotypes of klebsiellae examined, including K2, K12, K36 and K43. However, there was no homology between such genes and the chromosome of E. coli. The ability of these rcs genes to induce a mucoid phenotype explains the apparent conjugative transfer from klebsiellae to E. coli of the ability to produce K21 or other Klebsiella capsular polysaccharides that are structurally and antigenically related to colanic acid.     

Plasmid-mediated conjugative transfer of Klebsiella sp. rcs genesable to induce colanic acid capsular polysaccharide biosynthesis in Escherichia coli

Regulation of capsular biosynthesis (rcs) genes, encoding the ability to induce the production of a colanic acid polysaccharide capsule, were transferred to Escherichia coli by conjugation with Klebsiella pneumoniae (aerogenes) of capsular serotype K36. Transfer was mediated by a 58.4-MDa conjugative plasmid of incompatibility group IncM, which carried a copy of Tn7 (specifying resistance to trimethoprim and streptomycin) together with determinants for several further resistances. This plasmid did not carry the rcs genes itself, but mediated the conjugative recA-dependent transfer of part of the Klebsiella chromosome to E. coli. Once resident in E. coli, the rcs gene(s) could not be mobilised to other strains of E. coli, and the mobilising plasmid could be cured from capsulate transconjugants without loss of the ability to produce colanic acid. All such cured transconjugants contained an insertion of Tn7 in the chromosome, suggesting that the transposon might be involved in mobilisation of the rcs genes from Klebsiella sp. to E. coli. These findings explain previous observations that the ability to manufacture capsular polysaccharide could be transferred by plasmids between Klebsiella sp. and E. coli.     

Assay of N-acetylheparosan deacetylase with a capsular polysaccharide from Escherichia coli K5 as substrate

A new substrate for the deacetylase which catalyzes the removal of the N-acetyl groups from N-acetylheparosan in the course of heparin biosynthesis has been prepared. The capsular polysaccharide from Escherichia coli 010:K5:H4, which is structurally identical to N-acetylheparosan, was partially N-deacetylated by hydrazinolysis and was then radioactively labeled by N-acetylation with [3H]acetic anhydride. Upon incubation of the labeled polysaccharide with microsomes from the Furth mastocytoma, [3H]acetyl groups were released, demonstrating that the bacterial polysaccharide was a substrate for the N-deacetylase. Reaction conditions were established which permitted the quantitative assay of N-deacetylase activity; a Km of 74 mg polysaccharide/liter was determined, which corresponds to 2.1 X 10(-4) M, expressed as concentration of uronic acid; Vmax was 3.4 nmol/mg protein/liter. In confirmation of previous results, it was observed (a) that the reaction was stimulated by 3'-phosphoadenylylsulfate (up to a maximum of 45% at a concentration of 0.5 mM), suggesting that N-sulfation occurred which facilitated continued action of the N-deacetylase, and (b) that NaCl and KCl inhibited the enzyme, with 50% reduction of activity at a concentration of 25 mM. In the course of this work, a simple, single-vial assay procedure was used. Released [3H]acetate was extracted from the acidified reaction mixture with a toluene- or xylene-based scintillation fluid containing 10% isoamyl alcohol and measured directly by scintillation spectrometry.     

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.