Cyanobacterial Heterocysts Research Articles

Hopanoid lipids compose the Frankia vesicle envelope, presumptive barrier of oxygen diffusion to nitrogenase.

Biological nitrogen fixation in aerobic organisms requires a mechanism for excluding oxygen from the site of nitrogenase activity. Oxygen exclusion in Frankia spp., members of an actinomycetal genus that forms nitrogen-fixing root-nodule symbioses in a wide range of woody Angiosperms, is accomplished within specialized structures termed vesicles, where nitrogen fixation is localized. The lipidic vesicle envelope is apparently a functional analogue of the cyanobacterial heterocyst envelope, forming an external gas-diffusion barrier around the nitrogen-fixing cells. We report here that purified vesicle envelopes consist primarily of two hopanoid lipids, rather than of glycolipids, as is the case in cyanobacteria. One envelope hopanoid, bacteriohopanetetrol phenylacetate monoester, is vesicle-specific. The Frankia vesicle envelope thus represents a layer specific to the locus of nitrogen fixation that is biosynthetically uniquely derived.

Genetic analysis of functional differences among distinct ferredoxins in Rhodobacter capsulatus

Rhodobacter capsulatus has been known to possess two ferredoxins (I and II) with distinct physicochemical and structural properties: ferredoxin I is a 2[4Fe-4S] type and the other is a [3Fe-4S] [4Fe-4S] type. To analyze their possible functional differences, their genes (fdxN and fdxA) were cloned, sequenced, and subjected to interposon mutagenesis experiments. The former gene was adjacent to a gene encoding a chloroplast-type [2Fe-2S] ferredoxin (fdxC). Mutants with inactivated fdxN and/or fdxC were obtained, and they showed virtually no growth under nitrogen-fixing conditions. Complementation experiments confirmed that both fdxN and fdxC were required for nitrogen fixation. On the other hand, we have not been able to disrupt fdxA under the screening conditions surveyed, including conditions that do not require nitrogenase activity for growth, suggesting that ferredoxin II could have an unknown essential role(s). These indicate functional differences among multiple ferredoxins in one bacterium other than in cyanobacterial heterocysts and indispensability of certain ferredoxins in nitrogen fixation other than Rhizobium meliloti FdxN.

Occurrence of the 32-kDa QB-binding protein of photosystem II in vegetative cells, heterocysts and akinetes ofAzolla carotiniana cyanobionts

Transmission electron microscopy and immunocytological labeling were used to localize the 32-kilodalton (kDa) protein (DI polypeptide) of photosystem II in different cell types of the cyanobionts within leaf cavities ofAzolla caroliniana Willd. The 32-kDa protein binds the secondary electron acceptor QB, and is highly conserved between plants and cyanobacteria. Three antisera, specific for different epitopes of the 32-kDa protein, were used as primary antibodies. Immunologically recognizable 32-kDa protein was localized on membranes ofAzolla chloroplasts, vegetative cyanobacterial cells, akinetes, and heterocysts that were at all stages of the differentiation process. The 32-kDa protein was not detected in nonphotosynthetic endosymbiotic bacteria found within leaf cavities. The amount of the 32-kDa protein observed in different cyanobacterial cell types was dependent upon the primary antiserum used and membrane orientation within a cell with respect to the plane of sectioning. Therefore, although 32-kDa protein was present in all three cyanobacterial cell types and clear trends in labeling patterns could be elucidated, it was not possible to quantitate the amounts of protein with respect to either cell type or leaf-cavity age.

Occurrence of the 32-kDa QB-binding protein of photosystem II in vegetative cells, dheterocysts and akinetes of Azolla carotiniana cyanobionts

Transmission electron microscopy and immunocytological labeling were used to localize the 32-kilodalton (kDa) protein (DI polypeptide) of photosystem II in different cell types of the cyanobionts within leaf cavities of Azolla caroliniana Willd. The 32-kDa protein binds the secondary electron acceptor QB, and is highly conserved between plants and cyanobacteria. Three antisera, specific for different epitopes of the 32-kDa protein, were used as primary antibodies. Immunologically recognizable 32-kDa protein was localized on membranes of Azolla chloroplasts, vegetative cyanobacterial cells, akinetes, and heterocysts that were at all stages of the differentiation process. The 32-kDa protein was not detected in nonphotosynthetic endosymbiotic bacteria found within leaf cavities. The amount of the 32-kDa protein observed in different cyanobacterial cell types was dependent upon the primary antiserum used and membrane orientation within a cell with respect to the plane of sectioning. Therefore, although 32-kDa protein was present in all three cyanobacterial cell types and clear trends in labeling patterns could be elucidated, it was not possible to quantitate the amounts of protein with respect to either cell type or leaf-cavity age.

Immunofluorescence detection and characterization of N2-fixing microorganisms from aquatic environments

The role of N2-fixing microheterotrophs in marine nitrogen budgets is poorly understood, primarily due to the absence of appropriate technology for assessing the in situ distribution and abundance of these microorganisms. We describe here the application of immunofluorescence microscopy for identifying and characterizing marine N2-fixing microorganisms. Polyclonal antibodies raised against the Fe-S protein of the nitrogenase enzyme complex from the bacterium Rhodospirillum rubrum were demonstrated to recognize nitrogenase from both marine and freshwater N2-fixing bacteria and cyanobacteria. Initial tests of the antibodies were conducted with immunoblots of cell lysates and indicated monospecificity for nitrogenase. We also conducted immunofluorescence assays on paraformaldehyde-fixed whole bacterial cells made permeable with lysozyme, allowing microscopic examination and identification of cells containing the enzyme. Immunofluorescence microscopy also revealed the localization of nitrogenase within cyanobacterial heterocysts or filaments. The approach and techniques described here provide reliable means to determine the presence, relative abundance, and distribution of N2-fixing microorganisms in natural microbial assemblages.

Differences in fatty acid composition between vegetative cells and N 2 -fixing vesicles of Frankia sp. strain CpI1

When growing on N(2), actinomycetes from the genus Frankia form multicellular structures that contain nitrogenase. The structures are referred to as vesicles and are indistinguishable from vesicles formed when Frankia sp. are in root-nodule symbioses. Vesicles isolated from N(2)-grown cells of Frankia sp. strain CpI1 had a significantly higher amount and different composition of fatty acids than did vegetative cells recovered from NH(4) (+)-containing medium. Lipids from vesicles, whole cells grown on N(2), and whole cells grown on NH(4) (+) were fractionated by silicic acid chromatography into neutral lipids, glycolipids, and polar lipids. The fatty acids were transesterified by methanolysis and analyzed by gas chromatography and mass spectrometry. Vesicles had considerably higher amounts of fatty acids in the neutral and glycolipid fractions but lower amounts of polar lipid fatty acids than did vegetative cells. Polar lipids from vesicles had a higher proportion of mono-unsaturated and cyclopropane fatty acids and a lower proportion of isobranched fatty acids than did polar lipids from NH(4) (+)-grown or N(2)-grown cells. The neutral lipid and glycolipid fractions contained several long-chain compounds with molecular ions at m/z 408 and 410. The proportions of these compounds were significantly higher in the lipids from vesicles than from vegetative cells. These results suggest that lipids in vesicles might be involved in the protection of nitrogenase from O(2) and suggest a parallel with the glycolipids involved in protecting nitrogenase from O(2) in the cyanobacterial heterocysts.

Rearrangement of nif genes during cyanobacterial heterocyst differentiation

Anabaena is a filamentous cyanobacterium that produces specialized cells, called heterocysts, at regular intervals along each filament when deprived of fixed nitrogen under aerobic conditions. Heterocysts are anaerobic factories for nitrogen fixation. In Anabaena vegetative-cell DNA, the nifD gene, encoding the α subunit of nitrogenase, is interrupted by an 11 000 base pair DNA element. During the differentiation of heterocysts from vegetative cells, this 11 kilobase (kb) element is excised by site-specific recombination between short, directly repeated DNA sequences present at the ends of the element. The excision results in restoration of the nifD coding sequence and of the entire nifHDK transcription unit. A gene has been identified, within the 11 kb element, that is believed to encode the site-specific recombinase responsible for excision of the element during heterocyst differentiation. A second developmentally regulated gene arrangement has also been observed in Anabaena . This event occurs close to the nifS gene and involves a different set of repeated sequences, implying a different site-specific recombination system.

Monitoring energy conversion in photosystem I of cyanobacterial heterocysts by photoacoustic spectroscopy

Photoacoustic spectroscopy was used to monitor photosynthetic energy storage in heterocysts isolated from the cyanobacterium Anabaena 7120. Three main energy-conversion pathways have been identified in these specialized cells: cyclic electron transport in photosystem I, nitrogen fixation, and respiration. The aim of this study was to define the conditions for estimating their interdependence. The gas phase (air, Ar, N2, H2) under which the sample was prepared or incubated, as well as the presence of electron donors (NADPH, NADH, H2) and KCN (an inhibitor of respiration), determined the relative flux through the pathways. Heterocysts in which respiration and nitrogen fixation are inhibited have afforded a useful system for specific study of cyclic electron transport around photosystem I. Under optimal conditions for photosystem I turnover, photochemical losses of above 20% were found.

Glutamine synthetase: activity and localization in cyanobacteria of the cycadsCycas revoluta andZamia skinneri

Nitrogen-fixing cyanobacteria inhabit the zone between the inner and outer cortex of cycad coralloid roots. In the growing tip of such roots the cyanobacterial heterocyst frequency, nitrogenase activity (C2H2-reduction) and glutamine synthetase activity (both transferase and biosynthetic) were comparable to those found in freeliving cyanobacteria. The relative level of glutamine synthetase protein and its pattern of cellular/subcellular localization in heterocysts and vegetative cells were also similar to those of free-living cyanobacteria. However, there was a progressive decline in nitrogenase activity along the coralloid root with maximum reduction occurring in the regions farthest from the growing tip. A similar but less pronounced pattern was observed for glutamine synthetase activity. Distribution of glutamine synthetase protein in cyanobacteria in the first 2-3 mm of the root tip indicated a slight decrease in the heterocysts and vegetative cells. However, the overall level of cyanobacterial glutamine synthetase protein did not change because of a drastic increase in the numbers of heterocysts, which contain a proportionally higher level of glutamine synthetase than the vegetative cells.

Concentration and Function of Membrane-Bound Cytochromes in Cyanobacterial Heterocysts

Membranes isolated from heterocysts and vegetative cells of Anabaena 7120 were assayed for content of cytochrome f, cytochrome b-563, cytochrome b-559(HP), cytochrome b-559(LP), and cytochrome aa(3) by use of reduced-minus-oxidized difference spectra. The level of cytochrome aa(3) in heterocyst membranes was 4 to 100 times higher than that in vegetative cells of Anabaena 7120 or other species of cyanobacteria. Heterocyst membranes lack cytochrome b-559(HP) but contain cytochrome b-559(LP) (E(m7.5) = +77 millivolts, n = 1) at approximately the same concentration as cytochrome f. The role of cytochrome b-559(LP) in the hydrogenase-dependent electron transfer pathway was investigated with the inhibitor 2-(n-heptyl)-4-hydroxyquinoline N-oxide which blocks electron flow from hydrogenase to acceptors reacting with the plastoquinone pool. Addition of inhibitor elicited no change in the reduction level of cytochrome b-559(LP) indicating that this cytochrome is not directly involved in this pathway.

Reconstitution of photosynthetic electron transfer in cyanobacterial heterocyst membranes

Photosynthetic membranes have been isolated from heterocysts of the cyanobacterium Anabaena 7120. Electron transfer reactions associated with photosystem I have been investigated using single turnover flashes. Turnover rates, which were dependent on reconstitution with cytochrome c-553 or plastocyanin, were maximal when a suitable electron donor system was provided. Hydrogen (via an endogenous uptake hydrogenase), NADH (via a dehydrogenase) or the ascorbate/dichlorophenolindophenol couple were all effective donors. NADPH could also serve as a donor but this system was dependent on addition of ferredoxin and ferredoxin—NADP reductase. Turnovers of P700, soluble cytochrome c-553 and cytochrome b-563 could be demonstrated.

Factors influencing the stability of nitrogenase activity in isolated cyanobacterial heterocysts

Factors influencing the stability of nitrogenase activity in isolated cyanobacterial heterocysts

Factors influencing the stability of nitrogenase activity in isolated cyanobacterial heterocysts

Factors influencing the stability of nitrogenase activity in isolated cyanobacterial heterocysts