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The literature suggests that the cyanobacterium Rivularia is found at sites where organic phosphorus (P), at times forms a high proportion of total P in the environment. Its ability to utilise organic P through "surface" phosphatase activity may be important in its success. The aim of this thesis was to investigate this in detail using both field material (from a freshwater stream and from a marine intertidal zone) and axenic isolates of Rivulariaceae. At both sites inorganic P concentrations peaked: in March/May (1992-4) at the freshwater site, and in June (1992-3) at the marine site (although in 5 of the pools containing Rivularia, inorganic P was maximal in February/March 1993). Pools associated with rotting seaweed had higher concentrations of inorganic P (which made up most of the total P) during peaks. It is likely that the high tide resulted in the mixing of weed pool water with Rivularia pool water, slightly lower down the eulittoral zone, and also influenced the retreating seawater. Organic P was a greater proportion of total P in the pools containing Rivularia and was found to increase in these pools during the tidal cycle, suggesting internal generation. At the freshwater site organic P concentrations were higher in pools associated with peat than in stream water. At the freshwater site phosphorus fractions were often below detection limits, but combined nitrogen was rarely this low; the reverse was the case at the marine site. At the freshwater site phosphomonoesterase activity of Rivularia was generally high, except when hormogonia were present in the colonies. At the marine site, phosphatase activity was usually low, with a peak using p-nitrophenyl phosphate (pNPP) as a substrate in July/August and, using 4-methyIumbelliferyl phosphate (4-MUP) as a substrate in September/October, especially in 1992. Phosphatase activity of Rivularia at both sites was negatively influenced by inorganic P and positively correlated with the presence of hairs. K(_m) (Michaelis-Menten constant) was lower using 4-MUP than pNPP in all organisms assayed. Apparent negative cooperativity was found in 7-day cultures of Calothrix parietina D550 using 4-MUP and in whole colonies of freshwater Rivularia using pNPP. These results were discussed with reference to the relationship between nitrogen (N) and P at the sites and the enzyme kinetics of field organisms and axenic isolates. Rivularia is successful in these apparently different environments probably because organic P can be utilised when phosphorus is limiting and colonies are able to fix N2 when N is limiting. Seasonal peaks in inorganic P probably allow regeneration of the Rivularia population.

The aquatic filamentous bacteria (Cyanobacterium) Lyngbya majuscula is a nitrogen-fixer found in coastal waters often attached or adjacent to sea grass, algae and coral. It is characterized by phycobiliproteins, unique pigments found only in cyanobacteria. To sustain photosynthesis and nitrogen fixation, L. majuscula requires iron proteins and is therefore sensitive to the availability of this metal. The hypothesis tested in this study concerns the potential use of hyperspectral imaging in detecting L. majuscula in coastal regions as biological indicators for the presence of iron debris or metal objects in the littoral environment. This concept would have potential benefits and applications in mine detection and countermeasure techniques. Using a USB2000 field spectroradiometer, a spectral library was developed for the benthic substrates of Midway Atoll, Northwest Hawaiian Islands, spectrally characterizing L. majuscula and the surrounding coral reef substrates. The data was analyzed to determine unique spectral characteristics of the benthic cyanobacteria in a mixed coral environment and evaluated against the resampled spectral resolution of a number of hyperspectral sensors: Airborne Visible/Infrared Imaging Spectrometer (AVIRIS), Hyperspectral Mapper (HyMap) and Compact Airborne Spectrographic Imager (CASI). The results of the in situ spectroscopy suggest a strong potential for all three sensors to detect these cyanobacteria in a mixed coral reef environment at four distinct wavelengths attributable to phycobiliprotein pigment absorptions unique to cyanobacteria. Of these four discriminative absorption ranges, the phycoerythrin absorption of 565-576 nm shows the greatest potential for segregating cyanobacteria from a mixed algal/ coral / sand environment so long as the coral Montipora spp. is not present within the scene, since it has an overlapping absorption in those wavelengths. In the presence of Montipora corals, these cyanobacteria are more difficult to detect. However, in a mixed environment composed of L. majuscula and Montipora corals, the cyanobacteria can be distinguished by a different phycocyanin absorption, at 615-632 nm.

Jagals, P., Prof. du Preez, H.H., Prof.

Decomposing barley straw (Hordeum vulgare) and oak leaves (Quercus robur) have previously been shown to inhibit the growth of a limited number of algae and cyanobacteria. Bioassays were conducted on a range of algae and cyanobacteria to evaluate their relative sensitivities to litter-derived inhibitor(s). A range of sensitivities were found, including some species that were stimulated by litter-derived inhibitor(s). Susceptibility to decomposing plant litter did not appear to be related to general taxonomic or structural features since susceptibility differed widely, even amongst members of the same genus. A microcystin-producing strain of Microcystis aeruginosa was very susceptible to decomposing barley straw. No specific effect on cell structure or morphology could be attributed solely to the litter-derived inhibitor(s). Evidence suggested that cell division, rather than cell expansion, was slowed or inhibited. Bioassays using Euglena gracilis showed the inhibitory compounds were not derived from the phototransformation of litter decomposition products and were not acting primarily by inhibiting photosynthesis. Barley straw inhibited the growth of filamentous algae in a drainage channel and, subsequently, the channel was recolonized by macrophytes. A one-year treatment with barley straw inhibited algal growth, but this reduction in growth was not maintained when the straw was removed. After three-years treatment with barley straw, macrophytes retained dominance after the removal of the straw. No inhibition of algal growth was observed in a different straw-treated drainage channel. The implications for water management are discussed.

Pseudomonas aeruginosa is an opportunistic bacterium that can infect immunocompromised people, and is especially prevalent in patients with cystic fibrosis. Treatment of P. aeruginosa is complicated due to resistance to many classes of antibiotics. This is partly due to biofilm formation, which is not simply a diffusion barrier, but also has a distinct mechanism for resisting the action of antibiotics. P. aeruginosa quinolone signal (PQS) has an important role in quorum sensing, which is involved in biofilm formation. PqsBC is a condensing enzyme in the biosynthesis of the PQS. The crystal structures of PqsBCC129A and PqsBCC129A-Fe3+ were collected to 2.04 Å and to 2.3 Å, respectively. The crystal structure showed that PqsB and PqsC have a pseudo 2-fold symmetry that mimics the FabH homodimer as well as the presence of a catalytic diad instead of the typical catalytic triad in PqsBC, seen in other FabH family enzymes. The PqsCC129A active site volume is twice the volume as those of FabH enzymes or PqsD, with a calculated volume of 761Å3, compared to 389Å3 for PqsD and 367Å3 for FabH.The PqsBCC129A-Fe3+ crystal structure shows that Fe3+ binds to nitrogen atoms from PqsB His282 and PqsC His2 along with oxygen atoms from PqsB Glu48 and Glu280. Therefore, there are four bonds involved in the interaction between Fe3+ and PqsBCC129A. These bond lengths are very similar to those observed in the structures of Azurin and FutA1 complexed with iron. These crystal structures of PqsBC provide a unique insight into substrate recognition and establish a scaffold for structure guided drug design of novel antimicrobial agents.

This thesis details research in three areas, in attempts to produce more effective inhibitors of the staphylococcal agr quorum sensing system. The non-ribosomal peptide synthase biosynthetic cluster responsible for the production of the aureusimines represents roughly 0.5% of the S. aureus genome. However, their function is yet to be elucidated. Research detailed herein develops a new reliable method for synthesis for these natural products. Efforts to discover the biological target or function of these compounds using affinity chromatography is reported. Further biological investigations revealed that the aureusimines are mild antagonists of the agr system. Weak inhibition of CCL-2 mediated chemotaxis of monocytes and staphylococcal biofilm formation is also observed. However, the main biological function of these natural pyrazinones is yet to be discovered. The staphylococcal bioreporter assays were used to eludicdate the structure-activity relationship of a series of truncated AIP-based antagonists against the AgrC1 receptor. Promising inhibitors are then evaluated against AgrC2, AgrC3 and AgrC4. Several compounds were found to be potent low nanomolar inhibitors across all four agr groups. A bioreporter assay based on the mutant receptor A101T T104V AgrC1 in which (Ala5)AIP1 is an agonist was also used to evaluate the panel of compounds. This revealed that most of these truncated AIP-based compounds are agonists of the mutant receptor, similar to (Ala5)AIP1. However, (Ala2, Leu4, Tfh5)trAIP1 (3.23) effectively inhibited activation of this bioreporter by AIP1. Compound 3.23 was also a sub-nanomolar inhibitor of AgrC1 and a low nanomolar inhibitor in other agr groups. Compound 3.23 is the most potent AgrC1 inhibitor discovered to date and, furthermore, its effects are likely to be less susceptible to mutations within the AgrC receptor. The depsipeptide natural products, solonamides, were synthesised using two uniquely different strategies. Development of a new synthetic strategy produced analogues with a high yield and diastereomeric excess in contrast with previous low yielding or non-stereoselective strategies. Their previously reported inhibition of the agr system was confirmed and fully quantified. Solonamide A and B inhibit activity in all four agr groups. Expression of toxic shock syndrome toxin-1 (TSST-1) and α-hemolysin were reduced in Staphylococcus aureus KH1187A. Schild analysis of data from agr bioreporters revealed that the inhibition is not competitive (as previously reported), but the solonamides act as negative allosteric modulators of both the AgrC1 and AgrC2 receptors, interacting with a new putative conserved allosteric binding site. Weak agonism at high concentrations was also discovered, which has not been previously observed. A panel of analogues was produced to assess the SAR with AgrC1. Modifications of the solonamide scaffold achieved mild improvements of physical characteristics and potency.

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 diverse array of natural products with significant potential in many biotechnological, clinical and commercial applications. These include pharmaceuticals, such as antitumour products, antibiotics, immunosuppressants, anticholesterolemics and anti-parasitic agents, as well as veterinary therapies and agrochemicals. These compounds are synthesised by complex secondary metabolism pathways involving polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS), both of which require an essential phosphopantetheinyl transferase (PPT) for their activity. PPTs activate the acyl, aryl and peptidyl carrier proteins within various biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. Phylogenetic analysis of the large superfamily of PPTs has revealed two separate families based on substrate specificity, which have been designated AcpS and Sfp-like. The AcpS PPT family activate acyl carrier proteins of fatty acid synthesis, while the Sfp-like PPT family, typified by the Bacillus subtilis PPT Sfp, has diverse roles in primary and secondary metabolism. The majority of cyanobacterial PPTs are of the Sfp-like PPT family and occur in genomes lacking an AcpS PPT. Phylogenetic analysis uncovered a distinct clade of cyanobacterial PPTs involved in heterocyst differentiation. Heterologous expression and functional analysis of NsPPT, the heterocyst-associated PPT in Nodularia spumigena NSOR10, represented the first characterisation of a cyanobacterial PPT. PCR-based screening was utilised to identify NsPPT and Southern hybridisation suggested this was the only PPT encoded by the N.spumigena NSOR10 genome. Enzymatic analyses demonstrated the ability of NsPPT to phosphopantetheinylate PKS and NRPS carrier proteins from a range of metabolism pathways and cyanobacterial species. Nostoc punctiforme ATCC 29133 encodes three PPTs. One of these PPTs, NgcS, is also a heterocyst-associated PPT and is homologous to NsPPT of N.spumigena NSOR10. Expression and enzymatic analysis of NgcS from N. punctiforme ATCC 29133, revealed contrasting phosphopantetheinylation activity to that seen for NsPPT, and indicated that NgcS may have evolved to have a strict specificity for the glycolipid biosynthesis pathway. Although the Sfp-like family of PPTs are normally associated with secondary metabolite biosynthesis, Synechocystis sp. PCC 6803 harbours a unique Sfp-like PPT (Sppt) but does not produce NRPS or PKS compounds. Genetic disruption of Sppt was attempted and expression of Sppt allowed the characterisation of its enzyme kinetics. Sppt displayed the ability to activate non-cognate cyanobacterial carrier proteins from NRPS and PKS biosynthetic pathways, although only at a low level of activity. This suggested that wild-type Synechocystis sp. PCC6803 would not be suitable for heterologous expression of cyanobacterial secondary metabolites. These results have important implications regarding the expression and manipulation of cyanobacterial bioactive compounds in heterologous hosts. Applications of this research may provide a biotechnological platform for the sustainable production of cyanobacterial natural products.

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.