Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

This thesis details my investigations of marine cyanobacterial natural products that resulted in the discovery of thirteen new secondary metabolites, the isolation of over fifteen previously reported metabolites and the biosynthetic investigation of two additional cyanobacterial compounds. Two novel lipopeptides were identified from a Lyngbya majuscula and Schizothrix sp. assemblage collected in the Fiji Islands. Somamide A is a depsipeptide consisting of a hexanoate moiety extended by seven amino acids, including two nonstandard units characteristic of cyanobacterial peptides. In contrast, somocystinamide A is a unique linear disulfide dimer displaying potent cytotoxicity against a mammalian neuroblastoma cell line. The organic extract from a Puerto Rican L. majuscula proved remarkably rich in chemistry, producing twelve known compounds as well as four new secondary metabolites. Among these new isolates were the novel sodium channel blocker antillatoxin B, a new chlorinated quinoline derivative and the new ��-pyrone malyngamide T. A collection of L. majuscula from Antany Mora, Madagascar, led to the isolation of the previously reported antineoplastic agent dolastatin 16 and the discovery of a new series of lipopeptides, the antanapeptins. These new molecules are characterized by the presence of the unique ��-hydroxy acid 3-hydroxy-2-methyloctynoate, or its reduced double- or single-bond equivalent. Wewakazole is a novel cyclic dodecapeptide isolated from a Papua New Guinea collection of L. majuscula. This large molecule contains both a methyloxazole and two oxazoles, residues rarely observed in marine cyanobacterial metabolites. Extensive utilization of 1D and 2D NMR techniques were required to elucidate the structure of this distinctive peptide. Biosynthetic investigations of two halogenated cytotoxins were also conducted on a cultured L. majuscula strain originally isolated from Hector Bay, Jamaica. Stable isotope feeding experiments demonstrated that both jamaicamide A and hectochlorin derive from mixed PKS and NRPS biosynthetic origins but are comprised of primary precursors unique to each molecule. / Graduation date: 2003

The toxicity and adverse health effects of arsenic are widely known. It is generally accepted that sorption/desorption reactions with oxy-hydroxide minerals (iron, manganese) control the fate and transport of inorganic arsenic in surface waters through adsorption and precipitation-dissolution processes. In terrestrial environments with limited reactive iron, recent data suggest organoarsenicals are potentially important components of the biogeochemical cycling of arsenic in near-surface environments. Elevated arsenic levels are common in South Texas from geogenic processes (weathering of As-containing rock units) and anthropogenic sources (a byproduct from decades of uranium mining). Sediments collected from South Texas show low reactive iron concentrations, undetectable in many areas, making oxy-hydroxide controls on arsenic unlikely. Studies have shown that eukaryotic algae isolated from arsenic-contaminated waters have increased tolerance to arsenate toxicity and the ability to uptake and biotransform arsenate. In this experiment, net uptake of arsenic over time by a freshwater cyanobacterium never previously exposed to arsenate was quantified as a function of increasing As concentrations and increasing N:P ratios. Toxic effects were not evident when comparing cyanobacterial growth, though extractions indicate accumulation of intracellular arsenic by the cyanobacterium. Increasing N:P ratios has minimal effect on net arsenate uptake over an 18 day period. However, cyanobacteria were shown to reduce arsenate at rates faster than the system can re-oxidize the arsenic suggesting gross arsenate uptake may be much higher. Widespread arsenate reduction by cyanobacterial blooms would increase arsenic mobility and potential toxicity and may be useful as a biomarker of arsenic exposure in oxic surface water environments.

Circadian rhythms of behavior have been well characterized in organisms including mammals, plants, insects, fungi, and photosynthetic bacteria. Cyanobacteria, such as the unicellular Synechococcus elongatus PCC 7942, display near 24-h circadian rhythms of gene expression. These rhythms persist in the absence of external cues, can be reset by the same stimuli to which they entrain, and are relatively insensitive to changes in ambient temperature within their physiological range. Key components have been identified as belonging to the central oscillator that comprises the timekeeping units, output pathways that relay temporal information to clock-controlled processes, and input pathways that synchronize the oscillator with local time. The emerging model of the cyanobacterial clock depicts the internal timekeeping elements KaiA, KaiB, and KaiC interacting with one another to form a large, multimeric complex that assembles and disassembles over the course of a day. Information is sent into and out of the oscillator via signal transduction pathways that include proteins involved in bacterial twocomponent systems. The research presented in this dissertation explores the regulatory mechanisms that exist at each level of the clock system. New components were identified that interact with an important protein in the input pathway; these new players are involved in clock-associated phenomena, such as resetting the internal oscillation to external stimuli and maintaining proper circadian periodicity, as well as the process of cell division. The model formerly associated with the temporal, transcriptional regulation of the kai genes was redefined to reflect the unique properties of the prokaryotic oscillator. The differential output of the clock was examined by studying the circadian regulation of the psbA gene family. Overall, these data provide insight into the complex molecular events that occur to create a circadian timing circuit in S. elongatus.

The toxicity and adverse health effects of arsenic are widely known. It is generally accepted that sorption/desorption reactions with oxy-hydroxide minerals (iron, manganese) control the fate and transport of inorganic arsenic in surface waters through adsorption and precipitation-dissolution processes. In terrestrial environments with limited reactive iron, recent data suggest organoarsenicals are potentially important components of the biogeochemical cycling of arsenic in near-surface environments. Elevated arsenic levels are common in South Texas from geogenic processes (weathering of As-containing rock units) and anthropogenic sources (a byproduct from decades of uranium mining). Sediments collected from South Texas show low reactive iron concentrations, undetectable in many areas, making oxy-hydroxide controls on arsenic unlikely. Studies have shown that eukaryotic algae isolated from arsenic-contaminated waters have increased tolerance to arsenate toxicity and the ability to uptake and biotransform arsenate. In this experiment, net uptake of arsenic over time by a freshwater cyanobacterium never previously exposed to arsenate was quantified as a function of increasing As concentrations and increasing N:P ratios. Toxic effects were not evident when comparing cyanobacterial growth, though extractions indicate accumulation of intracellular arsenic by the cyanobacterium. Increasing N:P ratios has minimal effect on net arsenate uptake over an 18 day period. However, cyanobacteria were shown to reduce arsenate at rates faster than the system can re-oxidize the arsenic suggesting gross arsenate uptake may be much higher. Widespread arsenate reduction by cyanobacterial blooms would increase arsenic mobility and potential toxicity and may be useful as a biomarker of arsenic exposure in oxic surface water environments.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (M.S. in environmental science)--Washington State University, December 2009. / Title from PDF title page (viewed on Feb. 18, 2010). "School of Earth and Environmental Sciences." Includes bibliographical references (p. 40-45).

Not available / text

Cyanobacteria are globally widespread and ecologically highly significant photoautotrophic microorganisms, with diverse geno- and phenotypic characters unprecedented among prokaryotes. This phylum embraces representatives with an exclusive adaptability in highly specialized environments, from oligotrophic ocean waters to the interior of cells in symbiotic plants, the most extreme being the chloroplasts. Insertion sequences (ISs) are short (~1000 bp) mobile genetic elements prevalent in microbial genomes, potentially representing potent adaptive forces. In this thesis, hypotheses tested that ISs play significant roles in both reductive and adaptive evolution in physiologically versatile cyanobacteria, using two model systems. First, the genome of an obligate plant (Azolla) symbiont, the cyanobacterium ‘Nostoc azollae 0708’, was sequenced, which led to the discovery of a highly ‘eroding’ genome (5,48 Mbp), loaded with ISs covering 14% of the genome, a situation likely caused by the relaxed selection pressure within the plant. The ISs were located in close proximity to the extremely numerous pseudogenes identified, although genes with key functions in a symbiotic context escaped IS mediated erosion (e.g. nitrogen fixation and differentiation genes). Some ISs were shown to have transposed short distances within the genome (‘local hoping’), and to be likely causative agents in pseudogene formation, and thus pivotal actors in the reductive evolution discovered. To widen the scope of ISs further, additionally 66 phylogenetically diverse microorganisms with a variety of life styles (free-living, symbionts, pathogens) were examined in regards to ISs influence. The data verified their over-all importance in shaping microbial genomes. Finally, natural microbial populations in the Baltic Sea, a semi-enclosed geologically young (~10,000 years) brackish water body offering steep gradients in salinity and nutrient loads, were examined using metatranscriptomics and metagenomics. A large proportion of the metagenome was devoted to ISs and most importantly a large fraction of the metatranscriptome consisted of IS transcripts (~1%), which may be suggestive of a high IS activity. These phenomena were most apparent in cyanobacteria in central parts of the Baltic Sea. The presence of an especially rich abundance of ISs in brackish waters was further substantiated by their low frequency (&lt; 0.1%) in microbes of marine waters. Hence, ISs may facilitate both adaptations (short term) and adaptive evolution (long term) in microbes entering brackish water, otherwise unable to cross the distinct limnic-to-marine salinity-divide. Together, the data reveal high genomic loads of ISs in cyanobacteria subject to highly demanding conditions and stress the importance of locally migrating ISs (and pseudogenization) as important facilitators in adaptation and evolution, being a more rapid process than hitherto expected. The findings strongly support current theories stating a crucial role of ISs in shaping microbial genomes to render fitness. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Lineages of the cyanobacteria Prochlorococcus marinus have diverged into two genetically distinct ‘ecotypes,’ high-light adapted (HL) and low-light adapted (LL), which thrive under different environmental conditions. This type of niche differentiation in prokaryotes is often accompanied by genetic and genomic divergence. Differential selection pressure associated with ecotype divergence can be analyzed using models of codon evolution. However, some characteristics of the Prochlorococcus genome violate underlying assumptions of these models. For example, high levels of recombination between bacterial strains are known to cause false positives for codon models. Therefore, it is important that statistical methods for detecting recombination be reliable. In Chapter 2, I evaluate a set of recombination detection methods under four different scenarios related to functional divergence: 1) varying tree shape, 2) positive selection, 3) non-stationary evolution, and 4) varying levels of recombination and divergence.