lfnA from Pseudomonas aeruginosa O12 and wbuX from Escherichia coli O145 encode membrane-associated proteins and are required for expression of 2,6-dideoxy-2-acetamidino-L-galactose in lipopolysaccharide O antigen.

The rare sugar 2,6-dideoxy-2-acetamidino-L-galactose (L-FucNAm) is found only in bacteria and is a component of cell surface glycans in a number of pathogenic species, including the O antigens of Pseudomonas aeruginosa serotype O12 and Escherichia coli O145. P. aeruginosa is an important opportunistic pathogen, and the O12 serotype is associated with multidrug-resistant epidemic outbreaks. O145 is one of the classic non-O157 serotypes associated with Shiga toxin-producing, enterohemorrhagic E. coli. The acetamidino (NAm) moiety of L-FucNAm is of interest, because at neutral pH it contributes a positive charge to the cell surface, and we aimed to characterize the biosynthesis of this functional group. The pathway is not known, but expression of NAm-modified sugars coincides with the presence of a pseA homologue in the relevant biosynthetic locus. PseA is a putative amidotransferase required for synthesis of a NAm-modified sugar in Campylobacter jejuni. In P. aeruginosa O12 and E. coli O145, the pseA homologues are lfnA and wbuX, respectively, and we hypothesized that these genes function in L-FucNAm biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and nuclear magnetic resonance analysis of the lfnA mutant O-antigen structure indicated that the mutant expresses 2,6-dideoxy-2-acetamido-L-galactose (L-FucNAc) in place of L-FucNAm. The mutation could be complemented by expression of either His(6)-tagged lfnA or wbuX in trans, confirming that these genes are functional homologues and that they are required for NAm moiety synthesis. Both proteins retained their activity when fused to a His(6) tag and localized to the membrane fraction. These data will assist future biochemical investigation of this pathway.

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.

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.

Homologs of the Rml enzymes from Salmonella enterica are responsible for dTDP-beta-L-rhamnose biosynthesis in the gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155.

The glycan chains of the surface layer (S-layer) glycoprotein from the gram-positive, thermophilic bacterium Aneurinibacillus (formerly Bacillus) thermoaerophilus strain DSM 10155 are composed of L-rhamnose- and D-glycero-D-manno-heptose-containing disaccharide repeating units which are linked to the S-layer polypeptide via core structures that have variable lengths and novel O-glycosidic linkages. In this work we investigated the enzymes involved in the biosynthesis of thymidine diphospho-L-rhamnose (dTDP-L-rhamnose) and their specific properties. Comparable to lipopolysaccharide O-antigen biosynthesis in gram-negative bacteria, dTDP-L-rhamnose is synthesized in a four-step reaction sequence from dTTP and glucose 1-phosphate by the enzymes glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC), and dTDP-4-dehydrorhamnose reductase (RmlD). The rhamnose biosynthesis operon from A. thermoaerophilus DSM 10155 was sequenced, and the genes were overexpressed in Escherichia coli. Compared to purified enterobacterial Rml enzymes, the enzymes from the gram-positive strain show remarkably increased thermostability, a property which is particularly interesting for high-throughput screening and enzymatic synthesis. The closely related strain A. thermoaerophilus L420-91(T) produces D-rhamnose- and 3-acetamido-3,6-dideoxy-D-galactose-containing S-layer glycan chains. Comparison of the enzyme activity patterns in A. thermoaerophilus strains DSM 10155 and L420-91(T) for L-rhamnose and D-rhamnose biosynthesis indicated that the enzymes are differentially expressed during S-layer glycan biosynthesis and that A. thermoaerophilus L420-91(T) is not able to synthesize dTDP-L-rhamnose. These findings confirm that in each strain the enzymes act specifically on S-layer glycoprotein glycan formation.

Biosynthesis pathway of ADP-L-glycero-beta-D-manno-heptose in Escherichia coli.

The steps involved in the biosynthesis of the ADP-L-glycero-beta-D-manno-heptose (ADP-L-beta-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-beta-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-beta-D-heptose. Our results demonstrate that the synthesis of ADP-D-beta-D-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional D-beta-D-heptose 7-phosphate kinase/D-beta-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-beta-D-heptose precursor utilized in the assembly of inner core LPS.