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

Non-homologous end joining as an important mutagenic process in cell cycle-arrested cells

Resting cells experience mutations without apparent external mutagenic influences. Such DNA replication-independent mutations are suspected to be a consequence of processing of spontaneous DNA lesions. Using experimental systems based on reversions of frameshift alleles in Saccharomyces cerevisiae, we evaluated the impact of defects in DNA double-strand break (DSB) repair on the frequency of replication-independent mutations. The deletion of the genes coding for Ku70 or DNA ligase IV, which are both obligatory constituents of the non-homologous end joining (NHEJ) pathway, each resulted in a 50% reduction of replication-independent mutation frequency in haploid cells. Sequencing indicated that typical NHEJ-dependent reversion events are small deletions within mononucleotide repeats, with a remarkable resemblance to DNA polymerase slippage errors. Experiments with diploid and RAD52- or RAD54-deficient strains confirmed that among DSB repair pathways only NHEJ accounts for a considerable fraction of replication-independent frameshift mutations in haploid and diploid NHEJ non-repressed cells. Thus our results provide evidence that G(0) cells with unrepressed NHEJ capacity pay for a large-scale chromosomal stability with an increased frequency of small-scale mutations, a finding of potential relevance for carcinogenesis

Biosynthesis of 2-acetamido-2,6-dideoxy-L-hexoses in bacteria follows a pattern distinct from those of the pathways of 6-deoxy-L-hexoses

6-Deoxy-L-hexoses have been shown to be synthesized from dTDP-D-glucose or GDP-D-mannose so that the gluco/galacto-configuration is converted into the manno/talo-configuration, and manno/talo is switched to gluco/galacto. Our laboratory has been investigating the biosynthesis of 2-acetamido-2,6-dideoxy-L-hexoses in both Gram-positive and Gram-negative bacteria, and in a recent paper we described the biosynthesis of the talo (pneumosamine) and galacto (fucosamine) derivatives from UDP-D-N-acetylglucosamine a 2-acetamido sugar [Kneidinger, O'Riordan, Li, Brisson, Lee and Lam (2003) J. Biol. Chem. 278, 3615-3627]. In the present study, we undertake the task to test the hypothesis that UDP-D-N-acetylglucosamine is the common precursor for the production of 2-acetamido-2,6-dideoxy-L-hexoses in the gluco-, galacto-, manno- and talo-configurations. We present data to reveal the steps for the biosynthesis of the gluco (quinovosamine)- and manno (rhamnosamine)-configured compounds. The corresponding enzymes WbvB, WbvR and WbvD from Vibrio cholerae serotype O37 have been overexpressed and purified to near homogeneity. The enzymic reactions have been analysed by capillary electrophoresis and NMR spectroscopy. Our data have revealed a general feature of reaction cascades due to the three enzymes. First, UDP-D-N-acetylglucosamine is catalysed by the multi-functional enzyme WbvB, whereby dehydration occurs at C-4, C-6 and epimerization at C-5, C-3 to produce UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose. Secondly, this intermediate is converted by the C-4 reductase, WbvR, in a stereospecific reaction to yield UDP-2-acetamido-L-rhamnose. Thirdly, UDP-2-acetamido-L-rhamnose is epimerized at C-2 to UDP-2-acetamido-L-quinovose by WbvD. Interestingly, WbvD is also an orthologue of WbjD, but not vice versa. Incubation of purified WbvD with UDP-2-acetamido-2,6-dideoxy-L-talose and analysing the reaction products by capillary electrophoresis revealed the same product peak as when WbjD was used. This sugar nucleotide is a specific substrate for WbjD and is a C-4 epimer of UDP-2-acetamido-L-rhamnose.NRC publication: Ye

Biosynthesis Pathway of ADP-l-glycero-β-d-manno-Heptose in Escherichia coli

The steps involved in the biosynthesis of the ADP-l-glycero-β-d-manno-heptose (ADP-l-β-d-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-d-β-d-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-d-β-d-heptose. Our results demonstrate that the synthesis of ADP-d-β-d-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional d-β-d-heptose 7-phosphate kinase/d-β-d-heptose 1-phosphate adenylyltransferase), and GmhB (d,d-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-l-β-d-heptose precursor utilized in the assembly of inner core LPS