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22 Feuillet, C., Schachermayr, G. and Keller, B. (1997) Molecular cloning of a
new receptor-like kinase gene encoded at the Lr10 disease resistance locus of
wheat, Plant J. 11, 45–52
23 Zhou, J. et al. (1995) The tomato gene Pti1 encodes a serine/threonine kinase
that is phosphorylated by Pto and is involved in the hypersensitive response,
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24 Zhou, J., Tang, X. and Martin, G.B. (1997) The Pto kinase conferring
resistance to tomato bacterial speck disease interacts with proteins that bind a
cis-element of pathogenesis-related genes, EMBO J. 16, 3207–3218
25 Ligterink, W. et al. (1997) Receptor-mediated activation of a MAP kinase in
pathogen defense of plants, Science 276, 2054–2057
26 Xing, T. Higgins, V.J. and Blumwald, E. (1996) Regulation of plant defense
response to fungal pathogens: two types of protein kinases in the reversible
phosphorylation of the host plasma membrane H+-ATPase, Plant Cell 8, 555–564
27 Roberts, D.M. and Harmon, A.C. (1992) Calcium-modulated proteins: targets
of intracellular calcium signals in higher plants, Annu. Rev. Plant Physiol.
Plant Mol. Biol. 43, 375–414
28 Sheen, J. (1996) Ca2+-dependent protein kinase and stress signal transduction,
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29 Tavernier, E. et al. (1995) Involvement of free calcium in action of cryptogein,
a proteinaceous elicitor of hypersensitive reaction in tobacco cells,
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30 Xu, H. and Heath, M.C. (1998) Role of calcium in signal transduction during
the hypersensitive response caused by basidiospore-derived infection of the
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31 Atkinson, M.M. and Baker, C.J. (1989) Role of the plasmalemma H+-ATPase
in Pseudomonas syringae-induced K+/H+ exchange in suspension-cultured
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32 De Wit, P.J.G.M. (1995) Fungal avirulence genes and plant resistance genes:
unravelling the molecular basis of gene for gene interactions, Adv. Bot. Res.
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33 De Koninck, P. and Schulman, H. (1998) Sensitivity of CaM Kinase II to the
frequency of Ca2+ oscillations, Science 279, 227–230
34 Allen, G.J. and Sanders, D. (1997) Vacuolar ion channels, Adv. Bot. Res. 25,
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35 Mehdy, M.C. (1994) Active oxygen species in plant defense against
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36 Segal, A.W. and Abo, A. (1993) The biochemical basis of the NADPH
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37 Dwyer, S.C. et al. (1996) Plant and human neutrophil oxidative burst
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Eduardo Blumwald*, Gilad S. Aharon and Bernard C-H. Lam are at
the Dept of Botany, University of Toronto, 25 Willcocks St,
Toronto, Ontario, Canada M5S 3B2.
*Author for correspondence (tel +1 416 978 2378;
fax +1 416 978 5878; e-mail [email protected]).
Regulation of nitrogen fixation in
heterocyst-forming cyanobacteria
Herbert Böhme
Some cyanobacteria are able to reduce atmospheric dinitrogen to ammonia – a process
where oxygen evolved by photosynthetic activity in the same cell is detrimental to nitrogen
fixation. Strategies to avoid oxygen range from temporal separation of nitrogen fixation and
oxygen evolution (in unicellular and filamentous, non-heterocystous strains) to spatial separation and cellular differentiation into nitrogen fixing heterocysts (in filamentous cyanobacteria).
Recent research has begun to clarify the genes involved in nitrogen fixation, the mechanisms
that regulate the expression of proteins involved in the assimilation of nitrogen from different
sources and the way in which the cell is able to sense its nitrogen status.
C
yanobacteria (blue-green algae) are a diverse group of prokaryotes. A common feature is their oxygenic photosynthesis, which is similar to that in algae and higher plants
and is the most important biological mechanism for capturing solar
energy. As sunlight is their energy source and water the reductant,
they generate oxygen in the light. Energy and reductant generated
by photosynthesis are usually used for carbon dioxide reduction.
Some strains are strict photoautotrophs, whereas others can use
exogenous carbon sources such as fructose and glucose.
Nitrogen fixation occurs only in prokaryotes and one line of
evidence for the common origin of the nitrogen fixation mechanism is the similar physical, chemical and biological characteristics of the nitrogen-fixing enzyme system in otherwise dissimilar
organisms. Many cyanobacteria are able to reduce atmospheric
346
September 1998, Vol. 3, No. 9
dinitrogen to ammonia. In some filamentous cyanobacteria nitrogen-fixing heterocysts are formed. Heterocysts are terminally differentiated cells whose interior becomes anaerobic, mainly as a
consequence of respiration, allowing the oxygen-sensitive process
of nitrogen fixation to continue. Heterocysts are spaced at semiregular intervals along the filament with approximately 7% of the
cells differentiating into heterocysts in free-living Anabaena/Nostoc
species. The regulation of dinitrogen fixation has been extensively
studied in the heterocyst system of diazotrophic cyanobacteria1.
Nitrogen fixation and heterocyst formation
During differentiation of a vegetative cell into a heterocyst, major
structural and biochemical changes occur that affect nitrogen fixation. Upon nitrogen deprivation phycobiliproteins are broken
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 1360 - 1385/98/$19.00 PII: S1360-1385(98)01290-4
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Heterocyst
Vegetative
cells
Vegetative
cells
Gln + 2-OG
H+
Glycogen
FdxH
N2
Nitrogenase
G6P
FNR
6PG
NADPH
Glucose
CO2
ADP/Pi
H2
ATP
PSI
Glu
Glu + Glu
2-OG
NADPH
?
Isocitrate
H2O
Citrate
Cyanophycin
Asp + Arg
O2
oxidative PPC
F6P
NH3
CO2 2-OG
b/f RET
R5P
Fructose
PetF
ATP
NH3
Maltose
Sucrose
Gln
?
OAA + AcCoA
NADH
GAP
FBP
PetF
CO2
?
PGA
ATP
PEP
Pyr
Carbohydrates
ATP
DAP
Fig. 1. Heterocyst metabolism and nitrogen fixation. The scheme of a heterocyst with adjacent vegetative cells is shown. The outer and inner
layers of the heterocyst envelope consist of polysaccharides and glycolipids, respectively. In this scheme the pore region is not drawn to scale
and shown enlarged to accommodate metabolite exchange between the cells. Cell wall and cell membranes are not drawn separately.
Heterocysts import carbohydrates from vegetative cells, with glutamine moving in the opposite direction. In a cell-free system derived from
heterocysts, the following substrates supported nitrogenase activity43: glycogen, maltose, sucrose (less active), glucose and fructose; glucose
6-phosphate (G6P) and other intermediates of the oxidative pentose-phosphate cycle (PPC), including dihydroxyacetone phosphate (DAP),
glyceraldehyde 3-phosphate (GAP) and fructose-1,6-bisphosphate (FBP), were particularly active. Glycolytic substrates, such as phosphoenolpyruvate (PEP) and pyruvate (Pyr) were inactive or inhibitory in acetylene reduction by the heterocyst extract. In the dark, reductant for
nitrogen and oxygen is generated by the activity of the oxidative PPC and possibly by isocitrate dehydrogenase. NADPH thus formed donates
electrons via ferredoxin:NADP reductase (FNR) to a heterocyst-specific ferredoxin (FdxH) and then to the two components of nitrogenase
(Fe-protein and FeMo-protein) as indicated. NAD(P)H and hydrogen are also electron donors to the respiratory electron transport (RET) generating the necessary ATP for the nitrogenase reaction. In the light, ATP is formed by cyclic photophosphorylation mediated by photosystem I
(a PSI-dimer, as indicated). Ferredoxin could be also photoreduced by PSI at the expense of hydrogen and NAD(P)H as electron donors.
Abbreviations: AcCoA, acetyl coenzyme A; Arg, arginine; Asp, aspartate; b/f, cytochrome b6f complex; F6P, fructose 6-phosphate; PetF,
vegetative cell type ferredoxin; Glu, glutamate; Gln, glutamine; OAA, oxaloacetate; 2-OG, 2-oxoglutarate; 6PG, 6-phosphogluconate; PGA,
3-phosphoglycerate; Pi, inorganic phosphate; R5P, ribose 5-phosphate.
down. At the same time, around the outer membrane of the heterocyst a double layered envelope is formed, which decreases the diffusion of oxygen. Connection to vegetative cells occurs through a
pore, equipped with microplasmodesmata. Heterocysts import
carbohydrates – these act as reductant and energy sources for
nitrogen fixation – an in turn, export glutamine. Changes in the
thylakoid structure of heterocysts are associated with a photosystem II that lacks oxygen evolving activity and Rubisco (the main
enzyme complex responsible for CO2 fixation). Therefore, reductant is almost exclusively channelled to the reduction of nitrogen
to ammonia, which in turn reacts with glutamate derived from the
imported carbohydrates. Both oxygen and nitrogen diffuse into
the cells, but increased respiratory activity in membranes near to
the polar ends of heterocysts depletes the oxygen concentration.
Hydrogen produced by the nitrogenase reaction feeds into an uptake hydrogenase system, which is induced upon heterocyst formation. This reacts with oxygen to produce water, contributing to
the ATP pool required for biosynthetic reactions such as nitrogen
fixation1.
Requirements for dinitrogen fixation
All diazotrophs, including nitrogen-fixing cyanobacteria, have the
same general requirements for nitrogen fixation: a nitrogenase
complex; ATP; a source of low-potential electrons and a partially
anaerobic environment (Fig. 1). It has been shown that heterocysts
contain the nitrogenase complex, which consists of an iron protein
(dinitrogenase-reductase) and a iron-molybdenum protein (dinitrogenase, with the iron-molybdenum cofactor) the latter of which
catalyzes nitrogen reduction1. Subsequent studies led to the cloning
and sequencing of nifH, nifD and nifK genes in Anabaena, which
encode the structural genes of the nitrogenase complex. Sequence
comparison revealed that the nitrogenase proteins of Anabaena
were very similar to those of other diazotrophic bacteria2.
ATP formation in heterocysts is not completely understood. The
rate of N2-fixation in the dark by heterocystous cyanobacteria is a
fraction of the rate in the light. ATP can be produced in the light by
either cyclic photophosphorylation or oxidative phosphorylation,
the latter process consumes oxygen and uses pyridine nucleotides
or hydrogen as electron sources. Reduced pyridine nucleotides can
September 1998, Vol. 3, No. 9
347
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be generated from the metabolism of imported carbohydrates1. The
nature of these carbohydrates is unknown, but a disaccharide such
as sucrose might be involved. The putative disaccharide would be
phosphorylated, converted to glucose-6-phosphate and then further
degraded via enzymes of the oxidative pentose-phosphate cycle,
which are particularly active in heterocysts, to produce NADPH and
CO2 (Ref. 1). It has been suggested that NADPH provides electrons for nitrogenase reductase via a heterocyst-specific ferredoxin
(FdxH) and ferredoxin:NADP+ oxidoreductase (FNR) through reversed electron transport. For this to occur, the ratio of NADPH to
NADP+ must be high, consistent with the levels measured in isolated heterocysts under nitrogen-fixing conditions3. In heterocysts,
expression of FNR is at an enhanced rate, about tenfold higher
than in vegetative cells4. By the oxidative pentose-phosphate cycle,
glucose-6-phosphate can be almost completely oxidized to CO2,
yielding mainly NADPH for nitrogen reduction.
Inactivation of the zwf and opcA genes, encoding glucose-6phosphate dehydrogenase (and a nearby gene of unknown function)
has been shown to inactive nitrogen fixation in a mutant of Nostoc
sp. ATCC 29133. This underlines the importance of this enzyme
in the oxidative pentose-phosphate cycle of heterocysts in relation
to nitrogen fixation5.
Because of the incomplete tricarboxylic acid cycle in cyanobacteria, isocitrate dehydrogenase is the terminal step in carbon flow.
This enzyme generates 2-oxoglutarate in an NADP-dependent reaction. The icd gene, encoding isocitrate dehydrogenase, shows a
fivefold increase in transcription and enzymic activity under nitrogen-fixing conditions. Upstream of the translation start region is a
DNA binding site for NtcA, a global nitrogen regulator, which is required for the expression of genes involved in nitrogen assimilation6.
From 2-oxoglutarate and glutamine, two molecules of glutamate
are produced by glutamate synthase (GltS) in a ferredoxin-dependent
reaction. Glutamine synthetase (GlnA) converts glutamate to glutamine using ammonia, generated upon nitrogen reduction, and ATP.
The main route of nitrogen assimilation in heterocysts occurs via
glutamine which is exported to vegetative cells1. In an in vitro assay,
isolated glutamate synthase (GltS) with heterocyst ferredoxin
(FdxH) as electron source had only 10% of the electron transfer activity of vegetative cell ferredoxin (PetF). Flavodoxin, thought partly
to replace the functions of PetF under iron limiting-conditions,
was inactive in this reaction7. Western blots of glutamate synthase
indicated that this enzyme is only present at low concentrations in
heterocysts. Furthermore, the enzyme only has very low activity
in heterocysts, suggesting that glutamate is synthesized in vegetative cells and exported to heterocysts for glutamine biosynthesis (F.J. Florencio, unpublished).
Glycogen granules found in cyanobacteria and especially in heterocysts are used as carbohydrate reserves that can be phosphorylated and broken down to glucose-6-phosphate as a substrate for the
oxidative pentose-phosphate cycle. Another polymer is cyanophycin, consisting of asparagine and arginine; similar to phycobiliproteins, it is a nitrogen reserve polymer that can be mobilized quickly1.
Genes involved in nitrogen fixation
Most of the genes involved in nitrogen fixation (nif genes), originally described in Klebsiella, have also been detected in other diazotrophic organisms. The structural genes of the nitrogenase complex,
nifH, nifD and nifK, represent one of the most highly conserved gene
groups in bacteria. On the basis of sequence similarity to Klebsiella DNA probes, nifH,D,K and nifS were cloned and mapped in
Anabaena sp. PCC 7120 (Anabaena 7120). Subsequent sequence
analysis of adjacent genes and comparison to other nif genes from
Klebsiella, Azotobacter and Rhizobium species led to the current
picture of nif gene organization in Anabaena (Fig. 2)8.
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September 1998, Vol. 3, No. 9
Developmentally regulated genome rearrangements in
heterocysts
Heterocyst differentiation in Anabaena 7120 is accompanied by developmentally regulated genome rearrangements that affect fdxN,
nifD and hupL gene expression9. These rearrangements of the vegetative cell genome occur during late stages of differentiation at
about the same time that the nitrogen fixation genes begin to be
transcribed. All the DNA elements shown in Fig. 2a (fdxN is interrupted by a 55 kb DNA element, the nifD gene by an 11-kb element and at an unknown distance to the nif-genes the hupL gene
by 10.5 kb of chromosomal DNA) become excised upon heterocyst differentiation10. The corresponding genes encoding these
site-specific recombinases are xisF, xisA and xisC, respectively,
which are located within the excised DNA elements. Recently, it
was demonstrated that xisF alone is not sufficient for the heterocyst specific excision of the fdxN element and xisH and xisI are
also required11. Anabaena 7120 mutants for xisA or xisF formed
heterocysts but did not grow on nitrogen-free media12. The hupLrearrangement in Anabaena 7120 was independently found by
pulsed-field electrophoresis and by comparison of the restriction pattern of vegetative cell and heterocyst DNA (Ref. 13). The
10.5 kb hupL element is not present in Anabaena 29413 (T. Happe
and H. Böhme, unpublished).
In contrast to Anabaena 7120, the closely related cyanobacterium Anabaena 29413 does not contain the fdxN element in the
nif1 region; only the nifD element is present. In the nif2 region, the
nifD element and the fdxN gene are absent (Fig. 2c). Both Pseudoanabaena and Fischerella lack the 11-kb element1.
Functions of nif genes in Anabaena
The function of many nif genes in Anabaena has still not been
determined, but a possible function can be inferred by analysing
analogous genes described in other diazotrophic bacteria. The first
operon on the left (Fig. 2a) includes the genes nifB, fdxN, nifS and
nifU, which are required for biosynthesis of the iron-molybdenum
(FeMo)- or the iron-vanadium (FeV) cofactor, but fdxN, nifS and
nifU of Anabaena 29413 were not essential for nitrogen fixation
to take place14,15. The glbN gene of Nostoc commune was discovered between nifS and nifU. It encodes cyanoglobin, the only known
prokaryotic myoglobin that might scavenge for oxygen or act as a
component of the membrane-associated, microaerobically induced
terminal oxidase. Cyanoglobin was only detected in Nostoc, when,
in addition to microaerobiosis, the cells were starved of nitrogen16.
The next operon to nifB,S,U consists of nifH, nifD and nifK (Fig. 2a).
The nifH gene encodes the dinitrogenase reductase, a homodimer
(2 3 30 kDa) with one [4Fe-4S]-cluster at the interface; nifD and
nifK encode the a- and b-subunits of dinitrogenase, respectively,
an a2b2 tetramer of 240 kDa associated with two FeMo-cofactors
and two P-clusters. Because NifE and NifN show significant structural similarity to NifD and NifK, respectively, it has been suggested that NifE and NifN generate the scaffold on which the
FeMo-cofactor is assembled. NifE and NifN also form an a2b2
tetramer that binds the NifB cofactor, a small iron-sulfur-cluster
protein and a precursor of the FeMo-cofactor. The precise function of the nifX gene in cyanobacterial nitrogen fixation remains to
be determined.
The nifW gene is necessary for full stability or processing of the
FeMo-protein. The functions of hesA and hesB are not known,
although insertional inactivation of hesA impairs nitrogen fixation
by approximately 55% (Ref. 17). The fdxH gene, which is transcribed late during heterocyst development together with the
nitrogenase genes, encodes a unique [2Fe-2S]-ferredoxin, which
is a specific electron donor for nitrogenase in vitro18. Inactivation
of fdxH led to a delay in nitrogen fixation, showing that FdxH is
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55-kb element
(a)
S
B
Anabaena sp.
PCC 7120
(nif genes)
11-kb element
U
H
D
K
E
N
W
X
fdxN
hesA
11-kb element
(b)
S
B
Anabaena sp.
ATCC 29413
(nif1 genes)
(c)
U
H
D
K
E
N
S
ZT
B
Q
ORF3
hesB mop
fdxH
W
X
fdxN
B
V
hesA1 ORF3
hesB1 mop
fdxH1
U
H
D
K
E–N
W
X
Anabaena sp.
ATCC 29413
(nif2 genes)
hesA2 fdxB
hesB2
fdxH2
(d)
D–G
K
E
N
Anabaena sp.
ATCC 29413
(vnf genes)
(e)
J
H
D
K
T Y
E
N
X
U
S
V WZ M F
L
A
Klebsiella
pneumoniae
(nif genes)
Fig. 2. Arrangement of nitrogen fixation genes from Anabaena ssp. and Klebsiella pneumoniae. The nif1 and nif2 systems of (a) Anabaena
7120 (nif genes), (b) Anabaena sp. ATCC 29413 (nif1 genes), (c) Anabaena 29413 (nif2 genes), and (d) the vnf system, encoding the alternative
vanadium (V)-dependent nitrogenase. (e) The arrangement of genes involved in nitrogen fixation in Klebsiella pneumoniae. The major nif gene
cluster of Anabaena 7120 heterocysts encompassing genes from nifB (left) to the mop gene (right) is separated from the nifVZT gene region.
Vertical arrows indicate the positions of the 55-kb and the 11-kb DNA-elements of the vegetative cell genome of Anabaena 7120, which become excised during heterocyst differentiation. The nif2E-N and vnfD-G genes of Anabaena 29413 are fused into a single open reading frame27.
necessary for the magnitude of maximum nitrogenase activity and
optimal growth under nitrogen-fixing conditions, but that fdxH is
not essential for diazotrophic growth19.
The nifV, nifZ and nifT genes are separated from the main nif
gene region in Anabaena 7120. The nifV gene encodes homocitrate synthase and homocitrate is an integral component of the
FeMo- cofactor. The functions of nifZ and nifT are not clear; inactivation of nifV in Anabaena 7120 led to mutant strains that were
still capable of diazotrophic growth (nitrogenase activity reduced
by about 30–40%)20.
The nifJ gene encoding a pyruvate:flavodoxin oxidoreductase,
is not closely linked to other nif genes of Anabaena 7120. In Klebsiella, NifJ functions to degrade pyruvate and generate reduced
flavodoxin (NifF) as a specific electron donor to nitrogenase. An
Anabaena 7120 a nifJ mutant was unable to grow on medium depleted of both iron and combined nitrogen. However, this strain
was capable of diazotrophic growth when iron was present21. No
equivalent of the nifF gene has been found in Anabaena19.
Induction of the nitrogenase complex is accompanied by the
induction of the hydrogen uptake system. The hupL gene encodes
the large subunit of a membrane-bound [NiFe]-uptake hydrogenase and uses molecular hydrogen, a byproduct of nitrogenase
activity10. To improve the efficiency of nitrogen fixation, hydrogen becomes oxidized in a respiratory, ATP-forming reaction
(Fig. 1).
Wolk and co-workers used transposon mutagenesis, based on a
Tn5-derivative bearing luxA,B (encoding luciferase) of Vibrio fischeri as a transcriptional reporter, to identify mutants that exhibit
enhanced luciferase activity after removal of ammonia from the
medium. Visualization of gene activation in single cells was made
possible using constructs in which the promoter region of PnifHDK
and PrbcLS was fused to luxA,B (Ref. 22). Among the first genes to
be activated by nitrogen deprivation (within 0.5 h) were the nirAnrtA,B,C,D-narB genes of the nir operon, encoding the structural
genes for nitrite reductase (nirA), nitrate permease (nrtA,B,C,D) and
nitrate reductase (narB). Anabaena strains carrying a mutation in
nirA, nrtC or nrtD remained competent to make heterocysts and
fix nitrogen23.
Alternative nitrogenase systems
Recently it was shown with Anabaena 29413 that in anaerobic
conditions a second, Mo-dependent nitrogenase system (nif2) is
expressed in all vegetative cells some hours after induction and
long before heterocysts begin to develop. In contrast to the nif1
system of heterocysts, which functions under both anaerobic or
external aerobic conditions and is developmentally regulated, the
nif2 system is expressed in all cells only under anaerobic conditions and is regulated by environmental factors24,25. Anabaena
29413 has a very similar nif1 and nif2 gene arrangement (Fig.
2b,c)26. The environmentally regulated nif2 system lacks fdxN, but
September 1998, Vol. 3, No. 9
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contains the fdxB gene downstream of fdxH2. The fdxB gene encodes a 2[4Fe-4S]-ferredoxin of unknown function24, and is similar to the corresponding gene from Rhodobacter.
In addition to the nif1 and nif2 genes, which encode nitrogenase-1
and -2, respectively, and which require the same FeMo-cofactor,
Anabaena 29413 also contains vnf genes encoding a V-dependent
nitrogenase27, as found in some other diazotrophic organisms. The
alternative, V-nitrogenase-encoding vnfD,G,K genes of Anabaena
29413 are organized much like those of Azotobacter spp. However,
the gene for the d subunit of the V-nitrogenase, vnfG, is fused to the
vnfD gene in Anabaena 29413. Two genes, vnfE and vnfN, which are
similar to vnfE,N genes of Azotobacter vinelandii were found downstream from vnfD,G,K in Anabaena 29413 (Ref. 27). Insertional
inactivation of the vnfN gene produced a mutant that grew poorly
on a medium where vanadium replaced molybdenum (Ref. 28).
Genes involved in the regulation of nitrogen fixation
Sigma factors
Upon deprivation of combined nitrogen, photosynthesizing vegetative cells differentiate to form N2-fixing heterocysts. This requires
the coordinated regulation of many genes. These changes in gene
expression involve modification of the transcription apparatus,
although the nature of that modification remains unknown. In general, sigma factors play a major role in the progression of differentiation in prokaryotes. Sigma factors are modular components
and can modify the major RNA polymerase to respond to nitrogen
deficiency. In a search for similar factors in Anabaena, sigA, encoding the major sigma factor in vegetative cells, and sigB and sigC,
two nitrogen-regulated sigma factors, were isolated. However, inactivation of either sigB or sigC genes, which are expressed under
nitrogen deficiency, still led to mutant strains capable of heterocyst
differentiation and nitrogen fixation29. A new group 2 sigma-factor
gene, sigD, has been cloned recently. A sigD-minus mutant strain
showed impaired diazotrophic growth and the appearance of heterocysts was delayed (I. Khudyakov and J.W. Golden, unpublished).
NtcA as a global nitrogen regulator
In cyanobacteria, ammonium exerts a negative control on proteins
involved in the programme of assimilation of nitrogen from sources
other than ammonium, such as nitrate and dinitrogen. NtcA is a global nitrogen regulator required for the activation of gene expression
in response to removal of ammonia in diverse cyanobacteria. NtcA
belongs to the family of bacterial transcriptional regulators, of which
the cAMP receptor protein (Crp) of response regulators are the
prototype. The amino acid sequence near the C-terminus of NtcA
predicts a helix-turn-helix motif, characteristic of the formation of
DNA–protein interactions30,31. The bifA gene, discovered independently as a DNA trans-acting factor from Anabaena 7120 on sites
upstream of xisA, is identical to the ntcA gene32. An ntcA mutant of
Anabaena 7120 failed to induce the nir operon and to express the
major glnA transcript induced under conditions of nitrogen depletion. In addition, the mutant did not develop heterocysts and was unable to express nifH,D,K in response to nitrogen deprivation34–36. In
unicellular and filamentous strains, NtcA binds to target sequences
of glnA, nirA and ntcA, which have a palindromic GTA(N8)TAC
motif (where N is any nucleotide) upstream of the transcription start
site. This sequence replaces the -35 promoter site of E. coli (Ref. 33).
Using similar experiments, an alternative but overlapping BifA
(= NtcA) binding site has been proposed: TGT(N9–10)ACA(Ref. 31).
In the unicellular diazotrophic cyanobacterium, Cyanothece sp.,
ntcA transcripts were weakly expressed during N2-fixation, but
expression increased in nitrate-grown and especially ammoniumgrown cells. According to these data NtcA seems to be more important for nitrogen assimilation than nitrogen fixation35.
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September 1998, Vol. 3, No. 9
GlnB (PII-protein) as sensor kinase of NtcA?
In enterobacteria, regulation of nitrogen metabolism is mediated
by a two-component ntr-system (nitrogen regulation) in which the
GlnB protein has the role of transmitting the nitrogen-status of the
cell to Ntr-proteins. The GlnB protein controls both the activity
and synthesis of glutamine synthetase (GlnA), a key enzyme in
bacterial nitrogen assimilation. A cyanobacterial glnB gene has
been isolated that is very similar to its bacterial counterpart36. In
Synechococcus sp. PCC 7942, a unicellular, non-nitrogen-fixing
cyanobacterium, the homotrimeric PII-protein is modified by serinephosphorylation. In the presence of ammonium, PII is found in its
dephosphorylated state. A kinase- or phosphatase-activity can be
separated by biochemical methods. The kinase activity depends
on ATP as a phosphoryl donor and the presence of 2-oxoglutarate,
as carbon skeleton required for nitrogen assimilation, to sense the
nitrogen status of the cell37,38. ATP and 2-oxoglutarate were bound
by the PII-protein in a mutually dependent manner. Glutamine had
no effect on kinase or phosphatase activities. By studying insertional mutants of Synechococcus 7942 lacking the PII-protein and
mutants, where at the phosphorylation site serine was exchanged
for alanine showed that in the presence of a dephosphorylated form
of PII nitrate and nitrite transport was inhibited. However, a pleiotropic PII-deficient mutant suggested that PII is not essential for activation of NtcA-dependent transcription39. In Synechocystis 6803,
a unicellular, non-nitrogen-fixing cyanobacterium, glnB expression was specifically activated (tenfold) under nitrogen deprivation. Induction of expression of the glnB gene might be under the
control of NtcA. Constitutive levels of GlnB were detected from a
s70-dependent E. coli-like promoter. This would ensure basal levels
of the PII-protein were available to sense changes in environmental conditions at any time. Preliminary results indicate a correlation
between PII state and GlnA activity40. In the filamentous, nitrogenfixing cyanobacterium Nostoc 29133, glnB could not be insertionally inactivated41. In the nitrogen-fixing Calothrix 7504,
heterocyst differentiation correlated with the modified form of PII
(Ref. 42). More experiments concerning nitrogen fixation in
cyanobacteria are necessary to clarify the relation between NtcA
and PII.
Conclusions and future prospects
Although we have a fairly sophisticated level of understanding
of nitrogen fixation in cyanobacteria there are still a number of
uncertain aspects. For example, because photosynthetic oxygen
evolution is absent from heterocysts, reductant must be provided
by adjoining vegetative cells. However, the molecules that are
transported into heterocysts to provide reductant and carbon skeletons for fixed nitrogen are not known with any certainty. Another
important question is the identity of the permeases that are present
between heterocysts and vegetative cells and that participate in
this process.
An oxygen sensor responsible for the regulated expression nifgenes has also yet to be described in cyanobacteria. The different
pathways of electron-donation to nitrogenase are also not clearly
known – an fdxH-minus strain was impaired in nitrogen fixation,
but not completely inhibited. Finally, the nif-genes of Anabaena
7120 are interrupted and removed from the chromosome during
heterocyst differentiation, but this process is not ubiquitous in
cyanobacteria, and there is as yet no explanation for it. Clearly,
there is more to be done to unravel the mysteries of nitrogen fixation
in cyanobacteria.
Acknowledgements
The authors work was supported by DFG-grants. Thanks to
B. Masepohl for help in drawing Fig. 2.
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Herbert Böhme is at the Botanisches Institut, Universität Bonn,
Kirschallee 1, 53115 Bonn, Germany (tel +49 228 735515;
fax +49 228 735513; e-mail [email protected]).
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Eduardo Blumwald*, Gilad S. Aharon and Bernard C-H. Lam are at
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Regulation of nitrogen fixation in
heterocyst-forming cyanobacteria
Herbert Böhme
Some cyanobacteria are able to reduce atmospheric dinitrogen to ammonia – a process
where oxygen evolved by photosynthetic activity in the same cell is detrimental to nitrogen
fixation. Strategies to avoid oxygen range from temporal separation of nitrogen fixation and
oxygen evolution (in unicellular and filamentous, non-heterocystous strains) to spatial separation and cellular differentiation into nitrogen fixing heterocysts (in filamentous cyanobacteria).
Recent research has begun to clarify the genes involved in nitrogen fixation, the mechanisms
that regulate the expression of proteins involved in the assimilation of nitrogen from different
sources and the way in which the cell is able to sense its nitrogen status.
C
yanobacteria (blue-green algae) are a diverse group of prokaryotes. A common feature is their oxygenic photosynthesis, which is similar to that in algae and higher plants
and is the most important biological mechanism for capturing solar
energy. As sunlight is their energy source and water the reductant,
they generate oxygen in the light. Energy and reductant generated
by photosynthesis are usually used for carbon dioxide reduction.
Some strains are strict photoautotrophs, whereas others can use
exogenous carbon sources such as fructose and glucose.
Nitrogen fixation occurs only in prokaryotes and one line of
evidence for the common origin of the nitrogen fixation mechanism is the similar physical, chemical and biological characteristics of the nitrogen-fixing enzyme system in otherwise dissimilar
organisms. Many cyanobacteria are able to reduce atmospheric
346
September 1998, Vol. 3, No. 9
dinitrogen to ammonia. In some filamentous cyanobacteria nitrogen-fixing heterocysts are formed. Heterocysts are terminally differentiated cells whose interior becomes anaerobic, mainly as a
consequence of respiration, allowing the oxygen-sensitive process
of nitrogen fixation to continue. Heterocysts are spaced at semiregular intervals along the filament with approximately 7% of the
cells differentiating into heterocysts in free-living Anabaena/Nostoc
species. The regulation of dinitrogen fixation has been extensively
studied in the heterocyst system of diazotrophic cyanobacteria1.
Nitrogen fixation and heterocyst formation
During differentiation of a vegetative cell into a heterocyst, major
structural and biochemical changes occur that affect nitrogen fixation. Upon nitrogen deprivation phycobiliproteins are broken
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 1360 - 1385/98/$19.00 PII: S1360-1385(98)01290-4
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Heterocyst
Vegetative
cells
Vegetative
cells
Gln + 2-OG
H+
Glycogen
FdxH
N2
Nitrogenase
G6P
FNR
6PG
NADPH
Glucose
CO2
ADP/Pi
H2
ATP
PSI
Glu
Glu + Glu
2-OG
NADPH
?
Isocitrate
H2O
Citrate
Cyanophycin
Asp + Arg
O2
oxidative PPC
F6P
NH3
CO2 2-OG
b/f RET
R5P
Fructose
PetF
ATP
NH3
Maltose
Sucrose
Gln
?
OAA + AcCoA
NADH
GAP
FBP
PetF
CO2
?
PGA
ATP
PEP
Pyr
Carbohydrates
ATP
DAP
Fig. 1. Heterocyst metabolism and nitrogen fixation. The scheme of a heterocyst with adjacent vegetative cells is shown. The outer and inner
layers of the heterocyst envelope consist of polysaccharides and glycolipids, respectively. In this scheme the pore region is not drawn to scale
and shown enlarged to accommodate metabolite exchange between the cells. Cell wall and cell membranes are not drawn separately.
Heterocysts import carbohydrates from vegetative cells, with glutamine moving in the opposite direction. In a cell-free system derived from
heterocysts, the following substrates supported nitrogenase activity43: glycogen, maltose, sucrose (less active), glucose and fructose; glucose
6-phosphate (G6P) and other intermediates of the oxidative pentose-phosphate cycle (PPC), including dihydroxyacetone phosphate (DAP),
glyceraldehyde 3-phosphate (GAP) and fructose-1,6-bisphosphate (FBP), were particularly active. Glycolytic substrates, such as phosphoenolpyruvate (PEP) and pyruvate (Pyr) were inactive or inhibitory in acetylene reduction by the heterocyst extract. In the dark, reductant for
nitrogen and oxygen is generated by the activity of the oxidative PPC and possibly by isocitrate dehydrogenase. NADPH thus formed donates
electrons via ferredoxin:NADP reductase (FNR) to a heterocyst-specific ferredoxin (FdxH) and then to the two components of nitrogenase
(Fe-protein and FeMo-protein) as indicated. NAD(P)H and hydrogen are also electron donors to the respiratory electron transport (RET) generating the necessary ATP for the nitrogenase reaction. In the light, ATP is formed by cyclic photophosphorylation mediated by photosystem I
(a PSI-dimer, as indicated). Ferredoxin could be also photoreduced by PSI at the expense of hydrogen and NAD(P)H as electron donors.
Abbreviations: AcCoA, acetyl coenzyme A; Arg, arginine; Asp, aspartate; b/f, cytochrome b6f complex; F6P, fructose 6-phosphate; PetF,
vegetative cell type ferredoxin; Glu, glutamate; Gln, glutamine; OAA, oxaloacetate; 2-OG, 2-oxoglutarate; 6PG, 6-phosphogluconate; PGA,
3-phosphoglycerate; Pi, inorganic phosphate; R5P, ribose 5-phosphate.
down. At the same time, around the outer membrane of the heterocyst a double layered envelope is formed, which decreases the diffusion of oxygen. Connection to vegetative cells occurs through a
pore, equipped with microplasmodesmata. Heterocysts import
carbohydrates – these act as reductant and energy sources for
nitrogen fixation – an in turn, export glutamine. Changes in the
thylakoid structure of heterocysts are associated with a photosystem II that lacks oxygen evolving activity and Rubisco (the main
enzyme complex responsible for CO2 fixation). Therefore, reductant is almost exclusively channelled to the reduction of nitrogen
to ammonia, which in turn reacts with glutamate derived from the
imported carbohydrates. Both oxygen and nitrogen diffuse into
the cells, but increased respiratory activity in membranes near to
the polar ends of heterocysts depletes the oxygen concentration.
Hydrogen produced by the nitrogenase reaction feeds into an uptake hydrogenase system, which is induced upon heterocyst formation. This reacts with oxygen to produce water, contributing to
the ATP pool required for biosynthetic reactions such as nitrogen
fixation1.
Requirements for dinitrogen fixation
All diazotrophs, including nitrogen-fixing cyanobacteria, have the
same general requirements for nitrogen fixation: a nitrogenase
complex; ATP; a source of low-potential electrons and a partially
anaerobic environment (Fig. 1). It has been shown that heterocysts
contain the nitrogenase complex, which consists of an iron protein
(dinitrogenase-reductase) and a iron-molybdenum protein (dinitrogenase, with the iron-molybdenum cofactor) the latter of which
catalyzes nitrogen reduction1. Subsequent studies led to the cloning
and sequencing of nifH, nifD and nifK genes in Anabaena, which
encode the structural genes of the nitrogenase complex. Sequence
comparison revealed that the nitrogenase proteins of Anabaena
were very similar to those of other diazotrophic bacteria2.
ATP formation in heterocysts is not completely understood. The
rate of N2-fixation in the dark by heterocystous cyanobacteria is a
fraction of the rate in the light. ATP can be produced in the light by
either cyclic photophosphorylation or oxidative phosphorylation,
the latter process consumes oxygen and uses pyridine nucleotides
or hydrogen as electron sources. Reduced pyridine nucleotides can
September 1998, Vol. 3, No. 9
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be generated from the metabolism of imported carbohydrates1. The
nature of these carbohydrates is unknown, but a disaccharide such
as sucrose might be involved. The putative disaccharide would be
phosphorylated, converted to glucose-6-phosphate and then further
degraded via enzymes of the oxidative pentose-phosphate cycle,
which are particularly active in heterocysts, to produce NADPH and
CO2 (Ref. 1). It has been suggested that NADPH provides electrons for nitrogenase reductase via a heterocyst-specific ferredoxin
(FdxH) and ferredoxin:NADP+ oxidoreductase (FNR) through reversed electron transport. For this to occur, the ratio of NADPH to
NADP+ must be high, consistent with the levels measured in isolated heterocysts under nitrogen-fixing conditions3. In heterocysts,
expression of FNR is at an enhanced rate, about tenfold higher
than in vegetative cells4. By the oxidative pentose-phosphate cycle,
glucose-6-phosphate can be almost completely oxidized to CO2,
yielding mainly NADPH for nitrogen reduction.
Inactivation of the zwf and opcA genes, encoding glucose-6phosphate dehydrogenase (and a nearby gene of unknown function)
has been shown to inactive nitrogen fixation in a mutant of Nostoc
sp. ATCC 29133. This underlines the importance of this enzyme
in the oxidative pentose-phosphate cycle of heterocysts in relation
to nitrogen fixation5.
Because of the incomplete tricarboxylic acid cycle in cyanobacteria, isocitrate dehydrogenase is the terminal step in carbon flow.
This enzyme generates 2-oxoglutarate in an NADP-dependent reaction. The icd gene, encoding isocitrate dehydrogenase, shows a
fivefold increase in transcription and enzymic activity under nitrogen-fixing conditions. Upstream of the translation start region is a
DNA binding site for NtcA, a global nitrogen regulator, which is required for the expression of genes involved in nitrogen assimilation6.
From 2-oxoglutarate and glutamine, two molecules of glutamate
are produced by glutamate synthase (GltS) in a ferredoxin-dependent
reaction. Glutamine synthetase (GlnA) converts glutamate to glutamine using ammonia, generated upon nitrogen reduction, and ATP.
The main route of nitrogen assimilation in heterocysts occurs via
glutamine which is exported to vegetative cells1. In an in vitro assay,
isolated glutamate synthase (GltS) with heterocyst ferredoxin
(FdxH) as electron source had only 10% of the electron transfer activity of vegetative cell ferredoxin (PetF). Flavodoxin, thought partly
to replace the functions of PetF under iron limiting-conditions,
was inactive in this reaction7. Western blots of glutamate synthase
indicated that this enzyme is only present at low concentrations in
heterocysts. Furthermore, the enzyme only has very low activity
in heterocysts, suggesting that glutamate is synthesized in vegetative cells and exported to heterocysts for glutamine biosynthesis (F.J. Florencio, unpublished).
Glycogen granules found in cyanobacteria and especially in heterocysts are used as carbohydrate reserves that can be phosphorylated and broken down to glucose-6-phosphate as a substrate for the
oxidative pentose-phosphate cycle. Another polymer is cyanophycin, consisting of asparagine and arginine; similar to phycobiliproteins, it is a nitrogen reserve polymer that can be mobilized quickly1.
Genes involved in nitrogen fixation
Most of the genes involved in nitrogen fixation (nif genes), originally described in Klebsiella, have also been detected in other diazotrophic organisms. The structural genes of the nitrogenase complex,
nifH, nifD and nifK, represent one of the most highly conserved gene
groups in bacteria. On the basis of sequence similarity to Klebsiella DNA probes, nifH,D,K and nifS were cloned and mapped in
Anabaena sp. PCC 7120 (Anabaena 7120). Subsequent sequence
analysis of adjacent genes and comparison to other nif genes from
Klebsiella, Azotobacter and Rhizobium species led to the current
picture of nif gene organization in Anabaena (Fig. 2)8.
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September 1998, Vol. 3, No. 9
Developmentally regulated genome rearrangements in
heterocysts
Heterocyst differentiation in Anabaena 7120 is accompanied by developmentally regulated genome rearrangements that affect fdxN,
nifD and hupL gene expression9. These rearrangements of the vegetative cell genome occur during late stages of differentiation at
about the same time that the nitrogen fixation genes begin to be
transcribed. All the DNA elements shown in Fig. 2a (fdxN is interrupted by a 55 kb DNA element, the nifD gene by an 11-kb element and at an unknown distance to the nif-genes the hupL gene
by 10.5 kb of chromosomal DNA) become excised upon heterocyst differentiation10. The corresponding genes encoding these
site-specific recombinases are xisF, xisA and xisC, respectively,
which are located within the excised DNA elements. Recently, it
was demonstrated that xisF alone is not sufficient for the heterocyst specific excision of the fdxN element and xisH and xisI are
also required11. Anabaena 7120 mutants for xisA or xisF formed
heterocysts but did not grow on nitrogen-free media12. The hupLrearrangement in Anabaena 7120 was independently found by
pulsed-field electrophoresis and by comparison of the restriction pattern of vegetative cell and heterocyst DNA (Ref. 13). The
10.5 kb hupL element is not present in Anabaena 29413 (T. Happe
and H. Böhme, unpublished).
In contrast to Anabaena 7120, the closely related cyanobacterium Anabaena 29413 does not contain the fdxN element in the
nif1 region; only the nifD element is present. In the nif2 region, the
nifD element and the fdxN gene are absent (Fig. 2c). Both Pseudoanabaena and Fischerella lack the 11-kb element1.
Functions of nif genes in Anabaena
The function of many nif genes in Anabaena has still not been
determined, but a possible function can be inferred by analysing
analogous genes described in other diazotrophic bacteria. The first
operon on the left (Fig. 2a) includes the genes nifB, fdxN, nifS and
nifU, which are required for biosynthesis of the iron-molybdenum
(FeMo)- or the iron-vanadium (FeV) cofactor, but fdxN, nifS and
nifU of Anabaena 29413 were not essential for nitrogen fixation
to take place14,15. The glbN gene of Nostoc commune was discovered between nifS and nifU. It encodes cyanoglobin, the only known
prokaryotic myoglobin that might scavenge for oxygen or act as a
component of the membrane-associated, microaerobically induced
terminal oxidase. Cyanoglobin was only detected in Nostoc, when,
in addition to microaerobiosis, the cells were starved of nitrogen16.
The next operon to nifB,S,U consists of nifH, nifD and nifK (Fig. 2a).
The nifH gene encodes the dinitrogenase reductase, a homodimer
(2 3 30 kDa) with one [4Fe-4S]-cluster at the interface; nifD and
nifK encode the a- and b-subunits of dinitrogenase, respectively,
an a2b2 tetramer of 240 kDa associated with two FeMo-cofactors
and two P-clusters. Because NifE and NifN show significant structural similarity to NifD and NifK, respectively, it has been suggested that NifE and NifN generate the scaffold on which the
FeMo-cofactor is assembled. NifE and NifN also form an a2b2
tetramer that binds the NifB cofactor, a small iron-sulfur-cluster
protein and a precursor of the FeMo-cofactor. The precise function of the nifX gene in cyanobacterial nitrogen fixation remains to
be determined.
The nifW gene is necessary for full stability or processing of the
FeMo-protein. The functions of hesA and hesB are not known,
although insertional inactivation of hesA impairs nitrogen fixation
by approximately 55% (Ref. 17). The fdxH gene, which is transcribed late during heterocyst development together with the
nitrogenase genes, encodes a unique [2Fe-2S]-ferredoxin, which
is a specific electron donor for nitrogenase in vitro18. Inactivation
of fdxH led to a delay in nitrogen fixation, showing that FdxH is
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55-kb element
(a)
S
B
Anabaena sp.
PCC 7120
(nif genes)
11-kb element
U
H
D
K
E
N
W
X
fdxN
hesA
11-kb element
(b)
S
B
Anabaena sp.
ATCC 29413
(nif1 genes)
(c)
U
H
D
K
E
N
S
ZT
B
Q
ORF3
hesB mop
fdxH
W
X
fdxN
B
V
hesA1 ORF3
hesB1 mop
fdxH1
U
H
D
K
E–N
W
X
Anabaena sp.
ATCC 29413
(nif2 genes)
hesA2 fdxB
hesB2
fdxH2
(d)
D–G
K
E
N
Anabaena sp.
ATCC 29413
(vnf genes)
(e)
J
H
D
K
T Y
E
N
X
U
S
V WZ M F
L
A
Klebsiella
pneumoniae
(nif genes)
Fig. 2. Arrangement of nitrogen fixation genes from Anabaena ssp. and Klebsiella pneumoniae. The nif1 and nif2 systems of (a) Anabaena
7120 (nif genes), (b) Anabaena sp. ATCC 29413 (nif1 genes), (c) Anabaena 29413 (nif2 genes), and (d) the vnf system, encoding the alternative
vanadium (V)-dependent nitrogenase. (e) The arrangement of genes involved in nitrogen fixation in Klebsiella pneumoniae. The major nif gene
cluster of Anabaena 7120 heterocysts encompassing genes from nifB (left) to the mop gene (right) is separated from the nifVZT gene region.
Vertical arrows indicate the positions of the 55-kb and the 11-kb DNA-elements of the vegetative cell genome of Anabaena 7120, which become excised during heterocyst differentiation. The nif2E-N and vnfD-G genes of Anabaena 29413 are fused into a single open reading frame27.
necessary for the magnitude of maximum nitrogenase activity and
optimal growth under nitrogen-fixing conditions, but that fdxH is
not essential for diazotrophic growth19.
The nifV, nifZ and nifT genes are separated from the main nif
gene region in Anabaena 7120. The nifV gene encodes homocitrate synthase and homocitrate is an integral component of the
FeMo- cofactor. The functions of nifZ and nifT are not clear; inactivation of nifV in Anabaena 7120 led to mutant strains that were
still capable of diazotrophic growth (nitrogenase activity reduced
by about 30–40%)20.
The nifJ gene encoding a pyruvate:flavodoxin oxidoreductase,
is not closely linked to other nif genes of Anabaena 7120. In Klebsiella, NifJ functions to degrade pyruvate and generate reduced
flavodoxin (NifF) as a specific electron donor to nitrogenase. An
Anabaena 7120 a nifJ mutant was unable to grow on medium depleted of both iron and combined nitrogen. However, this strain
was capable of diazotrophic growth when iron was present21. No
equivalent of the nifF gene has been found in Anabaena19.
Induction of the nitrogenase complex is accompanied by the
induction of the hydrogen uptake system. The hupL gene encodes
the large subunit of a membrane-bound [NiFe]-uptake hydrogenase and uses molecular hydrogen, a byproduct of nitrogenase
activity10. To improve the efficiency of nitrogen fixation, hydrogen becomes oxidized in a respiratory, ATP-forming reaction
(Fig. 1).
Wolk and co-workers used transposon mutagenesis, based on a
Tn5-derivative bearing luxA,B (encoding luciferase) of Vibrio fischeri as a transcriptional reporter, to identify mutants that exhibit
enhanced luciferase activity after removal of ammonia from the
medium. Visualization of gene activation in single cells was made
possible using constructs in which the promoter region of PnifHDK
and PrbcLS was fused to luxA,B (Ref. 22). Among the first genes to
be activated by nitrogen deprivation (within 0.5 h) were the nirAnrtA,B,C,D-narB genes of the nir operon, encoding the structural
genes for nitrite reductase (nirA), nitrate permease (nrtA,B,C,D) and
nitrate reductase (narB). Anabaena strains carrying a mutation in
nirA, nrtC or nrtD remained competent to make heterocysts and
fix nitrogen23.
Alternative nitrogenase systems
Recently it was shown with Anabaena 29413 that in anaerobic
conditions a second, Mo-dependent nitrogenase system (nif2) is
expressed in all vegetative cells some hours after induction and
long before heterocysts begin to develop. In contrast to the nif1
system of heterocysts, which functions under both anaerobic or
external aerobic conditions and is developmentally regulated, the
nif2 system is expressed in all cells only under anaerobic conditions and is regulated by environmental factors24,25. Anabaena
29413 has a very similar nif1 and nif2 gene arrangement (Fig.
2b,c)26. The environmentally regulated nif2 system lacks fdxN, but
September 1998, Vol. 3, No. 9
349
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contains the fdxB gene downstream of fdxH2. The fdxB gene encodes a 2[4Fe-4S]-ferredoxin of unknown function24, and is similar to the corresponding gene from Rhodobacter.
In addition to the nif1 and nif2 genes, which encode nitrogenase-1
and -2, respectively, and which require the same FeMo-cofactor,
Anabaena 29413 also contains vnf genes encoding a V-dependent
nitrogenase27, as found in some other diazotrophic organisms. The
alternative, V-nitrogenase-encoding vnfD,G,K genes of Anabaena
29413 are organized much like those of Azotobacter spp. However,
the gene for the d subunit of the V-nitrogenase, vnfG, is fused to the
vnfD gene in Anabaena 29413. Two genes, vnfE and vnfN, which are
similar to vnfE,N genes of Azotobacter vinelandii were found downstream from vnfD,G,K in Anabaena 29413 (Ref. 27). Insertional
inactivation of the vnfN gene produced a mutant that grew poorly
on a medium where vanadium replaced molybdenum (Ref. 28).
Genes involved in the regulation of nitrogen fixation
Sigma factors
Upon deprivation of combined nitrogen, photosynthesizing vegetative cells differentiate to form N2-fixing heterocysts. This requires
the coordinated regulation of many genes. These changes in gene
expression involve modification of the transcription apparatus,
although the nature of that modification remains unknown. In general, sigma factors play a major role in the progression of differentiation in prokaryotes. Sigma factors are modular components
and can modify the major RNA polymerase to respond to nitrogen
deficiency. In a search for similar factors in Anabaena, sigA, encoding the major sigma factor in vegetative cells, and sigB and sigC,
two nitrogen-regulated sigma factors, were isolated. However, inactivation of either sigB or sigC genes, which are expressed under
nitrogen deficiency, still led to mutant strains capable of heterocyst
differentiation and nitrogen fixation29. A new group 2 sigma-factor
gene, sigD, has been cloned recently. A sigD-minus mutant strain
showed impaired diazotrophic growth and the appearance of heterocysts was delayed (I. Khudyakov and J.W. Golden, unpublished).
NtcA as a global nitrogen regulator
In cyanobacteria, ammonium exerts a negative control on proteins
involved in the programme of assimilation of nitrogen from sources
other than ammonium, such as nitrate and dinitrogen. NtcA is a global nitrogen regulator required for the activation of gene expression
in response to removal of ammonia in diverse cyanobacteria. NtcA
belongs to the family of bacterial transcriptional regulators, of which
the cAMP receptor protein (Crp) of response regulators are the
prototype. The amino acid sequence near the C-terminus of NtcA
predicts a helix-turn-helix motif, characteristic of the formation of
DNA–protein interactions30,31. The bifA gene, discovered independently as a DNA trans-acting factor from Anabaena 7120 on sites
upstream of xisA, is identical to the ntcA gene32. An ntcA mutant of
Anabaena 7120 failed to induce the nir operon and to express the
major glnA transcript induced under conditions of nitrogen depletion. In addition, the mutant did not develop heterocysts and was unable to express nifH,D,K in response to nitrogen deprivation34–36. In
unicellular and filamentous strains, NtcA binds to target sequences
of glnA, nirA and ntcA, which have a palindromic GTA(N8)TAC
motif (where N is any nucleotide) upstream of the transcription start
site. This sequence replaces the -35 promoter site of E. coli (Ref. 33).
Using similar experiments, an alternative but overlapping BifA
(= NtcA) binding site has been proposed: TGT(N9–10)ACA(Ref. 31).
In the unicellular diazotrophic cyanobacterium, Cyanothece sp.,
ntcA transcripts were weakly expressed during N2-fixation, but
expression increased in nitrate-grown and especially ammoniumgrown cells. According to these data NtcA seems to be more important for nitrogen assimilation than nitrogen fixation35.
350
September 1998, Vol. 3, No. 9
GlnB (PII-protein) as sensor kinase of NtcA?
In enterobacteria, regulation of nitrogen metabolism is mediated
by a two-component ntr-system (nitrogen regulation) in which the
GlnB protein has the role of transmitting the nitrogen-status of the
cell to Ntr-proteins. The GlnB protein controls both the activity
and synthesis of glutamine synthetase (GlnA), a key enzyme in
bacterial nitrogen assimilation. A cyanobacterial glnB gene has
been isolated that is very similar to its bacterial counterpart36. In
Synechococcus sp. PCC 7942, a unicellular, non-nitrogen-fixing
cyanobacterium, the homotrimeric PII-protein is modified by serinephosphorylation. In the presence of ammonium, PII is found in its
dephosphorylated state. A kinase- or phosphatase-activity can be
separated by biochemical methods. The kinase activity depends
on ATP as a phosphoryl donor and the presence of 2-oxoglutarate,
as carbon skeleton required for nitrogen assimilation, to sense the
nitrogen status of the cell37,38. ATP and 2-oxoglutarate were bound
by the PII-protein in a mutually dependent manner. Glutamine had
no effect on kinase or phosphatase activities. By studying insertional mutants of Synechococcus 7942 lacking the PII-protein and
mutants, where at the phosphorylation site serine was exchanged
for alanine showed that in the presence of a dephosphorylated form
of PII nitrate and nitrite transport was inhibited. However, a pleiotropic PII-deficient mutant suggested that PII is not essential for activation of NtcA-dependent transcription39. In Synechocystis 6803,
a unicellular, non-nitrogen-fixing cyanobacterium, glnB expression was specifically activated (tenfold) under nitrogen deprivation. Induction of expression of the glnB gene might be under the
control of NtcA. Constitutive levels of GlnB were detected from a
s70-dependent E. coli-like promoter. This would ensure basal levels
of the PII-protein were available to sense changes in environmental conditions at any time. Preliminary results indicate a correlation
between PII state and GlnA activity40. In the filamentous, nitrogenfixing cyanobacterium Nostoc 29133, glnB could not be insertionally inactivated41. In the nitrogen-fixing Calothrix 7504,
heterocyst differentiation correlated with the modified form of PII
(Ref. 42). More experiments concerning nitrogen fixation in
cyanobacteria are necessary to clarify the relation between NtcA
and PII.
Conclusions and future prospects
Although we have a fairly sophisticated level of understanding
of nitrogen fixation in cyanobacteria there are still a number of
uncertain aspects. For example, because photosynthetic oxygen
evolution is absent from heterocysts, reductant must be provided
by adjoining vegetative cells. However, the molecules that are
transported into heterocysts to provide reductant and carbon skeletons for fixed nitrogen are not known with any certainty. Another
important question is the identity of the permeases that are present
between heterocysts and vegetative cells and that participate in
this process.
An oxygen sensor responsible for the regulated expression nifgenes has also yet to be described in cyanobacteria. The different
pathways of electron-donation to nitrogenase are also not clearly
known – an fdxH-minus strain was impaired in nitrogen fixation,
but not completely inhibited. Finally, the nif-genes of Anabaena
7120 are interrupted and removed from the chromosome during
heterocyst differentiation, but this process is not ubiquitous in
cyanobacteria, and there is as yet no explanation for it. Clearly,
there is more to be done to unravel the mysteries of nitrogen fixation
in cyanobacteria.
Acknowledgements
The authors work was supported by DFG-grants. Thanks to
B. Masepohl for help in drawing Fig. 2.
trends in plant science
reviews
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Herbert Böhme is at the Botanisches Institut, Universität Bonn,
Kirschallee 1, 53115 Bonn, Germany (tel +49 228 735515;
fax +49 228 735513; e-mail [email protected]).
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