Thymidine diphosphate-6-deoxy-L-lyxo-4-hexulose reductase synthesizing dTDP-6-deoxy-L-talose from Actinobacillus actinomycetemcomitans

The serotype c-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans NCTC 9710 contains an unusual sugar, 6-deoxy-L-talose, which has been identified as a constituent of cell wall components in some bacteria. Two genes coding for thymidine diphosphate (dTDP)-6-deoxy-L-lyxo-4-hexulose reductases were identified in the gene cluster required for biosynthesis of serotype c-specific polysaccharide. Both dTDP-6-deoxy-L-lyxo-4-hexulose reductases were overproduced and purified from Escherichia coli transformed with the plasmids containing these genes. The sugar nucleotides converted by both reductases were purified by reversed-phase high performance liquid chromatography and identified by (1)H nuclear magnetic resonance and gas-liquid chromatography. The results indicated that one of two reductases produced dTDP-6-deoxy-L-talose and the other produced dTDP-L-rhamnose (dTDP-6-deoxy-L-mannose). The amino acid sequence of the dTDP-6-deoxy-L-lyxo-4-hexulose reductase forming dTDP-6-deoxy-L-talose shared only weak homology with that forming dTDP-L-rhamnose, despite the fact that these two enzymes catalyze the reduction of the same substrate and the products are determined by the stereospecificity of the reductase activity. Neither the gene for dTDP-6-deoxy-L-talose biosynthesis nor its corresponding protein product has been found in other bacteria; this biosynthetic pathway is identified here for the first time.     

Identification of a fourth gene involved in dTDP-rhamnose synthesis in Streptococcus mutans.

We had isolated three genes (rmlA, rmlB, and rmlC) involved in dTDP-rhamnose synthesis in Streptococcus mutans and found that three genes were insufficient for dTDP-rhamnose synthesis (Y. Tsukioka, Y. Yamashita, T. Oho, Y. Nakano, and T. Koga, J. Bacteriol. 179:1126-1134, 1997). The rmlD gene of S. mutans, encoding the enzyme which catalyzes the last step of dTDP-rhamnose synthesis, has been cloned and sequenced. The cell extract of Escherichia coli expressing the rmlD gene of S. mutans exhibited enzymatic activity corresponding to its counterpart in Shigella flexneri, a gram-negative bacterium. Rhamnose was not detected in the cell wall preparation purified from the mutant in which the cloned gene was insertionally inactivated. Rabbit antiserum against S. mutans serotype c-specific antigen did not react with autoclaved extracts from the mutant. The rmlD gene product of S. mutans compensated for the incompleteness of dTDP-rhamnose synthesis by the three previously isolated genes. These results indicate that the rmlD gene product is indispensable for the dTDP-rhamnose pathway and subsequently for the synthesis of serotype-specific antigen in S. mutans. Furthermore, conservation of the rmlD gene in Streptococcus species was demonstrated by Southern blot analysis.     

A gene cluster for the synthesis of serotype d-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans

The serotype d antigen of Actinobacillus actinomycetemcomitans consists of D-glucose, D-mannose, and L-rhamnose in a molar ratio of 1:2:1. A gene cluster involved in the synthesis of serotype-specific polysaccharide antigen was cloned from the chromosomal DNA of A. actinomycetemcomitans IDH 781 (serotype d). This cluster consisted of 12 open reading frames. Insertional inactivation of six genes in this cluster resulted in loss of ability of A. actinomycetemcomitans IDH 781 cells to produce the polysaccharide. Comparing the structure of the gene cluster with similar clusters from other serotypes of A. actinomycetemcomitans, showed that eight genes are unique to serotype d; the other four genes are involved in the biosynthesis of dTDP-L-rhamnose. These results suggest that the synthesis and structure of serotype d-specific polysaccharide of A. actinomycetemcomitans is quite different from those of other serotype strains.     

Genetic analysis of the gene cluster for the synthesis of serotype a-specific polysaccharide antigen in Aactinobacillus actinomycetemcomitans

The serotype a-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans consists of 6-deoxy-D-talose. A gene cluster associated with the biosynthesis of SPA was cloned and sequenced from the chromosomal DNA of A. actinomycetemcomitans SUNYaB 75 (serotype a). This cluster consisted of 14 open reading frames. Insertional inactivation of eight genes in this cluster resulted in loss of the ability of A. actinomycetemcomitans SUNYaB 75 cells to produce the polysaccharide. A protein database search revealed that the 11 sequential genes containing these eight genes were not found in SPA-associated gene clusters of the other serotypes of A. actinomycetemcomitans. These results suggest that the gene cluster is unique to serotype a and is essential to the synthesis of the SPA.     

The rkp-3 gene region of Sinorhizobium meliloti Rm41 contains strain-specific genes that determine K antigen structure.

The rkp-3 region is indispensable for capsular polysaccharide (K antigen) synthesis in Sinorhizobium meliloti Rm41. Strain Rm41 produces a K antigen of strain-specific structure, designated as the KR5 antigen. The data in this report show that the rkp-3 gene region comprises 10 open reading frames involved in bacterial polysaccharide synthesis and export. The predicted amino acid sequences for the rkpL-Q gene products are homologous to enzymes involved in the production of specific sugar moieties, while the putative products of the rkpRST genes show a high degree of similarity to proteins required for transporting polysaccharides to the cell surface. Southern analysis experiments using gene-specific probes suggest that genes involved in the synthesis of the precursor sugars are unique in strain Rm41, whereas sequences coding for export proteins are widely distributed among Sinorhizobium species. Mutations in the rkpL-Q genes result in a modified K antigen pattern and impaired symbiotic capabilities. On this basis, we suggest that these genes are required for the production of the KR5 antigen that is necessary for S. meliloti Rm41 exoB (AK631)-alfalfa (Medicago sativa) symbiosis.     

Characterization of glucosyltransferase expressed from a Streptococcus sobrinus gene cloned in Escherichia coli

The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, gtfI, previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage lambda vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtfI was efficiently expressed. The GTF-I expressed in E. coli was compared to the corresponding enzymes in S. sobrinus strains MFe28 (serotype h), B13 (serotype d) and 6715 (serotype g) and shown to resemble them closely in molecular mass and isoelectric point. The insoluble glucan produced by GTF-I from recombinant E. coli consisted of 1,3-alpha-D-glycosyl residues (approximately 90%). An internal fragment of the gtfI gene was used as a probe in hybridization experiments to demonstrate the presence of homologous sequences in chromosomal DNA of other streptococci of the mutans group.     

Guanosine diphosphate-4-keto-6-deoxy-d-mannose reductase in the pathway for the synthesis of GDP-6-deoxy-d-talose in Actinobacillus actinomycetemcomitans.

The serotype a-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans is an unusual sugar, 6-deoxy-d-talose. Guanosine diphosphate (GDP)-6-deoxy-d-talose is the activated sugar nucleotide form of 6-deoxy-d-talose, which has been identified as a constituent of only a few microbial polysaccharides. In this paper, we identify two genes encoding GDP-6-deoxy-d-talose synthetic enzymes, GDP-alpha-d-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose reductase, in the gene cluster required for the biosynthesis of serotype a-specific polysaccharide antigen from A. actinomycetemcomitans SUNYaB 75. Both gene products were produced and purified from Escherichia coli transformed with plasmids containing these genes. Their enzymatic reactants were analysed by reversed-phase HPLC (RP-HPLC). The sugar nucleotide produced from GDP-alpha-d-mannose by these enzymes was purified by RP-HPLC and identified by electrospray ionization-MS, 1H nuclear magnetic resonance, and GC/MS. The results indicated that GDP-6-deoxy-d-talose is produced from GDP-alpha-d-mannose. This paper is the first report on the GDP-6-deoxy-d-talose biosynthetic pathway and the role of GDP-4-keto-6-deoxy-d-mannose reductase in the synthesis of GDP-6-deoxy-d-talose.     

Genetic analysis of the gene cluster responsible for synthesis of serotype e-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans

A gene cluster associated with the biosynthesis of the serotype e-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans IDH1705 belonging to serotype e was cloned and sequenced. This cluster consisted of 18 open reading frames. Escherichia coli produced the polysaccharide that reacts with the serotype e-specific antiserum when transformed with a plasmid containing the cluster. Comparing the structure of the gene cluster with similar clusters from A. actinomycetemcomitans strains Y4 (serotype b) and NCTC9710 (serotype c) revealed that a 5.3-kb region containing the distal half of one gene and two entire genes in the cluster from strain IDH1705 replaced a 6.2-kb region containing eight genes in the cluster from strain Y4, and a 4.7-kb region containing four genes in the cluster from strain NCTC9710. These results suggest that this region is essential to the antigenic specificity of serotype e A. actinomycetemcomitans.     

A novel mechanism for glucose side-chain formation in rhamnose-glucose polysaccharide synthesis

We have cloned two genes (rgpH and rgpI) that encode proteins for the formation of the glucose side-chains of the Streptococcus mutans rhamnose-glucose polysaccharide (RGP), which consists of a rhamnan backbone with glucose side-chains. The roles of rgpH and rgpI were evaluated in a rhamnan-synthesizing Escherichia coli. An E. coli strain that harbored rgpH reacted with antiserum directed against complete RGP, whereas the E. coli strain that carried rgpI did not react with this antiserum. Although E. coli:rgpH reacted strongly with rhamnan-specific antiserum, co-transformation of this strain with rgpI increased the number of glucose side-chains and decreased immunoreactivity with the rhamnan-specific antiserum significantly. These results suggest that two genes are involved in side-chain formation during S. mutans RGP synthesis in E. coli: one gene encodes a glucosyltransferase, and the other gene probably controls the frequency of branching. This is the first report to identify a gene that is involved in regulation of branching frequency in polysaccharide synthesis.     

A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose.

The serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) consists of D-fucose and L-rhamnose. Thymidine diphosphate (dTDP)-D-fucose is the activated nucleotide sugar form of D-fucose, which has been identified as a constituent of structural polysaccharides in only a few bacteria. In this paper, we show that three dTDP-D-fucose synthetic enzymes are encoded by genes in the gene cluster responsible for the synthesis of serotype b-specific polysaccharide in A. actinomycetemcomitans. The first and second steps of the dTDP-D-fucose synthetic pathway are catalyzed by D-glucose-1-phosphate thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase, which are encoded by rmlA and rmlB in the gene cluster, respectively. These two reactions are common to the well studied dTDP-L-rhamnose synthetic pathway. However, the enzyme catalyzing the last step of the dTDP-D-fucose synthetic pathway has never been reported. We identified the fcd gene encoding a dTDP-4-keto-6-deoxy-D-glucose reductase. After purifying the three enzymes, their enzymatic activities were analyzed by reversed-phase high performance liquid chromatography. In addition, nuclear magnetic resonance analysis and gas-liquid chromatography analysis proved that the fcd gene product converts dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose. Moreover, kinetic analysis of the enzyme indicated that the Km values for dTDP-4-keto-6-deoxy-D-glucose and NADPH are 97.3 and 28.7 microM, respectively, and that the enzyme follows the sequential mechanism. This paper is the first report on the dTDP-D-fucose synthetic pathway and dTDP-4-keto-6-deoxy-D-glucose reductase.     

Linkage of genes essential for synthesis of a polysaccharide capsule in Sphingomonas strain S88.

Several structurally related capsular polysaccharides that are secreted by members of the genus Sphingomonas are being developed as aqueous rheological control agents for diverse industrial and food applications. They include gellan (S-60), welan (S-130), rhamsan (S-194), S-657, S-88, S-198, S-7, and NW-11. We refer to these polysaccharides as sphingans, after the genus name. This paper characterizes the first gene cluster isolated from a Sphingomonas species (S88) that is required for capsule synthesis. Overlapping DNA segments which spanned about 50 kbp of S88 DNA restored the synthesis of sphingan S-88 in capsule-negative mutants. The mutations were mapped into functional complementation groups, and the contiguous nucleotide sequence for the 29-kbp cluster was determined. The genetic complementation map and the DNA sequences were interpreted as an extended multicistronic locus containing genes essential for the assembly and secretion of polysaccharide S-88. Many of the deduced amino acid sequences were similar to gene products from other polysaccharide-secreting bacteria such as Rhizobium meliloti (succinoglycan), Xanthomonas campestris (xanthan gum), and Salmonella enterica (O antigen). The S88 locus contained a four-gene operon for the biosynthesis of dTDP-L-rhamnose, an essential precursor for the sphingans. Unexpectedly, there were also two genes for secretion of a lytic or toxin-like protein nested within the polysaccharide cluster. The conservation and linkage of genes that code for a defensive capsule and genes for secretion of an offensive lysin or toxin suggest a heretofore unknown pathogenic life history for Sphingomonas strain S88.     

Molecular analysis of the rfb O antigen gene cluster of Salmonella enterica serogroup O:6,14 and development of a serogroup-specific PCR assay

The Kauffmann-White scheme for serotyping Salmonella recognizes 46 somatic (O) antigen groups, which together with detection of the flagellar (H) antigens form the basis for serotype identification. Although serotyping has become an invaluable typing method for epidemiological investigations of Salmonella, it does have some practical limitations. We have been characterizing the genes required for O and H antigen biosynthesis with the goal of developing a DNA-based system for the determination of serotype in Salmonella. The majority of the enzymes involved in O antigen biosynthesis are encoded by the rfb gene cluster. We report the sequencing of the rfb region from S. enterica serotype Sundsvall (serogroup O:6,14). The S. enterica serotype Sundsvall rfb region is 8.4 kb in length and comprises six open reading frames. When compared with other previously characterized rfb regions, the serogroup O:6,14 sequence is most related to serogroup C(1). On the basis of DNA sequence similarity, we identified two genes from the mannose biosynthetic pathway, two mannosyl transferase genes, the O unit flippase gene and, possibly, the O antigen polymerase. The whole cluster is derived from a low-G+C-content organism. Comparative sequencing of an additional serogroup O:6,14 isolate (S. enterica serotype Carrau) revealed a highly homologous sequence, suggesting that O antigen factors O:24 and O:25 (additional O factors associated with serogroup O:6,14) are encoded outside the rfb gene cluster. We developed a serogroup O:6,14-specific PCR assay based on a region of the putative wzx (O antigen flippase) gene. This provides the basis for a sensitive and specific test for the rapid identification of Salmonella serogroup O:6,14.     

Molecular cloning of the ViaB region of Salmonella typhi

The ViaB region required for Vi antigen production in Salmonella typhi was cloned. The plasmid pGBM124 containing a 14-kb S. typhi chromosomal DNA fragment conferred the ability to produce Vi antigen on Escherichia coli HB101 and ViaB-deleted S. typhi GIFU10007-3. Tn5 insertion analysis showed that the 14-kb DNA was split into three regions. Region 1 and region 2 are involved in the biosynthesis of Vi polysaccharide. Region 3 is involved in translocation of the Vi polysaccharide to the cell surface. Southern blot hybridization showed that regions 2 and 3 but not region 1, were considerably homologous to the DNA of Vi-positive Citrobacter freundii.     

DNA homology of surface protein antigen A gene in mutans streptococci

1. A recombinant plasmid, pYA724, containing an 8.45 kb DNA fragment encoding surface protein antigen A (spaA) from Streptococcus sobrinus 6715 was used to examine the DNA homology of the spaA gene with chromosomal DNA of various mutans streptococci strains. 2. Restriction endonuclease BamHI-digested pYA724 DNA was radio-labeled by nick-translation, and a DNA-DNA hybridization experiment was carried out. pYA724 DNA hybridized with chromosomal DNA of serotypes a, c, d, e, f and g strains, but not with b by dot DNA-hybridization and Southern blot DNA hybridization. 3. Chromosomal DNAs were isolated from several serotype c Streptococcus mutans strains, digested with BamHI, and analyzed by Southern blot DNA hybridization. pYA724 DNA hybridized with different sizes and numbers of BamHI-digested DNA fragments of the chromosomal DNAs. 4. These data indicated that all mutans streptococci strains except serotype b have DNA homologous with the spaA gene, although within the same serotype strain the spaA gene has a diversity of arrangement within the chromosome.     

Molecular cloning and characterization of the spaB gene of Streptococcus sobrinus

A gene of Streptococcus sobrinus 6715 (serotype g) designated spaB and encoding a surface protein antigen was isolated from a cosmid gene bank. A 5.4 kb HindIII/AvaI DNA fragment containing the gene was inserted into plasmid pBR322 to yield plasmid pXI404. Analysis of plasmid-encoded gene products showed that the 5.4 kb fragment of pXI404 encoded a 195 kDa protein. Southern blot experiments revealed that the 5.4 kb chromosomal insert DNA had sequence similarity with genomic DNA of S. sobrinus 6715, S. sobrinus B13 (serotype d) and Streptococcus cricetus HS6 (serotype a). The recombinant SpaB protein (rSpaB) was purified and monospecific antiserum was prepared. With immunological techniques and the anti-rSpaB serum, we have shown: (1) that the rSpaB protein has physico-chemical and antigenic identity with the S. sobrinus SpaB protein, (2) the presence of cross-reactive proteins in the extracellular protein of serotypes a and d of the mutans group of streptococci and (3) that the SpaB protein is expressed on the surface of mutans streptococcal serotypes a, d and g.     

Gene products required for surface expression of the capsular form of the group 1 K antigen in Escherichia coli (O9a:K30)

The group 1 K30 antigen from Escherichia coli (O9a:K30) is present on the cell surface as both a capsular structure composed of high-molecular-weight K30 polysaccharide and as short K30 oligosaccharides linked to lipid A-core in a lipopolysaccharide molecule (K30LPS). To determine the molecular processes that are responsible for the two forms of K antigen, the 16 kb chromosomal cps region has been characterized. This region encodes 12 gene products required for the synthesis, polymerization and translocation of the K30 antigen. The gene products include four glycosyltransferases responsible for synthesis of the K30 repeat unit; a PST (1) exporter (Wzx), required to transfer lipid-linked K30 units across the plasma membrane to the periplasmic space; and a K30-antigen polymerase (Wzy). These gene products are typical of those seen in O-antigen biosynthesis gene clusters and they interact with the lipopolysaccharide translocation pathway to express K30LPS on the cell surface. The same gene products also provide the biosynthetic intermediates for the capsule assembly pathway, although they are not in themselves sufficient for synthesis of the K30 capsule. Three additional genes, wza, wzb and wzc, encode homologues to proteins that are encoded by gene clusters involved in expression of a variety of bacterial exopolysaccharides. Mutant analysis indicates that Wza and Wzc are required for wild-type surface expression of the capsular structure but are not essential for polymerization and play no role in the translocation of K30LPS. These surface expression components provide the key feature that distinguishes the assembly systems for O antigens and capsules.     

Homology between surface protein antigen genes of Streptococcus sobrinus and Streptococcus mutans

The structural gene (pag gene) for a 210 kDa protein antigen of Streptococcus sobrinus serotype g was cloned and compared with that (pac gene) of a 190 kDa protein antigen of Streptococcus mutans serotype c. Immunodiffusion analysis revealed that the product of the pag gene immunologically cross-reacted with that of the pac gene. Southern blot and nucleotide sequence analyses revealed that a significant homology existed between the middle regions of the two structural genes.     

Three highly conserved proteins catalyze the conversion of UDP-N-acetyl-D-glucosamine to precursors for the biosynthesis of O antigen in Pseudomonas aeruginosa O11 and capsule in Staphylococcus aureus type 5. Implications for the UDP-N-acetyl-L-fucosamine biosynthetic pathway

N-Acetyl-l-fucosamine is a constituent of surface polysaccharide structures of Pseudomonas aeruginosa and Staphylococcus aureus. The three P. aeruginosa enzymes WbjB, WbjC, and WbjD, as well as the S. aureus homologs Cap5E, Cap5F, and Cap5G, involved in the biosynthesis of N-acetyl-l-fucosamine have been overexpressed and purified to near homogeneity. Capillary electrophoresis (CE), mass spectroscopy (MS), and nuclear magnetic resonance spectroscopy have been used to elucidate the biosynthesis pathway, which proceeds in five reaction steps. WbjB/Cap5E catalyzed 4,6-dehydration of UDP-N-acetyl-d-glucosamine and 3- and 5-epimerization to yield a mixture of three keto-deoxy-sugars. The third intermediate compound was subsequently reduced at C-4 to UDP-2-acetamido-2,6-dideoxy-l-talose by WbjC/Cap5F. Incubation of UDP-2-acetamido-2,6-dideoxy-l-talose (UDP-TalNAc) with WbjD/Cap5G resulted in a new peak separable by CE that demonstrated identical mass and fragmentation patterns by CE-MS/MS to UDP-TalNAc. These results are consistent with WbjD/Cap5G-mediated 2-epimerization of UDP-TalNAc to UDP-FucNAc. A nonpolar gene knockout of wbjB, the first of the genes associated with this pathway, was constructed in P. aeruginosa serotype O11 strain PA103. The corresponding mutant produced rough lipopolysaccharide devoid of B-band O antigen. This lipopolysaccharide deficiency could be complemented with P. aeruginosa wbjB or with the S. aureus homolog cap5E. Insertional inactivation of either the cap5G or cap5F genes abolished capsule polysaccharide production in the S. aureus strain Newman. Providing the appropriate gene in trans, thereby complementing these mutants, fully restored the capsular polysaccharide phenotype.