Iron is an essential micronutrient that is required by some microorganisms in relatively large quantities. This is problematic for those inhabiting marine environments, where iron is highly insoluble and the dissolved fraction is predominantly strongly bound to organic compounds. Due to low supply and high demand, iron limits primary productivity in many oceanic waters, and may also limit growth of organisms in coastal waters under some circumstances. Recent incidents of explosive growth (???blooms???) of the noxious filamentous cyanobacterium Lyngbya majuscula in the coastal marine waters of Moreton Bay, Queensland, have prompted speculation that terrestrial human activities have increased iron availability to the organism, thus overcoming previous limitations on growth imposed by scarcity of the nutrient. This thesis describes work investigating the chemical form of iron in coastal waters under various environmental conditions and the way in which this influences its availability to L. majuscula. Chemical speciation of iron was investigated as a function of terrestrial-derived inputs of natural organic matter (NOM) of variable origin and sunlight in coastal marine waters, employing chemiluminescence-based and spectrophotometric techniques with high sensitivity and temporal resolution. These techniques allowed determination of iron and other chemical parameters at naturally occurring (typically nanomolar) concentrations. The mechanism of iron acquisition by L. majuscula was also investigated using a radioisotope-labelling labelling technique in addition to the other techniques described. Results indicated that iron speciation can be described by five classes: inorganic dissolved and organically complexed dissolved iron in both ferrous (reduced) and ferric (oxidised) forms, and precipitated inorganic iron. Simulation of laboratory results by numerical kinetic modelling of the processes investigated indicated that while the thermodynamic impetus is strongly towards precipitated iron, iron complexation by NOM and its reduction by sunlight-mediated processes and/or L. majuscula results in meta-stable dissolved species that are more readily available to L. majuscula. Superoxide is a critical intermediate in iron reduction by both sunlight and L. majuscula. Thus L. majuscula is capable of altering iron speciation to increase its availability, however uptake is also strongly dependent on environmental conditions and may be enhanced by increased inputs of iron, NOM and sunlight into coastal waters.

Cyanobacteria produce a myriad of structurally unique secondary metabolites with useful bioactive properties. Heterologous expression of a variety of microbial natural compounds has been used to harness their diversity and facilitate their combinatorial biosynthesis. However, these genetic techniques have not been developed for secondary metabolite-producing cyanobacteria. Therefore the genetically manipulable Escherichia coli and Synechocystis sp. PCC6803 were engineered in order to develop effective heterologous hosts and promoters for the expression of cyanobacterial-derived compounds. The phosphopanthetheinyl transferase (PPT), Sppt, from Synechocystis sp. PCC6803 was characterised to determine its ability to activate carrier proteins from secondary metabolite pathways. Despite in silico evidence which suggested Sppt was able to activate a wide range of carrier proteins, biochemical analysis revealed that it is dedicated for fatty acid synthesis. Consequently, E. coli and Synechocystis sp. PCC6803 were engineered to encode a broad-range PPT, from the filamentous cyanobacteria Nodularia spumigena NSOR10, for the activation of carrier proteins from nonribosomal peptide synthesis. Cyanobacterial natural product engineering was also explored with the characterisation of two relaxed specificity adenylation domains (A-domains) from the biosynthetic pathway of the toxin microcystin. The wide variety of microcystin compounds produced by cyanobacterial species suggests that multiple amino acids can be activated by the same A-domain. This was supported by preliminary ATP-[32P]PPi exchange assays and was subsequently harnessed in the production of a variety of dipeptides using two reconstituted modules in vitro. Transposition was investigated as a potential mechanism for the transfer of nonribosomal peptide synthetase gene clusters to heterologous hosts. This was performed via the characterisation of the putative transposase, Mat, physically linked with the microcystin synthetase gene cluster (mcyS). PCR screening, in silico analysis and nitrocellulose filter binding assays indicated that this transposase may have mediated mcyS gene cluster rearrangements but not entire gene cluster mobilisation between species. The potential role of transposases in the natural combinatorial biosynthesis of microcystin has evolutionary implications for the dynamic nature of cyanobacterial genomes and applications for use in the engineering of novel bioactive compounds. Therefore, the results from this study may provide a biotechnological platform for the transfer, expression and combinatorial biosynthesis of novel cyanobacterial-derived natural products.

Cyanobacteria produce a myriad of structurally unique secondary metabolites with useful bioactive properties. Heterologous expression of a variety of microbial natural compounds has been used to harness their diversity and facilitate their combinatorial biosynthesis. However, these genetic techniques have not been developed for secondary metabolite-producing cyanobacteria. Therefore the genetically manipulable Escherichia coli and Synechocystis sp. PCC6803 were engineered in order to develop effective heterologous hosts and promoters for the expression of cyanobacterial-derived compounds. The phosphopanthetheinyl transferase (PPT), Sppt, from Synechocystis sp. PCC6803 was characterised to determine its ability to activate carrier proteins from secondary metabolite pathways. Despite in silico evidence which suggested Sppt was able to activate a wide range of carrier proteins, biochemical analysis revealed that it is dedicated for fatty acid synthesis. Consequently, E. coli and Synechocystis sp. PCC6803 were engineered to encode a broad-range PPT, from the filamentous cyanobacteria Nodularia spumigena NSOR10, for the activation of carrier proteins from nonribosomal peptide synthesis. Cyanobacterial natural product engineering was also explored with the characterisation of two relaxed specificity adenylation domains (A-domains) from the biosynthetic pathway of the toxin microcystin. The wide variety of microcystin compounds produced by cyanobacterial species suggests that multiple amino acids can be activated by the same A-domain. This was supported by preliminary ATP-[32P]PPi exchange assays and was subsequently harnessed in the production of a variety of dipeptides using two reconstituted modules in vitro. Transposition was investigated as a potential mechanism for the transfer of nonribosomal peptide synthetase gene clusters to heterologous hosts. This was performed via the characterisation of the putative transposase, Mat, physically linked with the microcystin synthetase gene cluster (mcyS). PCR screening, in silico analysis and nitrocellulose filter binding assays indicated that this transposase may have mediated mcyS gene cluster rearrangements but not entire gene cluster mobilisation between species. The potential role of transposases in the natural combinatorial biosynthesis of microcystin has evolutionary implications for the dynamic nature of cyanobacterial genomes and applications for use in the engineering of novel bioactive compounds. Therefore, the results from this study may provide a biotechnological platform for the transfer, expression and combinatorial biosynthesis of novel cyanobacterial-derived natural products.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (M.S.)--Montana State University--Bozeman, 2004. / Typescript. Chairperson, Graduate Committee: David M. Ward. Includes bibliographical references (leaves 60-65).

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of New South Wales, 2005. / Also available online.

Thesis (M.S.)--Georgia State University, 2008. / Title from file title page. Zhi-Ren Liu, committee chair; Delon W. Barfuss, Jenny J. Yang, committee members. Electronic text (69 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed June 11, 2008. Includes bibliographical references (p. 61-67).

Thesis supervisor: Debashish Bhattacharya. Includes bibliographic references (p. 126-142).

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 196-212). Also available in electronic version. Access restricted to campus users.