Today, mankind faces a new challenge in energetic terms: a new Industrial Revolution is imperative, already called by some as an Energetic Revolution. This corresponds to a conversion to clean, environmentally friendly and renewable energy sources. In this context, hydrogen arises as a valid alternative, since its combustion produces a considerable amount of energy and releases solely water as a by-product. In the present thesis, two model cyanobacteria, namely Synechocystis sp. strain PCC 6803 and Anabaena/Nostoc sp. strain PCC 7120, were used to examine the hydrogen metabolism. The efforts were focused on to understand the transcription regulation of the hox genes, encoding the structural elements of the bidirectional hydrogenase enzyme. Here, it is shown that such regulation is operated in a very distinct and intricate way, with different factors contributing to its delicate tuning. While in Synechocystis sp. strain PCC 6803 the hox genes were shown to be transcribed as a single operon, in Anabaena/Nostoc sp. strain PCC 7120 they were shown to be transcribed as two independent operons (possibly three). Two transcription factors, LexA and AbrB-like protein, were identified and further characterized in relation to the hydrogen metabolism. Furthermore, different environmental conditions were demonstrated to operate changes on the transcription of the bidirectional hydrogenase genes. In addition, functional studies of three open reading frames found within the hox operon of Synechocystis sp. strain PCC 6803 suggest that this may be a stress responsive operon. However, based on the gained knowledge, it is still not possible to connect the signal transduction pathways, from the environmental signal, through the response regulator, to the final regulation of the hox genes. Nevertheless, the crucial importance of studying the transcription regulation of the different players involved in the hydrogen metabolism is now established and a new era seems to be rising.

Analysis of the genome of Anabaena sp. PCC7120 reveals that it lacks the gene, ccaA, which encodes the bonafide carboxysomal, β-class carbonic anhydrase (CA) CcaA. However, the carboxysome enriched fraction of Anabaena PCC7120 exhibits CA activity. Bioinformatic analysis reveals that the N-terminal region of the carboxysome protein CcmM has high sequence and structural similarity to the γ-class CA of Methanosarcina thermophila. Recombinantly expressed CcmM is found to be inactive in in-vitro CA assays. E. coli cell extracts containing an overexpressed form of CcmM comprised of the N-terminal 209 amino acids (CcmM209) are also inactive. However, CcmM209 displays CA activity after incubation with the thiol oxidizing agent diamide or when bound to an affinity matrix. It appears that CcmM is indeed a functional γ-CA which is active under oxidizing condition. It is hypothesized that the C-terminal RbcS like domain in CcmM may regulate activity by allowing CcmM activation only when sequestered within the carboxysome.

Analysis of the genome of Anabaena sp. PCC7120 reveals that it lacks the gene, ccaA, which encodes the bonafide carboxysomal, β-class carbonic anhydrase (CA) CcaA. However, the carboxysome enriched fraction of Anabaena PCC7120 exhibits CA activity. Bioinformatic analysis reveals that the N-terminal region of the carboxysome protein CcmM has high sequence and structural similarity to the γ-class CA of Methanosarcina thermophila. Recombinantly expressed CcmM is found to be inactive in in-vitro CA assays. E. coli cell extracts containing an overexpressed form of CcmM comprised of the N-terminal 209 amino acids (CcmM209) are also inactive. However, CcmM209 displays CA activity after incubation with the thiol oxidizing agent diamide or when bound to an affinity matrix. It appears that CcmM is indeed a functional γ-CA which is active under oxidizing condition. It is hypothesized that the C-terminal RbcS like domain in CcmM may regulate activity by allowing CcmM activation only when sequestered within the carboxysome.

Geosmin is an odorous secondary metabolite produced by some cyanobacteria during growth and released from the cells. Little is known about the biosynthesis of geosmin and the gene(s) required for its production have not been characterized. During late August and early September geosmin episodes due to planktonic cyanobacteria frequently occur in the northwest basin of Lake Ontario waters resulting in taste and odour episodes in drinking water that serves more than 5 million people. At high concentrations geosmin evades traditional drinking water treatment and reaches the tap. These episodes often elicit consumer concern and are wrongly construed to reflect impaired drinking water safety. Water quality managers in the region have generally been unable to prevent or control taste and odour episodes via a proactive approach due to the lack of knowledge of cyanobacterial communities in offshore waters as well as the inability to predict when geosmin will reach intake pipes due to downwelling, the process by which the surface waters mix with the hypolimnion. This study evaluated denaturing gradient gel electrophoresis (DGGE) as a molecular tool for proactive monitoring of potential taste and odour-causing cyanobacteria in environmental samples. The 16S rRNA gene was assessed for its ability to distinguish among geosmin-producing and non-producing strains. This study also examined the evolutionary relationships among geosmin-producing cyanobacteria using the full-length 16S rRNA gene and compared phylogenies with current taxonomy. <br /><br /> A DGGE standard using the V3 hypervariable region of the 16S rRNA gene was developed using geosmin-producing and non-producing isolates of cyanobacteria. Included in the standard was the suspected primary contributor to Lake Ontario taste and odour, <em>Anabaena lemmermannii</em> Richter. This standard was then applied to various environmental collections from Lake Ontario (August 2005) to examine the cyanobacterial community composition. DGGE profiles were consistent with the presence of <em>An. lemmermannii</em> at locations with increased geosmin concentrations (determined using gas chromatography-mass spectrometry), supporting hypothesis that <em>An. lemmermannii</em> is the primary contributor to northwestern Lake Ontario taste and odour. In addition, the application of DGGE in the identification of potential geosmin-producing species of cyanobacteria was deemed to be a potentially useful approach to monitoring cyanobacterial communities in source waters. The 16S rRNA-V3 region alone did not distinguish among geosmin-producing and non-producing strains, however with additional data (actual geosmin concentration) it was showed relationships. <br /><br /> In the phylogenetic analyses, geosmin-producing cyanobacteria did not group monophyletically and it was not possible to state that a single evolutionary event has led to the acquisition of the geosmin-producing trait. Phylogenies also showed that the taxonomy of the Cyanobacteria is largely unresolved. All five Sections (bacteriological classification)/four orders (Komárek & Anagnostidis classification) were paraphyletic, however the heterocystous cyanobacteria (Sections IV and V/Nostocales and Stigonematales) grouped separately from the non-heterocystous cyanobacteria (Sections I, III/Chroococcales and Oscillatoriales). Although both systems of classification compared in this study were similar, nomenclature and groupings were occasionally different among the groups. This demonstrates the incongruity between bacteriologists and phycologists and emphasizes the need for a consensus system of classification for the Cyanobacteria.

Cyanobacteria are an ancient lineage of gram-negative photosynthetic prokaryotes that play an important role in the nitrogen cycle in terrestrial and aquatic systems. Widespread cyanobacterial blooms have prompted numerous studies on the classification of this group, however defining species is problematic due to lack of clarity as to which characters best define the various taxonomic levels. The genera Anabaena, Aphanizomenon and Nostoc form one of the most controversial groups and are typically paraphyletic within phylogenetic trees and share similar morphological characters. This study’s purpose was to determine the taxonomic and phylogenetic relationships among isolates from these three genera using 16S rRNA and bacterial elongation factor P (efp) gene sequences as well as morphological analyses. These data confirmed the non-monophyly of Anabaena and Aphanizomenon and demonstrated that many of the isolates were intermixed among various clades in both gene phylogenies. In addition, the genus Nostoc was clearly not monophyletic and this finding differed from previous studies. The genetic divergence of the genus Nostoc was confirmed based on 16S rRNA gene sequence similarities (≥85.1%), and the isolates of Anabaena were genetically differentiated, contrary to previous studies (16S rRNA gene sequence similarities ≥89.4%). The morphological diversity was larger than the molecular diversity, since the statistical analysis ANOSIM showed that the isolates were morphologically well differentiated; however, the 16S rRNA gene sequence similarities showed some isolates as being related at the species level. Planktonic and benthic strains were not distinguished phylogenetically, although some well-supported clusters were noted. Cellular measurements (length and width of vegetative cells, end cells, heterocysts and akinetes) were noted to be the morphological characters that best supported the differentiation among isolates, more than qualitative characterization. Among the metric parameters, the length of akinetes resulted in better differentiation among isolates. The efp gene sequence analyses did not appear to be useful for the taxonomic differentiation at lower taxonomic levels, but gave well-supported clusters for Aphanizomenon that was supported by the morphological analyses. Both gene regions gave similar trees with the exception of the Aphanizomenon isolates which clustered together in phylogenetic trees based on the efp gene. This differed from the 16S rRNA gene in which this genus was paraphyletic with Anabaena species that were similar in morphology to Aphanizomenon. Hence, the application of multiple taxonomic criteria is required for the successful delineation of cyanobacterial species.

Geosmin is an odorous secondary metabolite produced by some cyanobacteria during growth and released from the cells. Little is known about the biosynthesis of geosmin and the gene(s) required for its production have not been characterized. During late August and early September geosmin episodes due to planktonic cyanobacteria frequently occur in the northwest basin of Lake Ontario waters resulting in taste and odour episodes in drinking water that serves more than 5 million people. At high concentrations geosmin evades traditional drinking water treatment and reaches the tap. These episodes often elicit consumer concern and are wrongly construed to reflect impaired drinking water safety. Water quality managers in the region have generally been unable to prevent or control taste and odour episodes via a proactive approach due to the lack of knowledge of cyanobacterial communities in offshore waters as well as the inability to predict when geosmin will reach intake pipes due to downwelling, the process by which the surface waters mix with the hypolimnion. This study evaluated denaturing gradient gel electrophoresis (DGGE) as a molecular tool for proactive monitoring of potential taste and odour-causing cyanobacteria in environmental samples. The 16S rRNA gene was assessed for its ability to distinguish among geosmin-producing and non-producing strains. This study also examined the evolutionary relationships among geosmin-producing cyanobacteria using the full-length 16S rRNA gene and compared phylogenies with current taxonomy. <br /><br /> A DGGE standard using the V3 hypervariable region of the 16S rRNA gene was developed using geosmin-producing and non-producing isolates of cyanobacteria. Included in the standard was the suspected primary contributor to Lake Ontario taste and odour, <em>Anabaena lemmermannii</em> Richter. This standard was then applied to various environmental collections from Lake Ontario (August 2005) to examine the cyanobacterial community composition. DGGE profiles were consistent with the presence of <em>An. lemmermannii</em> at locations with increased geosmin concentrations (determined using gas chromatography-mass spectrometry), supporting hypothesis that <em>An. lemmermannii</em> is the primary contributor to northwestern Lake Ontario taste and odour. In addition, the application of DGGE in the identification of potential geosmin-producing species of cyanobacteria was deemed to be a potentially useful approach to monitoring cyanobacterial communities in source waters. The 16S rRNA-V3 region alone did not distinguish among geosmin-producing and non-producing strains, however with additional data (actual geosmin concentration) it was showed relationships. <br /><br /> In the phylogenetic analyses, geosmin-producing cyanobacteria did not group monophyletically and it was not possible to state that a single evolutionary event has led to the acquisition of the geosmin-producing trait. Phylogenies also showed that the taxonomy of the Cyanobacteria is largely unresolved. All five Sections (bacteriological classification)/four orders (Komárek & Anagnostidis classification) were paraphyletic, however the heterocystous cyanobacteria (Sections IV and V/Nostocales and Stigonematales) grouped separately from the non-heterocystous cyanobacteria (Sections I, III/Chroococcales and Oscillatoriales). Although both systems of classification compared in this study were similar, nomenclature and groupings were occasionally different among the groups. This demonstrates the incongruity between bacteriologists and phycologists and emphasizes the need for a consensus system of classification for the Cyanobacteria.

Cyanobacteria are an ancient lineage of gram-negative photosynthetic prokaryotes that play an important role in the nitrogen cycle in terrestrial and aquatic systems. Widespread cyanobacterial blooms have prompted numerous studies on the classification of this group, however defining species is problematic due to lack of clarity as to which characters best define the various taxonomic levels. The genera Anabaena, Aphanizomenon and Nostoc form one of the most controversial groups and are typically paraphyletic within phylogenetic trees and share similar morphological characters. This study’s purpose was to determine the taxonomic and phylogenetic relationships among isolates from these three genera using 16S rRNA and bacterial elongation factor P (efp) gene sequences as well as morphological analyses. These data confirmed the non-monophyly of Anabaena and Aphanizomenon and demonstrated that many of the isolates were intermixed among various clades in both gene phylogenies. In addition, the genus Nostoc was clearly not monophyletic and this finding differed from previous studies. The genetic divergence of the genus Nostoc was confirmed based on 16S rRNA gene sequence similarities (≥85.1%), and the isolates of Anabaena were genetically differentiated, contrary to previous studies (16S rRNA gene sequence similarities ≥89.4%). The morphological diversity was larger than the molecular diversity, since the statistical analysis ANOSIM showed that the isolates were morphologically well differentiated; however, the 16S rRNA gene sequence similarities showed some isolates as being related at the species level. Planktonic and benthic strains were not distinguished phylogenetically, although some well-supported clusters were noted. Cellular measurements (length and width of vegetative cells, end cells, heterocysts and akinetes) were noted to be the morphological characters that best supported the differentiation among isolates, more than qualitative characterization. Among the metric parameters, the length of akinetes resulted in better differentiation among isolates. The efp gene sequence analyses did not appear to be useful for the taxonomic differentiation at lower taxonomic levels, but gave well-supported clusters for Aphanizomenon that was supported by the morphological analyses. Both gene regions gave similar trees with the exception of the Aphanizomenon isolates which clustered together in phylogenetic trees based on the efp gene. This differed from the 16S rRNA gene in which this genus was paraphyletic with Anabaena species that were similar in morphology to Aphanizomenon. Hence, the application of multiple taxonomic criteria is required for the successful delineation of cyanobacterial species.

Many multicellular cyanobacteria produce specialized nitrogen-fixing heterocysts. During diazotrophic growth of the model organism Anabaena (Nostoc) sp. strain PCC 7120, a regulated developmental pattern of single heterocysts separated by about 10 to 20 photosynthetic vegetative cells is maintained along filaments. Heterocyst structure and metabolic activity function to accommodate the oxygen-sensitive process of nitrogen fixation. This dissertation focuses on my research on heterocyst development, including morphogenesis, transport of molecules between cells in a filament, differential gene expression, and pattern formation. We using microarray experiments we found that conR (all0187) gene is necessary for normal septum-formation of vegetative cells, diazotrophic grow, and heterocyst morphogenesis. In our studies we characterized the expression of sigma factors genes in Anabaena PCC 7120 during heterocyst differentiation, and we found that the expression of sigC, sigG and sigE is localized primarily in heterocysts. Expression studies using sigE mutant showed that nifH is under the control of this specific sigma factor.

Iron is an essential nutrient for growth of photosynthetic microorganisms such as cyanobacteria and algae. Iron is required for proteins involved in the important processes of carbon and nitrogen assimilation. Low concentrations of iron in cultures or natural waters can lead to iron limitation which affects many aspects of algal metabolism. In natural waters, iron limitation can have effects on the patterns and rates of primary productivity. The cellular content of certain proteins can be affected by media iron concentrations. Methods have been used that assay components of the cell as an indirect measure of iron nutritional status. For example, spectroscopy can be performed to determine the cellular concentration of iron-containing proteins involved in photosynthesis. Organisms grown in media that imitate natural conditions, or organisms collected from their natural habitat are usually dilute. Methods that assay iron nutritional status such as spectroscopy and column chromatography require large sample sizes which are difficult to obtain from natural samples. In addition, methods that utilize techniques such as immunology or radioactive labelling are complex and time-consuming. These considerations led to the necessity of developing a technique that would be simple, rapid and effective on dilute samples. The method developed here utilized fast protein liquid chromatography (FPLC), which fulfilled these requirements. A complete analysis could be done within two to three hours with minimal sample treatment. The FPLC was simple to operate and was effective on a sample containing less than 100 μg of protein. Some photosynthetic organisms, when iron-depleted, can produce the flavin-containing protein flavodoxin (Flv). This protein substitutes for the iron-containing protein ferredoxin (Fd) in Fd-dependent reactions such as the light-induced reduction of NADP. The FPLC technique identified and quantified, in relative terms, Fd and Flv in the cell. Optical spectroscopy was used to verify FPLC retention time assignments. The results illustrated how the FPLC could be used to observe the changes in relative Fd and Flv content as a function of media iron concentration in cultures of the cyanobacterium Anabaena grown in the laboratory. It was found that Fd content decreased and Flv content increased with decreasing media iron concentration. In addition, samples of the cyanobacterium Trichodesmium collected from the ocean near Barbados were analyzed using FPLC to assay relative Fd and Flv content. By analogy with Anabaena, Fd and Flv retention times were identified. Using this technique conclusions could be drawn regarding the changing iron nutritional status of Trichodesmium in its natural habitat .

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