published_or_final_version / abstract / toc / Botany / Doctoral / Doctor of Philosophy

The transcriptional response of freshwater Synechococcus sp. PCC 7942 and oceanic Synechococcus sp. WH 7803 to ammonium, nitrate or iron was studied in single or multiple factor limited cultures. Both strains showed maximum production of NiR mRNA when grown in nitrate-containing media. When grown in ammonium-containing medium, they did not show any signal for NiR mRNA synthesis. The influence on the transcription of NiR mRNA by iron as sole limiting nutrient was also evaluated. Iron increased the NiR mRNA whether or not the positive effect of nitrate was already present. The hybridization signal of mRNA for the large subunit of ribulose bisphosphate carboxylase/oxygenase enzyme (rbcl), was increased in both strains when iron was added. The relative response of NiA was larger than that of rbcL to iron addition. In the final set of experiments, combinations of iron, ammonium, or nitrate nutrient limitation were studied to understand their interactive effect on NiR and rbcL mRNA production. The ammonium-grown cells were allowed to starve for iron and then transferred to +iron+nitrate, +iron-nitrate, or -iron+nitrate cultures. rbcL mANA was increased gradually up to 168 hr. NiR mRNA increased initially but, for unknown reasons, decreased after 24 hr to a minimum that was similar for +iron+nitrate, +iron-nitrate, and -iron+nitrate cultures. A comparison of the rbcL or NiR mRNA from multiple nutrient limitation with that of single nutrient limitation showed different time dependent patterns of synthesis. The response to iron starvation could be different when cells experience a co-limitation with nitrate. This response is also influenced by species specific differences by evolutionary adaptation to different environments. This difference is confirmed in the NiR and rbcL mRNA response in two strains of marine and freshwater Synechococcus for single (iron) or multiple (iron+nitrate) limitation. Physiological studies commonly assume a single factor will constrain cell growth at any moment and when conditions change, the limiting factor will switch. The differential response in the synthesis of NiA and rbcl mANA observed shows that cells exhibit unique responses to combined limitations.

In addition to nitrogen and phosphorus, iron is an essential nutrient for oceanic primary productivity. Unlike nitrogen and phosphorus however, negligible amounts of iron are supplied to surface waters through recycling or mixing but instead from the limited and sporadic input of aeolian particulate. The low concentration of iron that becomes biologically available from the dust places a serious constraint on the heavily iron-dependent processes of photosynthesis and nitrate reduction which affect primary productivity. As much as 47% of the total oceanic primary productivity can be attributed to cyanobacteria making them critical organisms in the biogeochemical cycles. This thesis addresses the effect of iron on primary productivity using a combined approach of physiological and molecular biology. The physiological response of three marine strains of Synechococcus to growth on different concentrations of FeEDTA was investigated. Cells grown with higher concentrations of iron had greater cell density, more Chl- and phycobiliproteins and higher carbon fixation rates than cells grown at limiting iron concentrations (l0-8 M Fe). Iron enrichment of iron limited cultures stimulated carbon fixation, growth rate, and pigment and protein synthesis. Iron limited cells spiked with SJ.l.M Nlf4Cl prior to short term incubations had higher dark carbon fixation than cells gro·wn at higher iron and also spiked to 5J.1M Nlf4Cl. The addition of ammonium relieves a restricted nitrogen assimilatory pathway in the low iron cells that is evidenced by increased dark carbon fixation. We propose that this measurement of enhanced dark carbon fixation could be a useful assay in supporting the contention that populations of Synechococcus in nitrate rich waters are iron limited. Molecular genetic techniques were used to look for the presence of an iron uptake gene in cyanobacteria. Preliminary results indicate that there is a gene that is homologous to the ferric uptake regulation (fur) gene in E. coli. This hybridization occurred in siderophore-producing cyanobacteria, but not in marine cyanobacteria that do not produce siderophores. The fact that marine Synechococcus do not produce siderophores and did not hybridize to the fur gene suggest that fundamentally different mechanisms for iron uptake operate in high biomass freshwater cyanobacteria and cyanobacteria from dilute oligotrophic waters.

Cyanobacterial blooms are becoming more frequent worldwide, with possible negative effects on human health. The effects of climate change and eutrophication have been associated with persistent cyanobacterial blooms becoming more frequent. Altered water characteristics, salinity in particular, influence ecosystem dynamics that may lead to conditions conducive to cyanobacterial blooms. The occurrence of an 18-month long Cyanothece sp. bloom (the longest for any cyanobacterium recorded so far worldwide and the first of the genus) from June 2009 to December 2010 in Africa’s largest estuarine lake, St Lucia, highlighted the susceptibility of ecosystems to anthropogenic alterations. This study investigated the long-term survival and physiological adaptations of Cyanothece sp. to various and dynamic environmental conditions that contributed towards its bloom persistence. The main findings are the high salinities at which Cyanothece sp. could perform important physiological processes such as N uptake, N2 fixation and photosynthesis. Nutrient uptake (both nitrogen and phosphorus) was observed over the full experimental salinity range (0-300) while N2 fixation was only observed up to a salinity of 120. Nutrient uptake rates significantly decreased at this threshold salinity of 120. Interestingly, photosystem II activity was not observed in Cyanothece sp. during this study, but photosystem I activity was robust. Salinity had a minor influence on electron transport rates by photosystem I, high temperature (> 30°C) did however increase electron transport rates. Rapid responses to hypo-osmotic shock (i.e. osmotic downshift during freshening events) by Cyanothece sp. cells also helped minimize cell rupture due to high turgor pressure. Zooplankton abundance within the St Lucia system was negatively correlated with salinity, while grazing experiments indicated that the typical estuarine zooplankton species are able to graze on Cyanothece sp. cells. Therefore, the disappearance of zooplankton at salinities above 60 must have been an important factor in the bloom persistence. Apart from the ecological factors that were at play in St Lucia during the bloom period, the persistence of the Cyanothece sp. bloom can be attributed to the robust nature of their nutrient uptake, nitrogen fixation and photosynthetic systems to maintain activity despite extreme hypersalinity levels.

No description available.

This research was an attempt to characterize previously constructed mutants with a specifically altered psbO gene which encodes a 33 kDa protein active in photosynthesis. This polypeptide was believed to function in stabilization of manganese ions during photolysis of water at the photosystem II. The initial phase of this work was concerned with determining the manganese content of the genetically manipulated PS II particles of the photosynthetically active cyanobacteria.We found however, that the results of the isolation procedure for PS II particles of photosynthetically active cyanobacteria as described by Burnap et al. was not reproducible in our research organism. This prevented the chemical characterization of function of these particles as had been planned.In the second phase of the research sequencing of the mutated gene was to be performed for several clones in order to determine the kinds of specific alterations that had been made. The mutated genes had been cloned into both pUC1 20 and pPGV5 vectors which were transformed into Escherichia OR (EQQJi) and the cyanobacterium Synechococcus PCC 7942, respectively.Several attempts were mad o isolate plasmid DNA from both the transformed E QQJI and cyanobacterium. Isolation of pUC120 DNA was not achieved due to the toxicity of the 33 kDa protein product of the psbO gene in sgJj. The pPGV5 plasmid isolation was successful and PCR-sequencing was performed. However, the sequencing did not result in a readable sequence. Instead, banding patterns showed more than one nucleotide per lane. Since pPGV5 contains a strong constitutive promoter, a large amount of mutant protein was being produced. Our findings suggested that transformed cyanobacteria may have been under pressure to revert the altered gene to wild-type. Thus, upon growth of a single colony to a larger volume, a heterogeneous population of cells with different sizes of plasmids may have resulted. Restriction analysis of isolated plasmid DNA confirmed the presence of multiple-sized plasmid molecules. Therefore, this research has shown that the previously constructed mutants are not stable enough to characterize for alterations in manganese binding. / Department of Biology

The 33 kDa manganese stabilizing protein (MSP) has been proposed to provide ligands to stabilize Mn ions in the water lysis reaction of photosystem II of photosynthesis. In previous research site-directed mutagenesis had been performed on regions of the psbO gene encoding two aspartic acid residues of MSP which were thought to have the potential to form carboxyl bridges with Mn ions. The purpose of this research was to analyze these mutants. Plasmids pUC120-33 (#1,3,5,7,9,11,15) containing mutant psbO genes could not be isolated from E.coli because the expressed MSP was toxic to the cells. However, a psbO mutant gene carried in pPGV5-33 (#7) was isolated from E.coli and transformed into cyanobacterium Svnechococcus PCC 7942. Cyanobacterial cells carrying the MSP mutant showed a susceptibility to intensive light (100 footcandles) with a decrease of 30% in the growth rate within the first 100 hours after inoculation. This result suggested a possible function of the MSP in protecting the oxygen evolving complex from intensive light exposure. However, the mutant appeared to revert after this time probably due to homologous gene recombination with the wild type gene. In order to further analyze the function of mutants without recombination occurring, the construction of an MSP deletion was attempted using insertion of a kanamycin cartridge into the middle of the psbO gene. The inactivated psbO gene was transformed into E.coli and transformants were selected by kanamycin resistance. However, plasmid DNA carrying the interrupted genes could not be isolated, probably due to toxicity of the expression product in E.coli cells. Thus, future studies should be directed to reconstruction of a deletion mutant by direct transformation into cyanobacterial cells. Once a deletion mutant has been constructed analyses of the site-directed mutations could be performed in cyanobacteria. / Department of Biology

Photosynthetic membranes were prepared from the cyanobacterium Synechococcus PCC 7942 with oxygen evolving specific activity of 250-300 µmoles 02/ mg chl/hr. The membranes retained activity with a half-life of 4-5 days when stored at 0°C, or when quickly frozen in liquid nitrogen, greater than 95% of the activity remained after 2 months. Attempts to purify homogeneous preparations of photosystem II complexes from these membranes by detergent extraction were unsuccessful as indicated by a lack of a significant increase in oxygen evolution specific activity of the detergent extracts. Photosynthetic membrane detergent extracts usually maintained the same oxygen evolution specific activity as the orginal membranes, and a considerable amount of Photosystem I activity (75 µmoles 02 consumed /mg chl/hr in the Mehler reaction) was still present. The donor side of the photosystem II particles in the detergent extract was intact since the artificial electron acceptor, 2,6-dichiorophenolindophenol (DCPIP), was reduced at a rate comparable to the oxygen evolving activity. All oxygen evolving activity of the detergent extracts was lost when ion-exchange chromatography was used to resolve the co-extracted photosystem II and photosystem I complexes. / Department of Biology

The investigations described in this paper are an attempt to further define and quantify the interrelationship of nitrogen fixation and iron nutritional physiology in these two species. Chapter II will present and compare data on nutritional ratios of field collected Trichodesmium colonies and laboratory Anabaena cultures, with the intent of examining possible correlations between observed iron levels and protein nitrogen and chlorophyll concentrations, as well as nitrogen fixation rates. Chapter Ill is an examination of nitrogen fixation and siderophore production in Anabaena with emphasis on the possible implications of hypothesized synergistic effects of these two physiological capabilities on cyanobacterial dominance and bloom formation. Chapter IV will deal with the possibility of gratuitous manganese repression of Anabaena siderophore production in the manner described by Hantke (1987) for regulation of siderophore production in E. coli. The Conclusions chapter (Chapter V) will present a discussion of the results of these experiments in the context of current problems in cyanobacterial physiology, ecology and evolution. It is hoped that a significant contribution can be made to our understanding of the related problems of cyanobacterial dominance in freshwater ecosysytems and the scarcity of cyanobacterial nitrogen fixation in marine ecosystems.

Thesis (Ph. D.)--Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2005. / Includes bibliographical references. / Prochlorococcus, a unicellular cyanobacterium, is the most abundant phytoplankton in the oligotrophic, oceanic gyres where major plant nutrients such as nitrogen (N) and phosphorus (P) are at nanomolar concentrations. Nitrogen availability controls primary productivity in many of these regions. The cellular mechanisms that Prochlorococcus uses to acquire and metabolize nitrogen are thus central to its ecology. One of the goals of this thesis was to investigate how two Prochlorococcus strains responded on a physiological and genetic level to changes in ambient nitrogen. We characterized the N-starvation response of Prochlorococcus MED4 and MIT9313 by quantifying changes in global mRNA expression, chlorophyll fluorescence, and Fv/Fm along a time-series of increasing N starvation. In addition to efficiently scavenging ambient nitrogen, Prochlorococcus strains are hypothesized to niche-partition the water column by utilizing different N sources. We thus studied the global mRNA expression profiles of these two Prochlorococcus strains on different N sources. The recent sequencing of a number of Prochlorococcus genomes has revealed that nearly half of Prochlorococcus genes are of unknown function. / (cont.) Genetic methods such as reporter gene assays and tagged mutagenesis are critical tools for unveiling the function of these genes. As the basis for such approaches, another goal of this thesis was to find conditions by which interspecific conjugation with Escherichia coli could be used to transfer plasmid DNA into Prochlorococcus MIT9313. Following conjugation, E. coli were removed from the Prochlorococcus cultures by infection with E. coli phage T7. We applied these methods to show that an RSF1010-derived plasmid will replicate in Prochlorococcus MIT9313. When this plasmid was modified to contain green fluorescent protein (GFP) we detected its expression in Prochlorococcus by Western blot and cellular fluorescence. Further, we applied these conjugation methods to show that Tn5 will transpose in vivo in Prochlorococcus. Collectively, these methods provide a means to experimentally alter the expression of genes in the Prochlorococcus cell. / by Andrew Carl Tolonen. / Ph.D.