NMR assignments of 1H, 13C and 15N resonances of the C-terminal subunit from Azotobacter vinelandii mannuronan C5-epimerase 6 (AlgE6R3)

The 19.9 kDa C-terminal module (R3) from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been 13C, 15N isotopically labelled and recombinantly expressed. We report here the 1H, 13C, 15N resonance assignment of AlgE6R3

Structural and mutational characterization of the catalytic A-module of the mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii

Alginate is a family of linear copolymers of (1→4)-linked β-d-mannuronic acid and its C-5 epimer α-l-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-Å resolution. AlgE4A folds into a right-handed parallel β-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The β-helix is composed of four parallel β-sheets, comprising 12 complete turns, and has an amphipathic α-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp(152), an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr(149), His(154), and Asp(178) as being essential for activity. Tyr(149) probably acts as the proton acceptor, whereas His(154) is the proton donor in the epimerization reaction

Characterization of the alginate biosynthetic gene cluster in Pseudomonas syringae pv. syringae

Alginate, a copolymer of D-mannuronic acid and L-guluronic acid, is produced by a variety of pseudomonads, including Pseudomonas syringae. Alginate biosynthesis has been most extensively studied in P. aeruginosa, and a number of structural and regulatory genes from this species have been cloned and characterized. In the present study, an alginate-defective (Alg2) mutant of P. syringae pv. syringae FF5 was shown to contain a Tn5 insertion in algL, a gene encoding alginate lyase. A cosmid clone designated pSK2 restored alginate production to the algL mutant and was shown to contain homologs of algD, alg8, alg44, algG, algX (alg60), algL, algF, and algA. The order and arrangement of the structural gene cluster were virtually identical to those previously described for P. aeruginosa. Complementation analyses, however, indicated that the structural gene clusters in P. aeruginosa and P. syringae were not functionally interchangeable when expressed from their native promoters. A region upstream of the algD gene in P. syringae pv. syringae was shown to activate the transcription of a promoterless glucuronidase (uidA) gene and indicated that transcription initiated upstream of algD as described for P. aeruginosa. Transcription of the algD promoter from P. syringae FF5 was significantly higher at 32°C than at 18 or 26°C and was stimulated when copper sulfate or sodium chloride was added to the medium. Alginate gene expression was also stimulated by the addition of the nonionic solute sorbitol, indicating that osmolarity is a signal for algD expression in P. syringae FF5.Peer reviewedPlant Patholog

Rapid reagentless quantification of alginate biosynthesis in Pseudomonas fluorescens bacteria mutants using FT-IR spectroscopy coupled to multivariate partial least squares regression

Alginate is an important medical and commercial product and currently is isolated from seaweeds. Certain microorganisms also produce alginate and these polymers have the potential to replace seaweed alginates in some applications, mainly because such production will allow much better and more reproducible control of critical qualitative polymer properties. The research conducted here presents the development of a new approach to this problem by analysing a transposon insertion mutant library constructed in an alginate-producing derivative of the Pseudomonas fluorescens strain SBW25. The procedure is based on the non-destructive and reagent-free method of Fourier transform infrared (FT-IR) spectroscopy which is used to generate a complex biochemical infrared fingerprint of the medium after bacterial growth. First, we investigate the potential differences caused by the growth media fructose and glycerol on the bacterial phenotype and alginate synthesis in 193 selected P. fluorescens mutants and show that clear phenotypic differences are observed in the infrared fingerprints. In order to quantify the level of the alginate we also report the construction and interpretation of multivariate partial least squares regression models which were able to quantify alginate levels successfully with typical normalized root-mean-square error in predictions of only approximately 14 %. We have demonstrated that this high-throughput approach can be implemented in alginate screens and we believe that this FT-IR spectroscopic methodology, when combined with the most appropriate chemometrics, could easily be modified for the quantification of other valuable microbial products and play a valuable screening role for synthetic biology