SummaryAzotobacter vinelandii NIFL is a nitrogen fixationspecific regulatory flavoprotein that modulates the activity of the transcriptional activator NIFA in response to oxygen and fixed nitrogen in vivo. NIFL is also responsive to ADP in vitro. Limited proteolysis of NIFL indicates that it comprises a relatively stable N-terminal domain and a C-terminal domain that is protected from trypsin digestion in the presence of adenosine nucleotides. ATP protects the protein from cleavage in the vicinity of potential nucleotide-binding sites in the C-terminus, whereas ADP protects the entire C-terminal domain. NIFL has an apparent K d of 130 M for ATP and 16 M for ADP. The purified N-terminal domain has an identical UV/visible absorption spectrum to the wild-type protein and is reduced by sodium dithionite, demonstrating that it is a flavinbinding domain. The isolated N-terminal domain does not inhibit NIFA activity. A subdomain fragment containing 160 residues of the C-terminal region, including the nucleotide-binding sites, is also not competent to inhibit NIFA. Removal of the first 146 residues of NIFL, which includes a conserved S-motif (PAS-like domain), found in a large family of sensory proteins from eubacteria, archea and eukarya eliminates the redox response. However, this truncated protein remains competent to inhibit NIFA activity in response to ADP in vitro and to the level of fixed nitrogen in vivo. The redox and nitrogen-sensing functions of A. vinelandii NIFL are therefore separable and are discrete functions of the protein.

The enhancer-binding protein NIFA is required for transcriptional activation of nif promoters by the alternative holoenzyme form of RNA polymerase, which contains the sigma factor 54 ( N ). NIFA hydrolyzes nucleoside triphosphates to catalyze the isomerization of closed promoter complexes to transcriptionally competent open complexes. The activity of NIFA is antagonized by the regulatory protein NIFL in response to oxygen and fixed nitrogen in vivo. We have investigated the requirement for nucleotides in the formation and stability of open promoter complexes by NIFA and inhibition of its activity by NIFL at the Klebsiella pneumoniae nifH promoter. Open complexes formed by 54 -containing RNA polymerase are considerably more stable to heparin challenge in the presence of GTP than in the presence of ATP. This differential stability is most probably a consequence of GTP being the initiating nucleotide at this promoter. Adenosine nucleosides are specifically required for Azotobacter vinelandii NIFL to inhibit open complex formation by native NIFA, and the nucleoside triphosphatase activity of NIFA is strongly inhibited by NIFL under these conditions. We propose a model in which NIFL modulates the activity of NIFA via an adenosine nucleotide switch.A distinct mechanism of transcriptional activation is observed among the family of prokaryotic enhancer-binding proteins which interact with the holoenzyme form of RNA polymerase containing the alternative sigma factor 54 (E 54 ) (19,26). The nitrogen fixation regulatory protein NIFA is a member of this family which binds to upstream activator sequences (UAS) and catalyzes the isomerization of closed promoter complexes to the open complex in a reaction which requires hydrolysis of a nucleoside triphosphate (25). Productive interactions between NIFA and E 54 are enabled by DNA loop formation, which is facilitated by the binding of integration host factor (IHF) (18,30). The amino acid sequence of NIFA conforms to the three-domain model for the structure of pneumoniae NIFA activates transcription in the absence of specific DNA binding and possesses nucleoside triphosphatase activity (6). In contrast to its K. pneumoniae counterpart, the native Azotobacter vinelandii NIFA protein has been purified in a soluble form, and its properties with respect to DNA binding and catalysis of open complex formation have been characterized in vitro (2).In both K. pneumoniae and A. vinelandii, the activity of NIFA is controlled by a second regulatory protein, NIFL, in response to the environmental effectors oxygen and fixed nitrogen (7,22). Although NIFL proteins show homology in their C-terminal domains to the histidine protein kinase family of two-component regulatory proteins (14), NIFL and NIFA appear to interact at stoichiometric levels (5, 15), and phosphotransfer between the two proteins has not been detected in vitro (2, 21). Moreover, although A. vinelandii NIFL shows greater homology to the canonical histidine protein kinases than does K. pneumoniae NIFL and contains a conserved...

Rates of carbon (C) specific growth and nitrogen (N2) fixation were monitored in cultures of Baltic Sea Nodularia and Aphanizomenon exposed to gradual limitation by inorganic phosphorus (P). Both cyanobacteria responded by decreased cellular P content followed by lowered rates of growth and N2 fixation. C-specific growth and cellular N content changed faster in Aphanizomenon both when inorganic P was lowered as well as during reintroduction of P. Aphanizomenon also showed a more rapid increase in N-specific N2 fixation associated with increased C-specific growth. When ambient concentrations of inorganic P declined, both cyanobacteria displayed higher rates of alkaline phosphatase (APase) activity. Lower substrate half-saturation constants (KM) and higher Vmax : KM ratio of the APase enzyme associated with Nodularia suggest a higher affinity for dissolved organic P (DOP) substrate than Aphanizomenon. Aphanizomenon, which appears more sensitive to changes in ambient dissolved inorganic P, may be adapted to environments with elevated concentrations of P or repeated intrusions of nutrient-rich water. Nodularia on the other hand, with its higher tolerance to increased P starvation may have an ecological advantage in stratified surface waters of the Baltic Sea during periods of low P availability.

Summary 449 I. INTRODUCTION 450 II. THE PARTNERS 451 1. Cyanobionts and their role 451 2. Hosts and their role 453 3. Location of cyanobionts in their hosts 455 III. INITIATION AND DEVELOPMENT OF SYMBIOSES 458 1. Initiation of symbioses 458 2. Geosiphon pyriforme 458 3. Cyanolichens 459 4. Liverworts and hornworts 460 5. Azolla 460 6. Cycads 461 7. Gunnera 461 IV. THE SYMBIOSES 462 1. Geographical distribution and ecological significance 462 2. Benefits to the partners 462 (a) Benefits to the cyanobionts 462 (b) Benefits to the hosts 463 3. Duration and stability 463 4. Mode of transmission and perpetuation 463 5. Recognition between the partners 464 6. Specificity and diversity 464 7. Symbiosis‐related genes 465 8. Modifications of the cyanobiont 466 (a) Growth and morphology 466 (b) Photosynthesis and carbon metabolism 467 (c) Glutamine synthetase 467 (d) Heterocysts 469 (e) N2fixation 470 9. Nutrient exchange 471 (a) Carbon 471 (b) Nitrogen 472 V. EVOLUTIONARY ASPECTS 472 VI. ARTIFICIAL SYMBIOSES 474 VII. FUTURE OUTLOOK AND PERSPECTIVES 475 1. Cryptic symbioses 476 2. Developmental profile of symbiotic tissues 476 3. Sensing and signalling 476 4. Genetic aspects 476 5. Physiological and biochemical aspects of nutrient exchange 477 6. Microaerobiosis 477 7. Potential applications 477 Acknowledgements 477 References 477 Cyanobacteria are an ancient, morphologically diverse group of prokaryotes with an oxygenic photosynthesis. Many cyanobacteria also possess the ability to fix N2. Although well suited to an independent existence in nature, some cyanobacteria occur in symbiosis with a wide range of hosts (protists, animals and plants). Among plants, such symbioses have independently evolved in phylogenetically diverse genera belonging to the algae, fungi, bryophytes, pteridophytes, gymnosperms and angiosperms. These are N2‐fixing symbioses involving heterocystous cyanobacteria, particularly Nostoc, as cyanobionts (cyanobacterial partners). A given host species associates with only a particular cyanobiont genus but such specificity does not extend to the strain level. The cyanobiont is located under a microaerobic environment in a variety of host organs and tissues (bladder, thalli and cephalodia in fungi; cavities in gametophytes of hornworts and liverworts or fronds of the Azolla sporophyte; coralloid roots in cycads; stem glands in Gunnera). Except for fungi, the hosts form these structures ahead of the cyanobiont infection. The symbiosis lasts for one generation except in Azolla and diatoms, in which it is perpetuated from generation to generation. Within each generation, multiple fresh infections occur as new symbiotic tissues and organs develop. The symbioses are stable over a wide range of environmental conditions, and sensing–signalling between partners ensures their synchronized growth and development. The cyanobiont population is kept constant in relation to the host biomass through controlled initiation and infection, nutrient supply and cell division. In most cases, the partners have remaine...