Identification and regulation of novel compatible solutes from hypersaline stromatolite-associated cyanobacteria.

Cyanobacteria are able to survive in various extreme environments via the production of organic compounds known as compatible solutes. In particular, cyanobacteria are capable of inhabiting hypersaline environments such as those found in intertidal regions. Cyanobacteria in these environments must possess regulatory mechanisms for surviving the changing osmotic pressure as a result of desiccation, rainfall and tidal fluxes. The objective of this study was to determine the compatible solutes that are accumulated by cyanobacteria from hypersaline regions, and specifically, the stromatolite ecosystems of Shark Bay, Western Australia. Previously, the cyanobacterial populations associated with these stromatolites were characterized in two separate studies. Compatible solutes were extracted from isolated cyanobacteria here and identified by nuclear magnetic resonance. As the media of isolation contained no complex carbon source, the solutes accumulated were likely synthesized by the cyanobacteria. The data indicate that from this one habitat taxonomically distinct cyanobacteria exposed to varying salinities accumulate a range of known compatible solutes. In addition, taxonomically similar cyanobacteria do not necessarily accumulate the same compatible solutes. Glucosylglycerol, a compatible solute unique to marine cyanobacteria was not detected; however, various saccharides, glycine betaine, and trimethylamine-N-oxide were identified as the predominant solutes. We conclude that the cyanobacterial communities from these hypersaline stromatolites are likely to possess more complex mechanisms of adaptation to osmotic stress than previously thought. The characterization of osmoregulatory properties of stromatolite microorganisms provides further insight into how life can thrive in such extreme environments.

A novel prokaryotic L-arginine:glycine amidinotransferase is involved in cylindrospermopsin biosynthesis.

We report the first characterization of an L-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 human L-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 an L-arginine:glycine amidinotransferase by (1) H 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 (T(max) 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 eukaryotic L-arginine:glycine amidinotransferases.

Community structure and antibiotic production of Streptomyces nodosus bioreactors cultured in liquid environments.

Immobilized bacteria are being assessed by industry for drug delivery, novel fermentation systems and the protection of organisms in harsh environments. Alginate bioreactors containing Streptomyces nodosus were examined for community structure, cell viability and amphotericin production under different growth conditions. When cell proliferation was encouraged, substrate hyphae were found inside the alginate matrix and within multicellular projections on the surface of the capsule. The periphery of these projections had erect and branched hyphae, morphologically identical to aerial hyphae. Antibiotic production from immobilized organisms was assessed using conditioned culture medium to eliminate the emergence of a free-dwelling population. These organisms sporulated with reduced antibiotic production compared with free-dwelling cultures. The commitment to sporulate was independent of a surface but dependent on community size and nutritional status. This is the first report of the sporulation of S. nodosus in liquid cultures and description of the multicellular community the organism adopts at a solid-liquid interface.

Characterization of the gene cluster responsible for cylindrospermopsin biosynthesis.

Toxic cyanobacterial blooms cause economic losses and pose significant public health threats on a global scale. Characterization of the gene cluster for the biosynthesis of the cyanobacterial toxin cylindrospermopsin (cyr) in Cylindrospermopsis raciborskii AWT205 is described, and the complete biosynthetic pathway is proposed. The cyr gene cluster spans 43 kb and is comprised of 15 open reading frames containing genes required for the biosynthesis, regulation, and export of the toxin. Biosynthesis is initiated via an amidinotransfer onto glycine followed by five polyketide extensions and subsequent reductions, and rings are formed via Michael additions in a stepwise manner. The uracil ring is formed by a novel pyrimidine biosynthesis mechanism and tailoring reactions, including sulfation and hydroxylation that complete biosynthesis. These findings enable the design of toxic strain-specific probes and allow the future study of the regulation and biological role of cylindrospermopsin.

Characterization of the 2-hydroxy-acid dehydrogenase McyI, encoded within the microcystin biosynthesis gene cluster of Microcystis aeruginosa PCC7806.

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thio-template 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 alpha-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.

Amplification of DNA encoding entire type I polyketide synthase domains and linkers from streptomyces species.

Polyketides are a group of bioactive compounds from bacteria, plants, and fungi. To increase the availability of analogs for testing, the active sites of polyketide synthases are often substituted with homologous domains having altered substrate specificities. This study reports the design of polymerase chain reaction primers that enables isolation of entire active site domains from type I polyketide synthases with native interdomain linkers. This bypasses the need for further genetic screening to obtain functional units for use in genetic engineering. This is especially important in bioprospecting projects exploring new environments for bioresources.

Structural characterization of organic multilayers on silicon(111) formed by immobilization of molecular films on functionalized Si-C linked monolayers.

Silicon(111)-H surfaces were derivatized with omega-functionalized alkenes in UV-mediated and thermal hydrosilylation reactions to give Si-C linked monolayers. Additional molecular layers of organic compounds were coupled either directly or via linker molecules to the functionalized alkyl monolayers. In the first instance, amino-terminated monolayers were prepared from a tert-butoxycarbonyl-protected omega-aminoalkene followed by removal of the protecting group. Various thiols were coupled to the monolayer using a heterobifunctional linker, which introduced maleimide groups onto the surface. In the second system, N-hydroxysuccinimide (NHS) ester-terminated monolayers were formed by reaction of Si-H with N-succinimidyl undecenoate. The reactivity of the NHS ester groups was confirmed by further modification of the monolayer. The stepwise assembly of these multilayer structures was characterized by X-ray reflectometry and X-ray photoelectron spectroscopy.

High frequency transformation of the Amphotericin-producing bacterium Streptomyces nodosus.

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 x 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.