Two marine, unicellular aerobic nitrogen-fixing cyanobacteria, Cyanothece strain BH63 and Cyanothece strain BH68, were isolated from the intertidal sands of the Texas Gulf coast in enrichment conditions designed to favor rapid growth. By cell morphology, ultrastructure, a GC content of 40%c, and aerobic nitrogen fitation ability, these strains were assigned to the genus Cyanothece. These strains can use molecular nitrogen as the sole nitrogen source and are capable of photoheterotrophic growth in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and glycerol. The strains demonstrated a doubling time of 10 to 14 h in the presence of nitrate and 16 to 20 h under nitrogen-fixing conditions. Rapid growth of nitrogen-fixing cultures can be obtained in continuous light even when the cultures are continuously shaken or bubbled with air. Under 12-h alternating light and dark cycles, the aerobic nitrogenase activity was confined to the dark phase. The typical rates of aerobic nitrogenase activity in Cyanothece strains BH63 and BH68 were 1,140 and 1,097 nmol of C2H2 reduced per mg (dry weight) per h, respectively, and nitrogenase activity was stimulated twofold by light. Ultrastructural observations revealed that numerous inclusion granules formed between the photosynthetic membranes in cells grown under nitrogen-fixing conditions. These Cyanothece strains possess many characteristics that make them particularly attractive for a detailed analysis of the interaction of nitrogen fixation and photosynthesis in an aerobic diazotroph.In all nitrogen-fixing organisms, the nitrogen fixation process is carried out by nitrogenase, an extremely oxygensensitive enzyme. Microorganisms have developed various strategies to protect their nitrogenase from oxygen inhibition (2, 5, 6). Among the nitrogen-fixing microorganisms, the cyanobacteria occupy a unique position because these are the only oxygenic photosynthetic organisms capable of nitrogen fixation under aerobic conditions (13). Nitrogen fixation has been reported for all three major morphological groups of cyanobacteria: heterocystous filamentous, nonheterocystous filamentous, and unicellular forms (5, 13). The oxygen protection mechanisms employed by these organisms vary considerably. In the heterocystous filamentous strains, about 5 to 10% of the cells undergo morphological differentiation into specialized cells called heterocysts under nitrogen-fixing conditions (13,49). In this arrangement, nitrogen fixation and photosynthetic oxygen evolution are spatially separated, so that oxygenic photosynthesis takes place in vegetative cells and nitrogen fixation occurs in heterocysts. The fixed nitrogen from these cells is exported to the neighboring vegetative cells, and the reductant for nitrogen fixation is imported from the vegetative cells (5, 49).There are many genera of nonheterocystous cyanobacteria, both filamentous and unicellular, that are capable of nitrogen fixation. The nonheterocystous filamentous forms have been placed in the genera Trichodesmium, Oscillatoria, an...

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It has been shown that some aerobic, unicellular, diazotrophic cyanobacteria temporally separate photosynthetic 02 evolution and oxygen-sensitive N2 fixation. Cyanothece sp. ATCC strain 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that fixes N2 during discrete periods of its cell cycle. When the bacteria are maintained under diurnal light-dark cycles, N2 fixation occurs in the darlk Similar cycling is observed in continuous light, implicating a circadian rhythm. Under N2-fixing conditions, large inclusion granules form between the thylakoid membranes. Maximum granulation, as observed by electron microscopy, occurs before the onset of N2 fixation, and the granules decrease in number during the period of N2 fixation.The granules can be purified from cell homogenates by differential centrifugation. Biochemical analyses of the granules indicate that these structures are primarily carbohydrate, with some protein. Further analyses of the carbohydrate have shown that it is a glucose polymer with some characteristics of glycogen. It is proposed that N2 fixation is driven by energy and reducing power stored in these inclusion granules. Cyanothece sp. strain ATCC 51142 represents an excellent experimental organism for the study of the protective mechanisms of nitrogenase, metabolic events in cyanobacteria under normal and stress conditions, the partitioning of resources between growth and storage, and biological rhythms.Some cyanobacteria use energy acquired through oxygenic photosynthesis to drive N2 fixation. Nitrogenase, the enzyme that catalyzes the six-electron reduction of dinitrogen to ammonia, is notoriously sensitive to molecular oxygen, so much so that it seems incompatible with the oxygen-evolving nature of photosynthetic organisms. However, cyanobacteria have devised remarkably elegant methods of protecting nitrogenase from contacting oxygen, including spatial separation, temporal separation, and efficient enzyme systems that destroy reactive 02 by-products (8).Aerobic, unicellular, diazotrophic cyanobacteria exhibit temporal separation of oxygenic photosynthetic activities and oxygen-sensitive N2 fixation (8) Dinitrogen fixation is an energy-demanding process, requiring several ATPs per molecule of combined nitrogen produced (41). Thus, for N2 fixation to continue in the dark, without light-driven energy production or exogenous nutrient sources, the cells must utilize internal stores of energy and reducing power. In fact, it has been shown that, under diazotrophic conditions, the aerobic, unicellular cyanobacteria accumulate large quantities of carbohydrates (9, 24-26). These pools of photosynthate accumulate during a phase of rapid CO2 fixation in the light and are then apparently utilized during the dark phase to drive N2 fixation (and presumably for maintenance energy). Work with both Synechococcus sp. strain Miami BG 043511 (24, 25) and Gloeothece spp. (9,26) has demonstrated that carbohydrates accumulate toward the end of the light phase and dissipate in the dark in cultures grown diazot...

A full-genome microarray of the (oxy)photosynthetic cyanobacterium Synechocystis sp. PCC 6803 was used to identify genes that were transcriptionally regulated by growth in iron (Fe)-deficient versus Fe-sufficient media. Transcript accumulation for 3,165 genes in the genome was analyzed using an analysis of variance model that accounted for slide and replicate (random) effects and dye (a fixed) effect in testing for differences in the four time periods. We determined that 85 genes showed statistically significant changes in the level of transcription (P Յ 0.05/3,165 ϭ 0.0000158) across the four time points examined, whereas 781 genes were characterized as interesting (P Յ 0.05 but greater than 0.0000158; 731 of these had a fold change Ͼ1.25ϫ). The genes identified included those known previously to be Fe regulated, such as isiA that encodes a novel chlorophyll-binding protein responsible for the pigment characteristics of low-Fe (LoFe) cells. ATP synthetase and phycobilisome genes were down-regulated in LoFe, and there were interesting changes in the transcription of genes involved in chlorophyll biosynthesis, in photosystem I and II assembly, and in energy metabolism. Hierarchical clustering demonstrated that photosynthesis genes, as a class, were repressed in LoFe and induced upon the re-addition of Fe. Specific regulatory genes were transcriptionally active in LoFe, including two genes that show homology to plant phytochromes (cph1 and cph2). These observations established the existence of a complex network of regulatory interactions and coordination in response to Fe availability.Fe is an essential element that is required for the growth and development of all organisms, including microorganisms (Hantke, 2001) and plants (Thimm et al., 2001;Negishi et al., 2002). Although Fe is abundant in nature, the availability of this element is very limited because of its poor solubility in aerobic environments. In the presence of oxygen at physiological pH, the rapid oxidation of the ferrous form to the ferric form leads to the precipitation of Fe and its essential unavailability. Thus, living organisms have developed various mechanisms to solubilize Fe to improve its bioavailability (Fox and Guerinot, 1998;Ratledge and Dover, 2000). Fe is of great importance for the growth of both pathogenic and nonpathogenic bacteria, and many strains devote a significant portion of their genome to the regulation of and the acquisition of Fe (Earhart, 1996;Paustian et al., 2001).Cyanobacteria are (oxy)photosynthetic organisms in which Fe stress has been studied in some detail (Straus, 1994;Behrenfeld and Kolber, 1999). Fe deficiency results in a variety of physiological and morphological changes in cyanobacteria, the most obvious of which is a significant change in cellular pigmentation. The overall changes include: loss of the light-harvesting phycobilisomes (Guikema and Sherman, 1983), changes in the fluorescence and absorption characteristics Sherman, 1983, 1984;Pakrasi et al., 1985aPakrasi et al., , 1985b, reduction in the numbe...

We utilized a full genome cDNA microarray to identify the genes that comprise the peroxide stimulon in the cyanobacterium Synechocystis sp. strain PCC 6803. We determined that a gene (slr1738) encoding a protein similar to PerR in Bacillus subtilis was induced by peroxide. We constructed a PerR knockout strain and used it to help identify components of the PerR regulon, and we found that the regulatory properties were consistent with the hypothesis that PerR functions as a repressor. This effort was guided by finding putative PerR boxes in positions upstream of specific genes and by careful statistical analysis. PerR and sll1621 (ahpC), which codes for a peroxiredoxin, share a divergent promoter that is regulated by PerR. We found that isiA, encoding a Chl protein that is induced under low-iron conditions, was strongly induced by a short-term peroxide stress. Other genes that were strongly induced by peroxide included sigD, sigB, and genes encoding peroxiredoxins and Dsb-like proteins that have not been studied yet in this strain. A gene (slr1894) that encoded a protein similar to MrgA in B. subtilis was upregulated by peroxide, and a strain containing an mrgA knockout mutation was highly sensitive to peroxide. A number of genes were downregulated, including key genes in the chlorophyll biosynthesis pathway and numerous regulatory genes, including those encoding histidine kinases. We used PerR mutants and a thioredoxin mutant (TrxA1) to study differential expression in response to peroxide and determined that neither PerR nor TrxA1 is essential for the peroxide protective response.