Cyanobacterial Nitrogen Fixation

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This ESF programme builds on the long- standing European research tradition in cyanobacterial nitrogen fixation. It is, however, the first coordinated European interdisciplinary programme in the field. The aim is to bring together researchers operating in a wide range of disciplines, from ecological to molecular and practical applications. This will allow gaps in knowledge to be bridged, synergism to be generated and scientific productivity to be thereby enhanced. Mobility of researchers among the various laboratories via fellowships and travel grants is therefore impor- Cyanobacterial Nitrogen Fixation (CYANOFIX) tant, as are other activities such as training summer schools and topical workshops, all An ESF scientific programme of which will be supported by CYANOFIX. At the end of the programme, one summarising symposium will be arranged. After water, it is the availability of nitrogen that limits crop productivity. Consequently the developed world commits a large portion of its energy budget to the production of nitrogen-based fertilisers. This underpins the impressive efficiency of modern inten- sive agriculture. However, overuse of nitrogenous fertiliser has caused problems such as the pollution of drinking and recreational water supplies. Paradoxically, Anabaenopsis sp. with terminal heterocysts. © Claudio Sili, CSMA-CNR. nitrogen is abundant in the Earth’s atmo- sphere (78%; 3.9 x 1015 tonnes), but very few organisms, all of them prokaryotes, are able to make use of atmospheric nitrogen. These organisms are termed nitrogen-fixers or diazotrophs. Nonetheless, some diazotrophs have been exploited in agriculture since classical times.

Many strains of cyanobacteria can fix N2 and these organisms make a major contribu- tion to the global nitrogen cycle. Cyanobacteria generate all of their cellular

The European Science carbon and nitrogen from atmospheric Foundation acts sources: they literally live on fresh air. as a catalyst Cyanobacteria possess unique mechanisms for the development of science by bringing for the protection of nitrogenase, the nitrogen-

together leading scientists fixing enzyme, against O2 and for nitrogen and funding agencies control of the expression of the N2-fixing to debate, plan and machinery and form symbioses with widely implement pan-European different organisms, and so have an impor- initiatives. tant role in modern N2 fixation research. 1 and ecology, to agronomy. Over 30 leading scientists from 11 different countries expressed a wish for their teams to be included in this programme and areas have been identified that are particularly suited to collaborative research. We predict that pooling the research investment currently being made by individual laboratories in Europe will have a synergistic effect on research progress. The various groups complement each other, bringing together key-laboratories and expertise in many different symbiotic and free- living model systems. Among the The ESF programme cyanobacterial-plant symbioses studied, expertise covers host plants from all major plant groups whilst Cyanobacterial N2 fixation has been exploited for fertilising purposes in groups that study free-living agriculture since antiquity. Several cyanobacteria cover all types of N2- diazotrophic cyanobacteria also form fixers. The groups also study differ- symbiotic associations with higher ent aspects of the progress of N2 plants, and references to the use in fixation: regulation, diversity, signal rice culture of one such symbiosis, transduction and the involvement of the water fern Azolla, go back 2000 specific genes and proteins in N2 years in the Chinese literature. fixation. In Europe, there is also a long- The five-year ESF programme (1998- standing tradition in the field of 2002) will focus on promoting mobility and transfer of knowledge cyanobacterial N2 fixation that goes back more than 100 years. And our among the European laboratories. To aim now is to build on this and achieve these goals, a steering com- establish the first coordinated Euro- mittee has been set up that has pean inter-disciplinary programme decided a series of actions that include short- and long-term travel on N2-fixing cyanobacteria. grants/fellowships, three training Cyanobacterial N2 fixation is a field summer schools, two workshops and that attracts researchers from many one symposium. disciplines, ranging from chemistry and chemical engineering, through biochemistry, physiology, genetics

Aims and objectives

The objectives of the collaborative research may be summarised under the following four headings: Physiology and molecular biology of

cyanobacterial N2 fixation.

The interrelations between N2

fixation and O2 have fascinated

2 products, the mechanisms that Anabaena cylindrica. regulate heterocyst differentiation © Claudio Sili, CSMA-CNR. will be clarified. Non-heterocystous cyanobacteria effect a temporal separation between

N2 fixation and photosynthetic O2 production. Typically, they fix N2 in the dark, and photosynthesise in the light. However, it is now emerging that different non-heterocystous cyanobacteria achieve this in different ways. In some, the nitrogenase proteins are turned over and can be detected only when cultures are scientists ever since it was discovered fixing N2 whilst, in others, nitroge- that nitrogenase was inactivated by nase persists, with activity coinciding with a transient activation of the Fe- exposure to O2. Since diazotrophic cyanobacteria have the ability simul- protein of this enzyme. Similarly, in some, the pattern of N2 fixation is taneously to generate O2 and fix N2, endogenous in that, once established the mechanisms that N2-fixers use in under alternating light and darkness, order to protect nitrogenase from O2 may be particularly well-developed in it persists under continuous illumi- cyanobacteria. nation: it may even be confined to a specific phase in the cell cycle. The Heterocystous cyanobacteria effect marine cyanobacterium a spatial separation between oxygenic Trichodesmium fixes N2 during the photosynthesis (in vegetative cells) light phase despite lacking hetero- and N2 fixation (in the non-photo- cysts but it seems to confine nitroge- synthetic heterocysts). Upon nitro- nase to a subset of cells of its fila- gen starvation, some filamentous ments. It is not clear whether or not cyanobacteria start a programme of these cells produce O2, so a detailed differentiation that leads to the examination of N2-fixing cells is formation of heterocysts. These cells needed. The precise mechanism(s) appear at semi-regular intervals along should be amenable to investigation The colony forming marine cyanobacterium Trichodesmium. the filaments and are the sites of N2 with mutants when proper genetics fixation. Heterocysts protect nitroge- © LM micrograph by have been developed. P. Lundgren, Department of Botany, nase from inactivation by O2 by Stockholm University. several mechanisms, including a high rate of respiration and decreased permeability to O2. Heterocysts do not evolve O2 and cannot fix CO2, so they rely on adjacent vegetative cells for a source of carbon. Studies on heterocyst development are being greatly facilitated by recent advances in the genetics of Anabaena. Genes involved in heterocyst development can now be tagged by transposon mutagenesis. By characterising mutants physiologically, by sequence analyses of tagged genes, and by biochemical analyses of the gene

3 Gloeothece In symbiotic cyanobacteria (typically membranacea, a Nostoc), release of nitrogen to the unicellular cyanobacterium that non-N2-fixing host plant is funda- photosynthesises mental for the survival of the during the day symbiotic association. However, the and fixes nitrogen ways in which the plant manages to at night. obtain fixed nitrogen from the N2- © Claudio Sili, CSMA-CNR. fixing Nostoc are largely unknown. Identification of such mechanisms, which may open ways for biotechnological applications, is a major goal. Cyanophycin is a copolymer of arginine and aspartate that consti- tutes a nitrogen-rich reserve material Nitrogenase consists of two pro- in N2-fixing cyanobacteria. Recent teins, a MoFe-protein and an Fe- progress on the enzymology of protein. In a few other diazotrophs, cyanophycin biosynthesis should be the Fe-protein of nitrogenase under- accompanied by an understanding of goes reversible covalent modification the turnover dynamics of (attachment of ADP-ribose) render- cyanophycin in relation to the ing the Fe-protein inactive. Covalent available nitrogen source. Further modification (inactivation) of investigation on this topic is of nitrogenase is stimulated by ammo- much interest. nium or sources, by transfer of cultures to the dark or exposure to The genetic regulation of nitrogen

O2. In a number of cyanobacteria the fixation in cyanobacteria differs Fe-protein can also be resolved into from that in other N2-fixers. The two components, but there is no presence of a source of combined evidence as yet that it can be co- nitrogen like nitrate or ammonium valently modified. Further investiga- in the environment generally pre- tion into this phenomenon will vents cyanobacteria from fixing N2. therefore be worthwhile. This behaviour makes bioenergetic sense, since assimilation of reduced The fate of the fixed nitrogen is forms of nitrogen is less expensive. another challenging aspect to Cyanobacteria fail to synthesise examine. Many N2-fixing nitrogenase when grown in the cyanobacteria release fixed nitrogen. presence of nitrate or ammonium, For example, under alternating light and it is clear that the genetic regula- and darkness, the unicellular tion of nitrogen metabolism in cyanobacterium Gloeothece fixes N2 cyanobacteria is different. For ex- in the dark, during which some of ample, expression of nitrogen the fixed nitrogen is released into its assimilation genes – including genes external slime capsule. This nitrogen involved in heterocyst differentia- is re-assimilated during the following tion and ammonia assimilation (e.g. light phase and the nitrogenase hetR and glnA) – is regulated by the activity may escape inhibition by recently discovered NtcA transcrip- accumulated nitrogen. In many tional regulator. Important aspects to cases, the release of amino acids is study in relation to NtcA function quite specific and transport systems are: a) how is NtcA activity modu- have been described and the lated in response to the nitrogen corresponding genes identified. status of the cell; b) which is the

4 earliest heterocyst differentiation Trichodesmium, Nostoc and Calothrix) gene that responds to NtcA; c) is some of which have been resistant to NtcA function important for regula- isolation of DNA and to genetic tion of N2 fixation in non-heterocys- transformation. Development of an tous and symbiotic cyanobacteria? appropriate genetic methodology in The PII protein (encoded by glnB), this type of cyanobacteria represents involved in the co-ordination of C- a high research priority in the and N-assimilation, has been programme. characterised in some N2-fixing Recent evidence suggests that some cyanobacteria. The interesting heterocystous cyanobacteria contains hypothesis that PII may be involved two molybdenum-based nitrogenase in nitrogen signalling in cyanobacteria systems, nif1 and nif2, with different can therefore be raised. However, as expression patterns. The vegetative PII can directly bind molecules cell nitrogenase (Nif2) has its coun- relevant to conditions of nitrogen terpart in non-heterocystous stress, it may not only act as a signal cyanobacteria that fix N only transducer but also as a sensor. 2 anaerobically. Furthermore, some Other stress responses may be cyanobacteria may produce Mo- elicited by exposure of cyanobacteria independent ‘alternative’ (V or Fe) to altered light, temperatures, desic- nitrogenases, encoded by the vnf and cation (osmotic stress) and nutrient anf genes. This is a topic amenable to starvation. Synthesis of stress pro- combining genetic work with bio- teins, such as molecular chaperones, chemical and ecological studies. is already being studied in some laboratories, others study the effects Symbiotic cyanobacteria Symbiotically competent of stresses on N2 fixation under natural conditions (light, tempera- cyanobacteria have some excellent ture, salinity, nutrients etc). In features that make them particularly addition, many of the responses that significant in any attempt to extend cyanobacteria show to environmen- the list of N2-fixing symbioses to tal stress are independent of the include plants of commercial nature of the stress itself, though interest, such as cereals. Unlike some responses are specific. The rhizobia (the symbionts of legume extent to which stress responses are plants), most symbiotic interlocked is currently a fascinating cyanobacteria carry their own area and one in which co-ordinated mechanism for protecting efforts will be valuable. nitrogenase from inactivation by oxygen (heterocysts). They have an Genetics of cyanobacteria unmatched host range (fungi to

One curious paradox concerning N2- angiosperms), are not restricted to fixing cyanobacteria is that, whilst roots but may form symbiosis with our understanding of the molecular various plant parts, and do not need genetics of some free-living hetero- to be located intracellularly within cystous strains (notably Anabaena) the host plant. However, before we has now outstripped our knowledge can establish new N2-fixing of their physiology and biochemistry, symbioses we may need to expand little is known about the genetics of our knowledge of those that already other cyanobacteria. We therefore exist. It is becoming increasingly need to expand our understanding of apparent that the symbionts the genetics of some hitherto rather communicate using chemical signals neglected free-living and symbiotic and during the establishment of a organisms, (e.g. Gloethece, functional N2-fixing cyanobacterial

5 Coralloid root of Cycas the warmer Mediterranean and revoluta; cyanobiont tropical areas. Heterocystous zone is visible in the cyanobacteria are clearly best adapted sectioned roots. © Maria Cristina for diazotrophic growth, but Margheri, CSMA-CNR. cyanobacteria in group b) also manage well without heterocysts. However, cyanobacteria in group c), constituting 30-40% of all non- heterocystous cyanobacteria, lack one or more O2 protective mechanisms. Sulfureta are environments characterised by high concentrations of sulphide and extended periods of symbiosis, it is likely anoxia. Some anaerobic N2-fixing that genes are induced cyanobacteria occur in such environ- in both partners, the ments. Sulphide is an inhibitor of products of which oxygenic photosynthesis and, at high would then assist in concentrations, cannot co-exist with the establishment of O2. In some systems, cyanobacteria the symbiosis. We will therefore perform anoxygenic study the nature of photosynthesis whilst, in others, such genes and their photosynthesis is temporarily The bright red stem products as well as communication switched off by sulphide, thereby glands on Gunnera between the symbionts throughout allowing N2 fixation to flourish. through which the symbiosis. Another aspect of vital Evidence has also been obtained for a cyanobacterium Nostoc enters when biotechnological importance to spatial separation of oxygenic photo- establishing the Nostoc- study, is the transfer of fixed synthesis and N2 fixation within Gunnera symbiosis. nitrogen to the host plant and the lamined benthic mats. Either fixed © C. Johansson, reciprocal flow of carbon to the nitrogen is transported or organisms Department of Botany, move through the system, changing Stockholm University. cyanobacterium. from N2 fixation to photosynthesis. Natural communities of N2-fixing cyanobacteria Microbial mats are typically built by

N2-fixing cyanobacteria can be non-heterocystous cyanobacteria subdivided into three groups: a) (e.g. Oscillatoria, Lyngbya, Symploca) filamentous cyanobacteria with though heterocystous species may heterocysts; b) non-heterocystous occasionally dominate. Many mats filamentous or unicellular are strongly diffusion limited and

cyanobacteria, capable of N2 fixation accumulate photosynthetic O2 under fully aerobic conditions; and c) during the day, while at night the non-heterocystous filamentous or system turns anaerobic, probably

unicellular cyanobacteria that induce permitting N2 fixation by some non- nitrogenase activity only anaerobi- heterocystous strains. Much more cally. Representatives of all of these work is needed to understand such groups (free-living and symbiotic) phenomena in natural systems. are found in a wide range of environments ranging from terrestrial to Planktonic cyanobacteria are com- aquatic environments, the latter mon in lakes as well as in open oceans including limnic (lakes and streams), and in the Baltic Sea. Recent calcula- brackish (e.g. the Baltic Sea) and fully tions indicate that marine N2 fixation marine habitats (oceans), and is in fact of major global importance. cyanobacteria may be found from Interestingly, the major N2-fixing cold polar regions (e.g. Svalbard) to cyanobacterium in oceans is an

6 aerobic, non-heterocystous species, Calothrix. © Arnaud Taton Trichodesmium. In contrast, freshwa- ter blooms of diazotrophic cyanobacteria consist of heterocystous species (e.g. Anabaena), and these are also dominant in brackish systems such as the Baltic Sea with blooms of Nodularia and Aphanizomenon. Heterocystous species are also found as symbionts in marine plankton (diatoms), so the perennials. There is also an urgent absence of free-living representatives need to identify the ecological niches in the open ocean remains a mystery. that are occupied by the different N2- There is a great need to fully under- fixing cyanobacteria. stand the behaviour of these bloom forming cyanobacteria in their Cyanobacterial systematics using natural environment. various genetic techniques will in addition be an extremely valuable In combination with classical mea- complement to studies on N2 fixa- surements of N2 fixation there is also tion. A precise taxonomic position of a need to study nif gene expression cyanobacteria living in different and the occurrence of nitrogenase in environments will allow comparisons natural systems. The regulation of of data collected in different ecologi- nitrogenase activity by environmen- cal niches and cultivated in different tal factors (e.g. N, P, Fe) and the laboratories. This is also required in transport of fixed nitrogen within order to evaluate the diversity of the the ecosystem, are other important N2-fixing machinery in areas to be addressed. Also, effects on cyanobacteria. A knowledge of the N2 fixation of changes in climate and biodiversity of symbiotic in anthropogenic activities are of cyanobacteria in relation to host immediate interest, also in a Euro- species, habitat and geographical pean perspective. Cyanobacteria location will contribute to an under- growing in terrestrial locations standing of the specificity of (including culturally important cyanobacterial symbioses and will archaeological sites) and in streams form a valuable basis for the future and rivers need to be studied. The elaboration of artificial symbioses. latter are particularly interesting because they differ from their counterparts in lakes by being

Funding

ESF scientific programmes are Nazionale delle Ricerche, Italy; principally financed by the Consejo Superior de Investigaciones Foundation’s Member Organisations Científicas, Oficina de Ciencia y on an à la carte basis. CYANOFIX is Tecnología de la Comisión supported by: Fonds National de la Interministerial de Ciencia y Recherche Scientifique, Belgium; Tecnología, Spain; Naturvetens- Suomen Akatemia/Finlands kapliga Forskningsrådet, Sweden. Akademi, Finland; Deutsche Forschungsgemeinschaft, Germany; Enterprise Ireland, Ireland; Consiglio

7 CYANOFIX Steering Committee

Professor Birgitta Bergman (Chair) Dr. Stefano Ventura Stockholm University Centro di Studio dei Microrganismi Dept of Botany Autotrofi CNR 10691 Stockholm p le delle Cascine 27 Sweden 50144 Florence Tel: +46 8 16 37 51 Italy Fax: +46 8 16 55 25 Tel: +39 055 350 542 E-mail: [email protected] Fax: +39 055 330 431 E-mail: [email protected] Dr. Enrique Flores (Vice-Chair) CSIC-University of Sevilla Dr. Annick Wilmotte Inst. Bioquimica Vegetal y Fotosintesis University of Liège Centro de Investigaciones Cientificas Isla Dept of Botany B22 de la Cartuja Lab. Algology, Mycology and Avda. Americo Vespucio s/n Experimental Systematics 41092 Sevilla 4000 Liège Spain Belgium Tel: +34 95 448 9523 Tel: +32 4 366 38 56 Fax: +34 95 446 0065 Fax: +32 4 366 28 53 E-mail: [email protected] E-mail: [email protected]

Professor Herbert Böhme Universität Bonn Dr. Annette Moth-Wiklund ESF Senior Scientific Secretary Botanisches Institut Kirschallee 1 Ms. Pat Cosgrove 53115 Bonn ESF Administrator Germany European Science Foundation Tel: +49 228 735 515 1 quai Lezay-Marnésia Fax: +49 228 735 513 67080 Strasbourg Cedex E-mail: [email protected] France WWW Home Page: Alternate member: http://www.esf.org Tel: +33 (0)3 88 76 71 06 Dr. Thomas Happe Universität Bonn Fax: +33 (0)3 88 37 05 32 Botanisches Institut E-mail: [email protected] Kirschallee 1 53115 Bonn Germany For the latest information on this Tel: +49 228 732 075 programme consult the CYANOFIX Fax: +49 228 735 513 home pages: E-mail: [email protected] http://www.esf.org/CYANOFIX Dr. Bruce Osborne AND University College Dublin http://www.area.fi.cnr.it/cyanofix/ Department of Botany Belfield Dublin 4

Ireland 993098 d’ordre : ° Tel: +353 1 706 22 49 Fax: +353 1 706 11 53 E-mail: [email protected]

Dr. Kaarina Sivonen University of Helsinki Dept Applied Chemistry and Microbiology PL 56, Viikki Biocenter Viikinkaari 9 00014 University of Helsinki Finland Tel: +358 9 708 59 270 Fax: +358 9 708 59 322 September 1999