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Energy restrictions appear to limit the growth of recombinant Zymomonas mobilis on xylose media. This yeast has been genetically engineered to convert xylose to ethanol.

The lipid portion of the photosynthetic non-sulphur bacterium Rhodospirillum rubrum generates a tricyclic diterpene, isogathenediol. This diterpene acts as a membrane fluidity modulator. The nuclear magnetic resonance spectrum and mass spectroscopy, used to determine the structure of isogathenediol, reveal the existence of six quaternary methyl groups, two hydroxyl groups and two methylene protons.

The gene cluster is characterized for the biosynthesis of the cyanobacterial toxin cylindrospermopsin (cyr) in Cylindrospermopsis raciborskii AWT205 and the complete biosynthetic pathway is described. The results have helped in the design of toxic strain-specific probes and also for the study of the regulation and biological role of cylindrospermopsin.

We report the first characterization of anl-arginine:glycine amidinotransferase from a prokaryote. The enzyme, CyrA, is involved in the pathway for biosynthesis of the polyketide-derived hepatotoxin cylindrospermopsin from Cylindrospermopsis raciborskii AWT205. CyrA is phylogenetically distinct from other amidinotransferases, and structural alignment shows differences between the active site residues of CyrA and the well-characterized humanl-arginine:glycine amidinotransferase (AGAT). Overexpression of recombinant CyrA in Escherichia coli enabled biochemical characterization of the enzyme, and we confirmed the predicted function of CyrA as anl-arginine:glycine amidinotransferase by 1H NMR. As compared with AGAT, CyrA showed narrow substrate specificity when presented with substrate analogs, and deviated from regular Michaelis–Menten kinetics in the presence of the non-natural substrate hydroxylamine. Studies of initial reaction velocities and product inhibition, and identification of intermediate reaction products, were used to probe the kinetic mechanism of CyrA, which is best described as a hybrid of ping-pong and sequential mechanisms. Differences in the active site residues of CyrA and AGAT are discussed in relation to the different properties of both enzymes. The enzyme had maximum activity and maximum stability at pH 8.5 and 6.5, respectively, and an optimum temperature of 32 °C. Investigations into the stability of the enzyme revealed that an inactivated form of this enzyme retained an appreciable amount of secondary structure elements even on heating to 94 °C, but lost its tertiary structure at low temperature ( Tmax of 44.5 °C), resulting in a state reminiscent of a molten globule. CyrA represents a novel group of prokaryotic amidinotransferases that utilize arginine and glycine as substrates with a complex kinetic mechanism and substrate specificity that differs from that of the eukaryoticl-arginine:glycine amidinotransferases. [ABSTRACT FROM AUTHOR]

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thiotemplate function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of D-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce α-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role. [ABSTRACT FROM AUTHOR]

This study has investigated DNA transformation in the Amphotericin-producing organism Streptomyces nodosus. Amphotericin B is an antifungal drug with severe side effects in humans and the availability of structural variants would aid investigations into the mode of action and cytotoxity of the drug. Analogs of related polyketide drugs have been rapidly made by genetic engineering of biosynthetic genes; however, this requires the introduction of foreign DNA into the host. Protocols for protoplast formation and regeneration were established; however, preparations were recalcitrant to DNA uptake. Electroporation-mediated methodologies also were not successful. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies of 5×10−5 exconjugants generated per recipient. Use of DNA methylation-impaired E. coli donor strains resulted in 100-fold higher transformation efficiencies, indicating that DNA methylation recognition systems are operable in the organism. This methodology will enable genetic and biochemical analysis of the gene cluster responsible for making Amphotericin B. [Copyright &y& Elsevier]

Examines xylose metabolism by recombinant Zymomonas mobilis ZM4(pZB5) using kinetic and nuclear magnetic resonance spectroscopy. Comparison of fungal production between northern and southern marshes; Sugar uptake and ethanol production of Z. mobilis; Reasons underlying decrease cell and ethanol yields.

In complementary experiments the metabolism of [1-²H]glucose in H2O and of unlabelled glucose in ²H2O by Zymomonas mobilis was examined. The utilization of [1-²H]glucose by Z. mobilis was monitored by high- resolution ²H NMR. The deuterium-labelling pattern and stereochemistry of the ethanols produced from the metabolism of [1-²H]glucose and unlabelled glucose in ²H2O were determined by a combination of 13C and ¹H NMR and selective enzyme action. The labelling patterns were explained in terms of enzyme mechanisms and stereo specificity, and metabolite enolization. [ABSTRACT FROM AUTHOR]

A novel polysaccharide has been observed in vivo by 13C nuclear magnetic resonance (NMR) spectroscopy of the gram-negative bacterium, Zymomonas mobilis. The polysaccharide, which was not removed from the Z. mobilis cell by washing with 0.86% saline, was extracted by mild acid treatment. The structure was elucidated by a combination of NMR techniques, including proton and proton-carbon two-dimensional methods. The structure was determined to be -α-fructofuranosyl-(2-1)-β-fructofuranosyl-(2-6)-. The polysaccharide is unlike levan, a β-(2-6)-fructose polymer which is produced by many bacteria, including Z.mobilis, when grown on sucrose. The polysaccharide isolated here is produced by cells cultured on glucose, fructose or sucrose. [ABSTRACT FROM AUTHOR]

Energy dispersive X-ray microanalysis was used to localize the two brominated natural products (aerothinonin and homoaerothionin) in the tissues of a marine demosponge, Aplysina fistularis. Virtually all of these compounds were localized within the spherules of the spherulous cells in the mesohyl. This is the first localization of any secondary metabolite at the cellular or sub-cellular level in any marine invertebrate. In Aplysina fistularis, as in other species of the same genus studied by Vacelet, the spherulous cells are concentrated just beneath the exopinacoderm and just beneath the endopinacoderm of the excurrent canals. Moreover, there is electron microscopic evidence for degeneration of some spherulous cells throughout the mesohyl. Presumably, this degeneration can release some aerothionin and homoaerothionin, which are known to have antibiotic properties. After release from the spherulous cells, these brominated natural products could function (1) within the mesohyl to exclude some types of bacteria or to aggregate ingested bacteria and/or (2) within the boundary layer of the surrounding seawater for defense or offense, as considered in the discussion section. [ABSTRACT FROM AUTHOR]

31p nuclear magnetic resonance spectra of tissue from the marine bivalve mollusc Tapes watlingi are presented. Spectra from foot muscle, adductor muscle and hearts contain resonances from the adenine nucleotides, inorganic phosphate and a phosphagen which is shown to be arginine phosphate. The intracellular pH of the tissue, measured from the chemical shift of the inorganic phosphate resonance, is 7.2, which is significantly lower than that of sea water, viz. 8.1. The importance of a correct choice of solution conditions for determining a correlation curve of chemical shift versus pH for tissue from marine organisms is emphasized. Spectra of perchloric acid extracts of muscle are used to determine levels of the major phosphorus-containing metabolites in the tissue. The time courses of changes in the phosphorus-containing metabolites of foot and adductor muscle are described. Under anaerobic conditions the sequence of events is similar to that observed in vertebrate skeletal muscle, but the overall rate of decay is much slower, e.g. arginine phosphate is present in foot muscle for almost 20 h at its physiological temperature. Oxygenation of the incubation medium prolongs the survival time of the tissue. Treatment with iodoacetamide and sodium azide under anaerobic conditions reduces the lifetime and changes the pattern of decay such that ATP begins to decay before the phosphagen is consumed, and most of the phosphate accumulates in sugar phosphates, mainly fructose 1,6-biphosphate. The effect of low salinity stress is also examined. Spectra of muscle from animals exposed to 50% and 75% normal sea water for periods of about four weeks are essentially identical with those of normal animals. Furthermore, the decay of intact muscle incubated in 50% sea water is similar to that in normal sea water. [ABSTRACT FROM AUTHOR]