Molecular characterization of cap3A, a gene from the operon required for the synthesis of the capsule of Streptococcus pneumoniae type 3: sequencing of mutations responsible for the unencapsulated phenotype and localization of the capsular cluster on the pneumococcal chromosome.

The complete nucleotide sequence of the cap3A gene of Streptococcus pneumoniae, which is directly responsible for the transformation of some unencapsulated, serotype 3 mutants to the encapsulated phenotype, has been determined. This gene encodes a protein of 394 amino acids with a predicted M(r) of 44,646. Twelve independent cap3A mutations have been mapped by genetic transformation, and three of them have been sequenced. Sequence comparisons revealed that cap3A was very similar (74.4%) to the hasB gene of Streptococcus pyogenes, which encodes a UDP-glucose dehydrogenase (UDP-GlcDH) that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, the donor substances in the pneumococcal type 3 capsular polysaccharide. Furthermore, a PCR-generated cap3A+ gene restored encapsulation in our cap3A mutants as well as in a mutant previously characterized as deficient in UDP-GlcDH (R. Austrian, H. P. Bernheimer, E.E.B. Smith, and G.T. Mills, J. Exp. Med. 110:585-602, 1959). These results support the conclusion that cap3A codes for UDP-GlcDH. We have also identified a region upstream of cap3A that should contain common genes necessary for the production of capsule of any type. Pulsed-field gel electrophoresis and Southern blotting showed that the capsular genes specific for serotype 3 are located near the genes encoding PBP 2X and PBP 1A in the S. pneumoniae chromosome, whereas copies of the common genes (or part of them) appear to be present in different locations in the genome.     

Evidence for horizontal transfer from streptococcus to escherichia coli of the kfid gene encoding the K5-specific UDP-glucose dehydrogenase

Capsular polysaccharides are important virulence factors both in Gram-positive and Gram-negative bacteria. A similar cluster organization of the genes involved in the synthesis of bacterial exopolysaccharides has been postulated in both cases, suggesting that these clusters evolved by module assembly. Horizontal gene transfer has been postulated to explain the polymorphism found in these cellular polymers. The cap1K and cap3A genes coding for the pneumococcal type 1 and type 3 UDP-glucose dehydrogenases, respectively, have been compared with other UDP-sugar dehydrogenases. We have observed that the evolutionary distance between Cap1K and Cap3A is approximately equal to that found between Cap1K (or Cap3A) and other UDP-GlcDH of families evolutionarily distant like KfiD, the dehydrogenase from Escherichia coli K5. On the basis of comparisons of G + C content, patterns of synonymous and nonsynonymous substitutions, dinucleotide frequencies, and codon usage bias, we conclude that the kfiD gene has been introduced into E. coli from an exogenous source, probably from a streptococcal species. Received: 26 May 1997 / Accepted: 30 July 1997     

Genome-wide analysis of the UDP-glucose dehydrogenase gene family in Arabidopsis, a key enzyme for matrix polysaccharides in cell walls

Arabidopsis cell walls contain large amounts of pectins and hemicelluloses, which are predominantly synthesized via the common precursor UDP-glucuronic acid. The major enzyme for the formation of this nucleotide-sugar is UDP-glucose dehydrogenase, catalysing the irreversible oxidation of UDP-glucose into UDP-glucuronic acid. Four functional gene family members and one pseudogene are present in the Arabidopsis genome, and they show distinct tissue-specific expression patterns during plant development. The analyses of reporter gene lines indicate gene expression of UDP-glucose dehydrogenases in growing tissues. The biochemical characterization of the different isoforms shows equal affinities for the cofactor NAD(+) ( approximately 40 microM) but variable affinities for the substrate UDP-glucose (120-335 microM) and different catalytic constants, suggesting a regulatory role for the different isoforms in carbon partitioning between cell wall formation and sucrose synthesis as the second major UDP-glucose-consuming pathway. UDP-glucose dehydrogenase is feedback inhibited by UDP-xylose. The relatively (compared with a soybean UDP-glucose dehydrogenase) low affinity of the enzymes for the substrate UDP-glucose is paralleled by the weak inhibition of the enzymes by UDP-xylose. The four Arabidopsis UDP-glucose dehydrogenase isoforms oxidize only UDP-glucose as a substrate. Nucleotide-sugars, which are converted by similar enzymes in bacteria, are not accepted as substrates for the Arabidopsis enzymes.     

Cloning and expression studies of the Dunaliella salina UDP-glucose dehydrogenase cDNA.

The enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) converts UDP-glucose to UDP-glucuronate. Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of primary cell walls. A cDNA, named DsUGDH, (GeneBank accession number: AY795899) corresponding to UGDH was cloned by RT-PCR approach from Dunaliella salina. The cDNA is 1941-bp long and has an open reading frame encoded a protein of 483 amino acids with a calculated molecular weight of 53 kDa. The derived amino acids sequence shows high homology with reported plants UGDHs, and has highly conserved amino acids motifs believed to be NAD binding site and catalytic site. Although UDP-glucose dehydrogenase is a comparatively well characterized enzyme, the cloning and characterization of the green alga Dunaliella salina UDP-glucose dehydrogenase gene is very important to understand the salt tolerance mechanism of Dunaliella salina. Northern analyses indicate that NaCl can induce the expression the DsUGDH.     

兽疫链球菌透明质酸合成相关基因hasB的克隆以及性质研究

透明质酸是链球菌荚膜的主要组成部分,有着重要的生理功能。UDP-葡萄糖脱氢酶(HasB)是透明质酸合成中的一个关键酶,而C类链球菌的UDP-葡萄糖脱氢酶编码基因(hasB)尚未被克隆。通过hasB基因的上下游序列设计引物从兽疫链球茵的基因组中克隆出一段序列,测序结果显示其包含一个由1206个碱基组成的开放阅读框,所编码的蛋白序列同化脓链球菌和乳链球菌的UDP-葡萄糖脱氢酶蛋白序列分别有63．1％和70．6％的相似性。将这段基因置于T7启动子下,并在大肠杆菌中进行表达,能够得到一个约47kDa的蛋白,酶活测定显示其具有UDP-葡萄糖脱氢酶活性。这些结果表明所克隆的基因是兽疫链球菌的UDP-葡萄糖脱氢酶编码基因。     

Versatility of the capsular genes during biofilm formation by Streptococcus pneumoniae

Streptococcus pneumoniae forms part of the natural microbiota of the nasopharynx. For the pneumococcus to cause infection, colonization needs to occur and this process is mediated by adherence of bacteria to the respiratory epithelium. Although the capsular polysaccharide (CPS) of S. pneumoniae is known to be important for infection to occur, its role in colonization is controversial. Biofilm models are starting to emerge as a promising tool to investigate the role of CPS during nasopharyngeal carriage, which is the first step in the dissemination and initiation of a pneumococcal infection. Using a well-defined model system to analyse in vitro biofilm formation in pneumococcus, here we explore the molecular changes underlying the appearance of capsular mutants using type 3 S. pneumoniae cells. Spontaneous colony phase variants show promoter mutations, as well as duplications, deletions and point mutations in the cap3A gene, which codes for a UDP-glucose dehydrogenase (UDP-GlcDH). Increased biofilm-forming capacity could usually be correlated with a reduction both in colony size and in the relative amount of CPS present on the cell surface of each colony variant. However, a mutation in Cap3A Thr83Ile (a strictly conserved residue in bacterial UDP-GlcDHs) that resulted in very low CPS production also led to impaired biofilm formation. We propose that non-encapsulated mutants of pneumococcal type 3 strains are essentially involved in the initial stages (the attachment stage) of biofilm formation during colonization/pathogenesis.     

Identification and characterization of UDP-glucose dehydrogenase from the hyperthermophilic archaon, Pyrobaculum islandicum

A gene encoding a UDP-glucose dehydrogenase homologue was identified in the hyperthermophilic archaeon, Pyrobaculum islandicum. This gene was expressed in Escherichia coli, and the product was purified and characterized. The expressed enzyme is the most thermostable UDP-glucose dehydrogenase so far described, with a half-life of 10 min at 90 °C. The enzyme retained its full activity after incubating in a pH range of 5.0-10.0 for 10 min at 80 °C. The temperature dependence of the kinetic parameters for this enzyme was examined at 37-70 °C. A decrease in K(m)s for UDP-glucose and NAD was observed with decreasing temperature. This resulted in the enzyme still retaining high catalytic efficiency (V(max)/K(m)) for the substrate and cofactor, even at 37 °C. These characteristics make the enzyme potentially useful for its application at a much lower temperature such as 37 °C than the optimum growth temperature of 100 °C for P. islandicum.     

6-Phosphogluconate dehydrogenase and the assay of uridine diphosphate glucose pyrophosphorylase in the cellular slime mould Dictyostelium discoideum

1. 6-Phosphogluconate dehydrogenase activity is present in all morphogenetic stages during cell differentiation in the cellular slime mould. 2. The different ratios of 6-phosphogluconate dehydrogenase/UDP-glucose pyrophosphorylase observed during this process can render spectrophotometric assays of UDP-glucose pyrophosphorylase inaccurate. 3. The disputed occurrence of increases in specific activity of UDP-glucose pyrophosphorylase during cell differentiation in the cellular slime mould is discussed in the light of these observations.     

Mechanism of the enzymatic reaction catalyzed by uridine diphosphate glucose dehydrogenase. The chemical synthesis of uridine diphosphate glucose-6-H3 and its oxidation by uridine diphosphate glucose dehydrogenase]

The synthesis of UDP-glucose-6-s-H was performed through condensation of alpha-D-glucopyranosyl phosphate-6-3-H and uridine 5'-phosphomorpholidate. Enzymic oxidation of UDP-glucose-6-3-H with calf liver UDP-glucose dehydrogenase was found to proceed with direct transfer of the hydrogen from C-6 of UDP-glucose onto NAD.     

Investigation of the UDP-glucose dehydrogenase reaction for a coupled assay of UDP-glucose pyrophosphorylase activities.

An optimized coupled enzyme assay for UDP-glucose pyrophosphorylase (EC 2.7.7.9) using UDP-glucose dehydrogenase (EC 1.1.1.22) is presented. This optimized assay was developed by a detailed investigation of the kinetics of the UDP-glucose dehydrogenase reaction. In addition the data provide a basis for the enzymatic synthesis of UDP-glucuronic acid. The results demonstrate that the two binding sites of the dehydrogenase differ since a different modulation of the enzyme activity and stability is observed after preincubation with UDP-glucose or NAD+ at various pH values. This is of general interest for the preparation of assay mixtures where UDP-glucose dehydrogenase is used as an auxiliary enzyme.     

Control of Uridine Diphosphate-Glucose Dehydrogenase Synthesis and Uridine Diphosphate-Glucuronic Acid Accumulation by a Regulator Gene Mutation in Escherichia coli K-12

Uridine diphosphate (UDP)-glucose dehydrogenase, the enzyme that converts UDP-glucose to UDP-glucuronic acid, was derepressed in a mucoid (capR9) strain of Escherichia coli K-12 and repressed in a nonmucoid (capR(+)) strain. A nonmucoid mutant (strain MC 152; capR9 non-2) derived from the mucoid strain accumulated large quantities of nucleotides. Among these nucleotides, UDP-glucuronic acid was identified as well as guanosine triphosphate and an adenosine diphosphate-sugar. UDP-glucose dehydrogenase was still derepressed in strain MC 152. When the nonmucoid mutant was transduced to the wild-type state for this regulator gene (capR(+)), the transductant was found to accumulate less total nucleotides, and the accumulation of UDP-glucuronic acid was abolished. UDP-glucose dehydrogenase was repressed in the capR(+)non-2 strain but not to the same extent that it was in the capR(+) strain.     

Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane

In this study, UDP-glucose dehydrogenase has been purified to electrophoretic homogeneity from sugarcane (Saccharum spp. hybrid) culm. The enzyme had a pH optimum of 8.4 and a subunit molecular mass of 52 kDa. Specific activity of the final preparation was 2.17 micromol/min/mg protein. Apparent K(m) values of 18.7+/-0.75 and 72.2+/-2.7 microM were determined for UDP-glucose and NAD(+), respectively. The reaction catalyzed by UDP-glucose dehydrogenase was irreversible with two equivalents of NADH produced for each UDP-glucose oxidized. Stiochiometry was not altered in the presence of carbonyl-trapping reagents. With respect to UDP-glucose, UDP-glucuronic acid, and UDP-xylose were competitive inhibitors of UDP-glucose dehydrogenase with K(i) values of 292 and 17.1 microM, respectively. The kinetic data are consistent with a bi-uni-uni-bi substituted enzyme mechanism for sugarcane UDP-glucose dehydrogenase. Oxidation of the alternative nucleotide sugars CTP-glucose and TDP-glucose was observed with rates of 8 and 2%, respectively, compared to UDP-glucose. The nucleotide sugar ADP-glucose was not oxidized by UDP-glucose dehydrogenase. This is of significance as it demonstrates carbon, destined for starch synthesis in tissues that synthesize cytosolic AGP-glucose, will not be partitioned toward cell wall biosynthesis.     

Regulatory aspects of glycosaminoglycan biosynthesis

The control of glycosaminoglycan biosynthesis was investigated by studying the kinetic and regulatory properties of some enzymes involved in the formation of UDP-sugar precursors: UDP-N-acetylglucosamine 4'-epimerase, catalyzing the interconversion of hexosamine precursors and UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase, utilizing UDP-glucose for the formation of uronic acid and galactose precursors. The study was carried out in tissues with different glycosaminoglycan production: bovine cornea, producing both chondroitin sulfate and keratan sulfate, and newborn-pig epiphysial-plate cartilage, producing mostly chondroitin sulfate. The biosynthesis of hexosamine precursors appeared to be regulated by the value of the NAD/NADH ratio. This control mechanism regulated also the activities of both UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase and, therefore, it could correlate the biosynthesis of glycosaminoglycan precursors with the redox activity of the cell. At the level of UDP-glucose utilization two other control mechanisms were demonstrated: the different affinities of UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase for UDP-glucose in tissues with different glycosaminoglycan production and the cellular concentration of UDP-xylose. This sugar-nucleotide inhibited UDP-glucose dehydrogenase, but did not affect the UDP-glucose 4'-epimerase activity; therefore, and increase of its cellular concentration may result in a decreased chondroitin sulfate synthesis and in an increased keratan sulfate formation.     

Characterization of the Type 8 Capsular Gene Cluster of Streptococcus pneumoniae

The complete nucleotide sequence of the capsular gene cluster (cap8) responsible for the biosynthesis of the capsular polysaccharide of Streptococcus pneumoniae type 8 has been determined. The cap8 gene cluster, located between the genes dexB and aliA, is composed of 12 open reading frames. A 14.7-kb DNA fragment embracing the cap8 genes was sufficient to transform an unencapsulated type 3 S. pneumoniae strain to a strain with the type 8 capsule. A possible scenario for the evolution of pneumococcal types 2 and 8 is outlined.     

Spontaneous sequence duplication within an open reading frame of the pneumococcal type 3 capsule locus causes high-frequency phase variation

The molecular genetic basis of high-frequency serotype 3 capsule phase variation in Streptococcus pneumoniae (the pneumococcus) was investigated. Pneumococci were grown in sorbarod biofilms at 34 degrees C to mimic nasopharyngeal carriage. Different type 3 pneumococci commonly associated with invasive disease generated apparently random tandem duplications of 11-239 bp segments within the cap3A gene of the type 3 capsule locus. These duplications alone were found to be responsible for high-frequency capsule phase variation, in which (phase off) acapsular variants possessed duplications within cap3A, and (phase on) capsular revertants possessed wild-type cap3A genes, indicating the precise excision of the duplication. Additionally, the frequency of phase reversion (off to on) was found to exhibit a linear relationship between (log) frequency of reversion and (log) length of duplication. This apparently random duplication giving rise to phase variation is in stark contrast to the 'preprogrammed' contingency genes in many Gram-negative organisms that possess homopolymeric sequence repeats or motifs for site-specific recombination.     

Depletion of glycogen synthetase and increase of glucose 6-phosphate dehydrogenase in livers of ethionine-treated mice

1. Ethionine-treated mice showed a marked depletion in liver glycogen, a decrease of glycogen-synthetase activity, an increase in activity of glucose 6-phosphate dehydrogenase and the solubilization of phosphorylase. 2. The administration of cortisol or glucose did not alleviate these changes but the effect of ethionine was completely prevented in animals given methionine as well as ethionine. 3. The activities of the following enzymes were unchanged: hexokinase, glucokinase, glucose 6-phosphatase, phosphoglucomutase, 6-phosphogluconate dehydrogenase, UDP-glucose pyrophosphorylase, UDP-glucose dehydrogenase and pyruvate kinase.     

Action of degradative enzymes on the light fraction of bovine septa protein polysaccharide

1. The administration of cortisol and of other glucocorticoid steroids to starved mice produced an increase in liver glycogen content, an elevation of glycogen-synthetase activity and a predominantly particulate localization of both phosphorylase and glycogen-synthetase enzymes. 2. Three daily doses of actinomycin D caused a marked glycogen depletion, a significant decrease in glycogen-synthetase activity, the solubilization of phosphorylase and glycogen synthetase and the following effects on the activities of various other enzymes: a decrease in UDP-glucose pyrophosphorylase and phosphoglucomutase, an increase in glucose 6-phosphate dehydrogenase and no change in glucose 6-phosphatase, 6-phosphogluconate dehydrogenase, pyruvate kinase and UDP-glucose dehydrogenase. 3. Glucose ingestion, but not cortisol administration, reversed the effects of actinomycin D on liver glycogen content and on the activities of phosphorylase and glycogen synthetase.