Heterocysts, sites of nitrogen fixation in certain filamentous cyanobacteria, are limited to a heterotrophic metabolism, rather than the photoautotrophic metabolism characteristic of cyanobacterial vegetative cells. The metabolic route of carbon catabolism in the supply of reductant to nitrogenase and for respiratory electron transport in heterocysts is unresolved. The gene (zwf) encoding glucose-6-phosphate dehydrogenase (G6PD), the initial enzyme of the oxidative pentose phosphate pathway, was inactivated in the heterocyst-forming, facultatively heterotrophic cyanobacterium, Nostoc sp. strain ATCC 29133. The zwf mutant strain had less than 5% of the wild-type apparent G6PD activity, while retaining wild-type rates of photoautotrophic growth with NH 4 ؉ and of dark O 2 uptake, but it failed to grow either under N 2 -fixing conditions or in the dark with organic carbon sources. A wild-type copy of zwf in trans in the zwf mutant strain restored only 25% of the G6PD specific activity, but the defective N 2 fixation and dark growth phenotypes were nearly completely complemented. Transcript analysis established that zwf is in an operon also containing genes encoding two other enzymes of the oxidative pentose phosphate cycle, fructose-1,6-bisphosphatase and transaldolase, as well as a previously undescribed gene (designated opcA) that is cotranscribed with zwf. Inactivation of opcA yielded a growth phenotype identical to that of the zwf mutant, including a 98% decrease, relative to the wild type, in apparent G6PD specific activity. The growth phenotype and lesion in G6PD activity in the opcA mutant were complemented in trans with a wild-type copy of opcA. In addition, placement in trans of a multicopy plasmid containing the wild-type copies of both zwf and opcA in the zwf mutant resulted in an approximately 20-fold stimulation of G6PD activity, relative to the wild type, complete restoration of nitrogenase activity, and a slight stimulation of N 2 -dependent photoautotrophic growth and fructose-supported dark growth. These results unequivocally establish that G6PD, and most likely the oxidative pentose phosphate pathway, represents the essential catabolic route for providing reductant for nitrogen fixation and respiration in differentiated heterocysts and for dark growth of vegetative cells. Moreover, the opcA gene product is involved by an as yet unknown mechanism in G6PD synthesis or catalytic activity.Cyanobacteria are a morphologically diverse but phylogenetically cohesive group of gram-negative eubacteria that are phenotypically united by their oxygen-evolving photosynthetic mechanism. All cyanobacteria are photoautotrophic, using ATP and NADPH generated in the light reactions for autotrophic CO 2 assimilation by enzymes of the Calvin-Benson, or reductive pentose phosphate, cycle. They synthesize glycogen as the storage material in the light, and this glycogen is subsequently catabolized to provide maintenance energy during the dark periods of natural day-night cycles.The vegetative cells of many filamentous cya...

The vegetative cells of the filamentous cyanobacterium Nostoc punctiforme can differentiate into three mutually exclusive cell types: nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogomium filaments. A DNA microarray consisting of 6,893 N. punctiforme genes was used to identify the global transcription patterns at single time points in the three developmental states, compared to those in ammonium-grown time zero cultures. Analysis of ammonium-grown cultures yielded a transcriptome of 2,935 genes, which is nearly twice the size of a soluble proteome. The NH 4 ؉ -grown transcriptome was enriched in genes encoding core metabolic functions. A steady-state N 2 -grown (heterocyst-containing) culture showed differential transcription of 495 genes, 373 of which were up-regulated. The majority of the up-regulated genes were predicted from studies of heterocyst differentiation and N 2 fixation; other genes are candidates for more detailed genetic analysis. Three days into the developmental process, akinetes showed a similar number of differentially expressed genes (497 genes), which were equally up-and down-regulated. The down-regulated genes were enriched in core metabolic functions, consistent with entry into a nongrowth state. There were relatively few adaptive genes up-regulated in 3-day akinetes, and there was little overlap with putative heterocyst developmental genes. There were 1,827 differentially transcribed genes in 24-h hormogonia, which was nearly fivefold greater than the number in akinete-forming or N 2 -fixing cultures. The majority of the up-regulated adaptive genes were genes encoding proteins for signal transduction and transcriptional regulation, which is characteristic of a motile filament that is poised to sense and respond to the environment. The greatest fraction of the 883 down-regulated genes was involved in core metabolism, also consistent with entry into a nongrowth state. The differentiation of heterocysts (steady state, N 2 grown), akinetes, and hormogonia appears to involve the up-regulation of genes distinct for each state.

Nostoc punctiforme is a phenotypically complex, filamentous, nitrogen-fixing cyanobacterium, whose vegetative cells can mature in four developmental directions. The particular developmental direction is determined by environmental signals. The vegetative cell cycle is maintained when nutrients are sufficient. Limitation for combined nitrogen induces the terminal differentiation of heterocysts, cells specialized for nitrogen fixation in an oxic environment. A number of unique regulatory events and genes have been identified and integrated into a working model of heterocyst differentiation. Phosphate limitation induces the transient differentiation of akinetes, spore-like cells resistant to cold and desiccation. A variety of environmental changes, both positive and negative for growth, induce the transient differentiation of hormogonia, motile filaments that function in dispersal. Initiation of the differentiation of heterocysts, akinetes and hormogonia are hypothesized to depart from the vegetative cell cycle, following separate and distinct events. N. punctiforme also forms nitrogen-fixing symbiotic associations; its plant partners influence the differentiation and behavior of hormogonia and heterocysts. N. punctiforme is genetically tractable and its genome sequence is nearly complete. Thus, the regulatory circuits of three cellular differentiation events and symbiotic interactions of N. punctiforme can be experimentally analyzed by functional genomics.

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Inclusions of neutral lipids termed lipid droplets (LDs) located throughout the cell were identified in the cyanobacterium Nostoc punctiforme by staining with lipophyllic fluorescent dyes. LDs increased in number upon entry into stationary phase and addition of exogenous fructose indicating a role for carbon storage, whereas high-light stress did not increase LD numbers. LD accumulation increased when nitrate was used as the nitrogen source during exponential growth as compared to added ammonia or nitrogen–fixing conditions. Analysis of isolated LDs revealed enrichment of triacylglycerol (TAG), - tochopherol, and C17 alkanes. LD TAG from exponential phase growth contained mainly saturated C16 and C18 fatty acids whereas stationary phase LD TAG had additional unsaturated fatty acids characteristic of whole cells. This is the first characterization of cyanobacterial LD composition and conditions leading to their production. Based upon their abnormally large size and atypical location these structures represent a novel sub-organelle in cyanobacteria.