88 research outputs found
Safety in numbers: multiple occurrences of highly similar homologs among Azotobacter vinelandii carbohydrate metabolism proteins probably confer adaptive benefits
Hexaene Derivatives of Nystatin Produced as a Result of an Induced Rearrangement within the nysC Polyketide Synthase Gene in S. noursei ATCC 11455
AbstractGenetic manipulation of the polyketide synthase (PKS) gene nysC involved in the biosynthesis of the tetraene antifungal antibiotic nystatin yielded a recombinant strain producing hexaene nystatin derivatives. Analysis of one such compound, S48HX, by LC-MS/MS suggested that it comprises a 36-membered macrolactone ring completely decorated by the post-PKS modification enzymes. Further characterization by bioassay has shown that S48HX exhibits antifungal activity. Genetic analysis of the hexaene-producing mutant revealed an in-frame deletion within the nysC gene via recombination between two homologous ketoreductase domain-encoding sequences. Apparently, this event resulted in the elimination of one complete module from NysC PKS, subsequently leading to the production of the nystatin derivative with a contracted macrolactone ring. These results represent the first example of manipulation of a PKS gene for the biosynthesis of a polyene antibiotic
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
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
Meeting report : 1st international functional metagenomics workshop May 7–8, 2012, St. Jacobs, Ontario, Canada
This report summarizes the events of the 1st International Functional Metagenomics Workshop. The workshop was held on May 7 and 8 in St. Jacobs, Ontario, Canada and was focused on building a core international functional metagenomics community, exploring strategic research areas, and identifying opportunities for future collaboration and funding. The workshop was initiated by researchers at the University of Waterloo with support from the Ontario Genomics Institute (OGI), Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Waterloo
1H, 13C and 15N resonances of the AlgE62 subunit from Azotobacter vinelandii mannuronan C5-epimerase
Special Issue: Recombinant Protein Expression in Microorganisms
Microorganisms are widely used in industrial biotechnology as cell factories for the sustainable production of a wide range of compounds and chemicals [...
A new and improved host-independent plasmid system for RK2-based conjugal transfer
-Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4 origin of transfer (oriT) are mostly mobilized using the E. coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. We have observed that large plasmids were occasionally modified after conjugal transfer when using E. coli S17-1 as a donor. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system
Regulation of the expression level of transcription factor XylS reveals new functional insight into its induction mechanism at the Pm promoter
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