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The nitrogen physiology of the
marine N2-fixing cyanobacteria
Trichodesmium spp.
Margaret R. Mulholland and Douglas G. Capone
Trichodesmium spp. have proved to be enigmatic organisms, and their ecology and physiology
are unusual among diazotrophs. Recent research shows that they can simultaneously fix N2
and take up combined nitrogen. The co-occurrence of these two processes is thought to be
incompatible, but they could be obligatory in Trichodesmium spp. if only a small fraction of
cells within a colony or along a filament are capable of N2 fixation. Combined nitrogen is
released from cells during periods of active growth and N2 fixation, and concomitantly taken up
by Trichodesmium spp. or cells living in association with colonies. Although the nitrogenase of
Trichodesmium spp. is affected by high concentrations of combined nitrogen, it might be relatively less sensitive to low concentrations of combined nitrogen typical of the oligotrophic
ocean and culture conditions. Nitrogenase activity and synthesis exhibits an endogenous
rhythm in Trichodesmium spp. cultures, which is affected by the addition of nitrogen.
I
n spite of the observation that nitrogen (N) is often the nutrient
that limits primary productivity in many oceanic environments,
marine N2 fixation has been largely overlooked as a source of
fixed N in these systems. This is probably because reports in the
early 1980s suggested that integrated rates of marine N2 fixation
were small compared with overall phytoplankton N demand.
However, a range of new evidence indicates that marine N2 fixation
does provide a significant source of new N to tropical marine
ecosystems. This evidence includes:
• Recognition of a more widespread occurrence and higher densities of the cyanobacteria Trichodesmium spp. than previously
reported1,2.
• Deficits and imbalances in the marine N budget indicating
unquantified N sources3–5.
• Particulate organic N and NO32 in surface waters with N isotopic signatures close to that of atmospheric N2 (low d15N)6,7.
• Measurements of rates of N2 fixation comparable to the flux and
uptake of fixed N (primarily NO32) into the euphotic zone1,2,11.
Furthermore, evidence is accumulating for other N2 fixers, which
could contribute substantially to the ocean’s fixed N budget8.
N2 fixation by Trichodesmium spp. is a significant source of new
N to tropical and subtropical marine systems where they occur1,9–11.
Species of the non-heterocystous, diazotrophic, cyanobacterial
genus Trichodesmium spp., are common throughout the N-depleted
tropical and subtropical oligotrophic ocean, and rates of N2 fixation
by Trichodesmium spp. have been measured in a variety of locations1. Although it is often assumed that Trichodesmium spp.
depend primarily on N2 fixation to meet their nutritional needs12,
they are also capable of taking up other nitrogenous compounds
(e.g. NH41, NO32 and some forms of organic N)13,14.
Much of what we know about the physiology and ecology of
Trichodesmium spp. derives from work on field populations because attempts to culture these species were unsuccessful for many
years. However, at least two Trichodesmium clones (IMS101 and
NIBB1067) have now been isolated from natural populations and
maintained for several years15,16. Both clones have been tentatively
identified as T. erythraeum based on their nifH gene sequence17–19.
Species-specific differences in N metabolism have not been
broadly investigated. In natural assemblages, T. thiebautii fixed
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April 2000, Vol. 5, No. 4
N2 at about twice the rate of T. erythraeum at light intensities
.500 mmol quanta m22 s21 and fixed N2 at ~1.6 times higher rates at
300 mmol quanta m22 s21 (Ref. 20). More rapid rates of N2 fixation
under high light intensities might explain the greater abundance of
T. thiebautii relative to T. erythraeum under such conditions. Low
light intensities were used for cultures during the isolation of strains
from natural populations, and might have selected for T. erythraeum.
Ecology of marine N2 fixation
Epiphytic, endosymbiotic and free-living unicellular and colonial
cyanobacterial species, as well as bacteria, have been shown to fix
N in pelagic marine systems21. However, to date, Trichodesmium
spp. appear to be the most quantitatively significant pelagic N2
fixers in marine systems. They are broadly distributed throughout
the marine tropics and sub-tropics, and surface aggregations can
occur over large expanses of ocean1,12. Besides providing important N inputs, these species can dominate primary productivity
and N cycling in the upper water column2,9,10, particularly when
they occur as episodic ‘blooms’1,4.
In the ocean, multi-cellular trichomes of Trichodesmium spp.
often form spherical aggregates called ‘puffs’ and fusiform bundles
called ‘tufts’, each containing up to several hundred trichomes20.
Free trichomes are also dispersed throughout the upper water column in tropical and subtropical seas11. Cultures of Trichodesmium
IMS101 and NIBB1067 occur largely as free filaments, forming
colonies only during stationary-phase growth13,15,16,22. The precise
causes of bundle formation, aggregation and the formation of
so-called ‘blooms’ are unknown, but many investigators have
suggested that oxygen protection of oxygen-labile nitrogenase, Fe
or other nutrient limitation might be important. Rates of N2 fixation
by Trichodesmium spp. colonies vary widely between sample
sites1,9,10,14 and this might be attributed to environmental and physiological factors affecting natural populations. Highly variable rates
of combined N uptake have also been measured14.
Nitrogenase synthesis and activity in natural23,24 and cultured22,25
populations of Trichodesmium spp. exhibits a daily cycle, and
nitrogenase activity is confined to the light portion of the day. This
daily cycle of N2 fixation corresponds with the pattern of photosynthetic C fixation. In natural populations of T. thiebautii, new
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Table 1. N and C-based growth rates for natural and cultured populations of Trichodesmium spp.
Location
Subtropical oceans
N. Pacific
Sargasso
N. Atlantic
Tropical oceans
Caribbean
Bahamas
N. Atlantic
Cultures
IMS101
Cell specific
N-based
N-based
Cell specific
C-based
C-based
Notes Ref.
N2 fixation rate turnover time doubling time CO2 fixation rate turnover time doubling time
(fmol cell21 h21)
(days)
(days)
(fmol cell21 h21)
(days)
(days)
33
5
2
21.0
133.0
350.0
14.7
92.0
243.0
54
85
18
93.0
59.0
286.0
64.0
41.0
198.0
13
50
14
56.0
14.0
52.0
39.0
9.7
36.0
165
530
935
30.0
9.0
5.5
21.0
6.5
3.8
32
6.1
2.8
515
1.3
2.2
a,b
a,b,c
a,b
50
51
52
a,b,c
51
a,b
d
a,b
e
f
Chlorophyll-based turnover time (days)
IMS101
IMS101
NIBB1067
NIBB1067
4.0–5.0
2.0–4.0
12.5–14.5
3.0–4.0
Assumed C biomass of 1 mmol C col21.
Assumed N biomass of 0.14 mmol N col21.
c
Data recalculated using 3:1 ratio of C2H2 reduction:N2 fixation.
d
D.G. Capone, unpublished.
e
E.J. Carpenter and D.G. Capone, unpublished.
f
M.R. Mulholland and D.G. Capone, unpublished.
a
b
nitrogenase is synthesized each morning23,26, the enzyme is modified and thereby inactivated during the afternoon and subsequently degraded through the night24. Peak N2 fixation occurs
around midday23, suggesting that nitrogenase activity might be
regulated by light. However, nifH (the gene encoding the nitrogenase protein) mRNA abundance in cells collected from natural
populations is highest before dawn, indicating that light itself does
not directly promote nitrogenase synthesis26. The metabolic signals
causing the modification and inactivation of nitrogenase have not
been identified23,24. An endogenous rhythm for N2 fixation and
the transcription, translation and activity of nitrogenase in
Trichodesmium IMS101 has been confirmed22,27.
Recently, it has been observed that not all cells within a colony
or along a filament of Trichodesmium spp. contain nitrogenase.
Immunological studies suggest that ,10–15% of cells clustered
along regions of filaments contain nitrogenase and are therefore
capable of fixing N2 (Refs 21,28–31). Cells within these clusters have
been named diazocytes. Although they have some ultrastructural similarities to heterocysts, they do not have thickened cell walls and retain many of the photosynthetic characteristics of vegetative cells21,30.
However, what is unclear is whether particular cells permanently differentiate to perform N2 fixation or if all cells retain the capacity but
do not simultaneously differentiate and fix N2. It follows that cells that
do not fix N2 require fixed N for growth. The observation that all
Trichodesmium spp. cells do not simultaneously have the ability to
fix N2 has important implications with regard to N utilization and N
metabolism in these species: some of these will be discussed here.
Nitrogen nutrition of Trichodesmium spp.
Low rates of 15N incorporation of combined N, which have been
reported previously, and low d15N of Trichodesmium spp. led to
the general conclusion that natural populations of Trichodesmium
spp. growing in N-depleted oligotrophic waters use N2 to meet the
majority of their cellular N requirements7. In this regard, estimates
of Trichodesmium spp. growth rates for natural populations
vary widely among and even within studies depending on whether
they are based on CO2 fixation (Table 1) or on N2 fixation
(Table 1; M.R. Mulholland and D.G. Capone, unpublished). Interestingly, doubling times are ~2–14 days for cultures of
Trichodesmium NIBB1067 and IMS101 growing exponentially
on seawater or on artificial media13–16,22, which is close to the Cbased doubling times for field populations (Table 1). The higher
growth rates estimated from C relative to N in natural populations
suggests that there are other sources of N. Alternatively, C-based
growth rates might be artifactually high (M.R. Mulholland and
D.G. Capone, unpublished).
Although surface waters in the tropical oligotrophic ocean
gyres are generally depleted in combined N – NH41 and dissolved
organic N (DON) are rapidly recycled to support much of the
apparent N demand of primary production by the phytoplankton
in these systems32,33. Dissolved pools of N (NH41, NO32 1 NO22
and DON) and PO423 can become enriched in and around blooms,
presumably because of N inputs from cells fixing N2 (Refs 4,34).
Trichodesmium spp. appear to simultaneously fix N2 and take
up combined N when it is available at low concentrations13,14.
Although low rates of NH41, NO32 and urea-uptake have been
reported in some studies using populations from the N Atlantic
Ocean and from the Caribbean Sea, there is considerable evidence
that Trichodesmium spp. are capable of taking up combined nitrogen (e.g. NH41, NO32 and DON)14. Natural populations of Trichodesmium spp. from the NW Pacific, the tropical N Atlantic, the
Caribbean Sea and the tropical Atlantic, as well as cultured populations, all have a capacity to take up NH41 at high rates14. High
rates of NH41 uptake have been correlated with NH41 concentrations
in exponentially growing cultured populations of Trichodesmium
NIBB1067 and IMS101 (Refs 13,14). A capacity for glutamate
(Glu) and glutamine (Gln) uptake has also been demonstrated in
natural and cultured populations of Trichodesmium spp.13,14,35,36.
April 2000, Vol. 5, No. 4
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3000
2500
2000
1500
1000
500
0
00.00
06.00
N Utilization (nmol N l–1 h–1)
(b) Exponential
12.00
Time (h)
18.00
24.00
18.00
24.00
18.00
24.00
3000
2500
2000
1500
1000
500
N2 fixation
0
00.00
06.00
(c) Stationary
N Utilization (nmol N l–1 h–1)
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N Utilization (nmol N l–1 h–1)
(a) Early exponential
12.00
Time (h)
3000
2500
2000
1500
1000
NH4+ uptake
500
0
00.00
06.00
12.00
Time (h)
Fig. 1. Pattern of NH41 uptake (light gray) and N2 fixation (dark gray)
over diel cycles during (a) early exponential or lag phase growth, (b)
exponential phase growth and (c) stationary phase growth. Note that
the total N uptake varies with the growth stage as does the proportion
of the total N turnover as a result of N2 fixation. N2 fixation accounts
for most of the N turnover during early exponential growth, less than
half the total N turnover during exponential growth and less than a
There are several possible explanations for the lack of N uptake
in some of the earlier studies. For example, when natural populations of Trichodesmium spp. are encountered in the field, there
is no way of knowing the prior nutritional history or of knowing
what stage of growth they are in at the time of sampling. In culture
systems, NH41 can accumulate to several mM in the growth
medium over the growth cycle and consequently cells take up this
recycled N (which has been measured using 15NH41 tracer additions) at rates comparable to rates of N2 fixation13,14. The relative
proportion of N utilization, derived from N2 fixation and N uptake,
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April 2000, Vol. 5, No. 4
changes over the course of the growth cycle as more N is fixed into the
system and becomes available through recycling33 (M.R. Mulholland
and D.G. Capone, unpublished). Because Trichodesmium spp. must
first fix N2 into the system to grow, rates of N regeneration might be
lower during the early growth stages. If natural populations are
encountered early in the growth cycle, when the proportion of N2
fixation is a higher fraction of the total N uptake, then measurements of NH41 uptake might be low. Alternatively, higher measured rates of N uptake and lower rates of N2 fixation might be
expected in populations sampled during late stationary phase
growth, when new biomass production is low (Fig. 1).
Another possible explanation for the variability in reported
NH41-uptake rates is that NH41 concentrations in the water in
interfilamental spaces of colonies can be higher relative to concentrations measured in surrounding waters. In field studies, although
ambient water column NH41 concentrations have always been at
the limits of analytical detection, Glu, Gln, NH41 and urea concentrations are enriched in the interfilamental spaces of colonies14,36.
Consequently, cells within or adjacent to these microenvironments
are exposed to higher ambient nutrient concentrations. Calculations of NH41 uptake using stable isotopes are sensitive to the
ambient concentrations of nutrients used in the calculations. If, in
actuality, cells are exposed to higher concentrations of N substrates
because of elevated N within the intrafilamental spaces, 15Nlabelled substrates diffusing in would be diluted relative to the
enrichment factor used in the calculation based on ambient seawater
concentrations. Using the extracolonial or ‘ambient’ concentrations
in making uptake-rate calculations might thereby result in an
underestimation of the true rate of uptake.
With respect to the natural abundance data, although low d15N signatures in aquatic communities typically indicate a N2 N source, this
does not preclude the utilization of other isotopically light N sources.
For instance, the NH41 and DON pools derived directly from the
release of recently fixed N2 would be isotopically lighter than
NH41 or DON derived from recycled material derived from nonatmospheric N sources. Rapid reassimilation of this isotopically light
pool of regenerated N would allow Trichodesmium spp. to retain
their light isotopic signature while using regenerated N sources.
If only a small fraction of Trichodesmium spp. cells contain nitrogenase, a high capacity for NH41 uptake and an ability to concurrently take up nitrogenous substrates and fix N2 during the light
period might be an important adaptation for the extracellular distribution of N among cells and filaments13,14,35. Examination of Trichodesmium spp. filaments has not produced evidence of structural
mechanisms for along-filament transport of fixed N (Ref. 30). Consequently, it is unlikely that fixed N is transferred between cells
along filaments. An extracellular mechanism for the transfer of fixed
N among Trichodesmium spp. cells that are fixing N2 and among
those that are not has been proposed. It is suggested that fixed N, primarily amino acids, are released by cells fixing N2 and these are
available for uptake by cells that are not fixing N2 (Ref. 35). High
rates of amino acid36, DON (Ref. 37) and NH41 (Ref. 13) release,
and a simultaneous uptake capacity for amino acids and NH41 by
Trichodesmium spp. colonies supports this contention (Fig. 2).
In cultures of Trichodesmium NIBB1067 and IMS101, growing exponentially on a defined seawater medium without added N
sources, N2 fixation accounts for ~15% whereas the sum of NH41
and glu uptake accounts for ~85% of the total-measured daily
N uptake13,14. This is also consistent with the idea that 85% of
Trichodesmium spp. cells along a filament or within a colony do
not fix N2, but instead must rely on the uptake of combined N.
Trichodesmium NIBB1067, an isolate from the Kuroshio current
near Japan, can grow on NH41, NO32 and urea-enriched media in
the laboratory38,39. However, nitrogenase synthesis and activity are
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affected by the N source. Cultures grown on
NO32, NH41 and urea do not exhibit nitrogeNH4+
Glu
N2
nase activity or reduced nitrogenase activity
13,22,38
during light periods
. In a study using
Trichodesmium NIBB1067, the modified,
inactive form of the nitrogenase Fe-protein
was found in cells grown on NO32 or NH41,
NH4+
but nitrogenase was absent entirely from cells
+
NH
4
Glu
grown on urea39. In Trichodesmium IMS101,
N2
the Fe-protein was absent from cells treated
N2
with 20 mM of either NH41 or urea. In another
NH4+
study of Trichodesmium NIBB1067, cells
cultured on medium containing excess NO32
Gln
or urea retained nitrogenase and a capacity to
fix N2 during at least a portion of the light
NH4+
period. However, they did so at rates lower
+
NH4 + Keto acid
N2
than those measured in cultures maintained
13
on medium without added N substrates .
Cultures growing in medium with excess
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urea turned over their cell N much more
Fig. 2. Conceptual diagram of nitrogen cycling within a colony of Trichodesmium. Four filarapidly than cultures growing on NO32ments are shown, with green cells representing vegetative or ‘non-N2-fixing’ cells. Blue cells
enriched medium or on medium without
represent N2-fixing cells clustered in regions along filaments. Dinitrogen diffuses into N2-fixing
added N sources13. However, growth rates
cells, where it is fixed in the form of NH41, Gln and Glu. Some of these compounds are released
were similar among cultures grown with or
directly into the growth medium where they are available for uptake by Trichodesmium cells or
without combined N sources13,38. The variother organisms and other non-assimilatory reactions (pink ovals). Organisms mediating other
ation in N turnover rates between cultures
possible fates of fixed N are depicted in red. Associated organisms with cell-surface amino acid
growing on different N sources could occur
oxidases might oxidize the amino acids, thereby liberating NH41 (depicted as red circles, lower
left). Additionally, NH41 might be released during grazing (red copepod in upper left).
because N2 fixation is confined to the light
period regardless of the N regime in natural
and cultured populations of Trichodesmium
spp. By contrast urea and NH41 are taken up
at comparable rates during both the dark and the light periods. Uptake might be satisfied by NH41 uptake supplied by N recycled within the
of NO32 occurs primarily during the light period13, and probably upper water column community13,14. Because Trichodesmium spp. are
competes with N2 fixation for photosynthetic energy and reductant. able to take up combined N, C-based productivity might not be a meaThe absolute growth rate (e.g. doubling of biomass) might be limited sure of new production in systems where allochthonous N is imported.
intrinsically.
Clearly, additional studies investigating the effect of combined N Nitrogen metabolism
on growth and the competitive advantage of Trichodesmium spp. Nitrogen assimilation in natural populations of Trichodesmium
need to be pursued. Although Trichodesmium NIBB1067 and spp. proceeds via the glutamine synthetase/glutamate synthase
IMS101 appear to be able to take up combined N and fix N2 simul- (GS/GOGAT) pathway35. GS is necessary for NH41 assimilation
taneously or alternately in cultures, little is known about the growth regardless of the primary form of N being used. High rates of GS
characteristics of Trichodesmium spp. under different N regimes in transferase activity (a reverse reaction assay that provides an
nature. Trichodesmium spp. can alleviate N limitation and therefore approximation of total GS active sites) relative to rates of total N
out-compete other species in N-limiting environments in nature, uptake have been observed in natural and cultured populations of
although blooms persist even after N concentrations become Trichodesmium spp. (Refs 13,14). Rates of both GS transferase
elevated. It is unclear how increasing availability of combined and GS biosynthetic activity (which approximates in vivo forward
N species affects the growth of Trichodesmium spp. and their reaction activity) in Trichodesmium spp. increase in the afternoon
during the period when rates of N2 fixation are highest. The ratio of
competitive success in natural systems.
The modification and expression of nitrogenase might be at least GS transferase:biosynthetic activity decreases during the period of
partially regulated by the N regime. NH41, Glu and Gln additions maximum N2 fixation, indicating that the proportion of the GS pool
have been shown to reduce N2 fixation over time, probably because that is biosynthetically active increases during the day.
The biosynthetic capacity of GS is sufficient to allow Triof feedback inhibition of nitrogenase by metabolite pools23 (M.R.
Mulholland et al. unpublished). From studies using metabolic chodesmium spp. colonies to turnover their cell N at least three times
inhibitors, it appears that metabolites that accumulate during N per day, suggesting that N assimilation does not limit the rate of N utiassimilation, are important in regulating nitrogenase activity and lization by cells, even during midday when N2 fixation rates are highsynthesis in natural and cultured populations of Trichodesmium est13,14. Cells appear to have sufficient capacity to assimilate all of the
spp.39. However, the presence of low concentrations of NH41 and intracellular N substrates derived from N2 fixation and N uptake in
amino acids in the culture medium during growth do not inhibit N2 cultures growing on media with or without added N (Ref. 13).
Excess GS activity is characteristic of cells limited by N or
fixation. Additions of combined N of 5–10 mM or more might be
necessary before nitrogenase synthesis or activity is affected. Such using N2 as their N source40. A positive correlation between GS and
high concentrations are not typical of the oligotrophic regions in nitrogenase enzyme abundance and distribution has been observed
which these species occur.
in a variety of heterocystous41,42 and non-heterocystous43,44 cyanoAlthough N2 fixation might supply the ‘new’ N necessary for alle- bacteria including Trichodesmium spp35. The GS enzyme is twice
viating N limitation of net growth, much of the gross N turnover as abundant in Trichodesmium spp. cells that contain nitrogenase
April 2000, Vol. 5, No. 4
151
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than in those that do not35. Although different types of GS might
be present in cells, different promoters for transcribing the gene
encoding GS during growth on fixed N and molecular N2 have
also been observed40. Thus, there might be both a constitutive pool
of GS, regulated for the general assimilation of N derived from
various N sources, and a nitrogenase-linked pool co-regulated
specifically with nitrogenase under low N conditions.
In support of two separately regulated GS pools in Trichodesmium
spp., glnA transcript abundance is elevated both during the pre-dawn
period and again in the late afternoon45. The early morning peak in
glnA transcript abundance corresponds with peak nifH (a gene encoding the Fe-protein of nitrogenase) transcript abundance26, whereas the
afternoon peak does not. In Trichodesmium NIBB1067 grown on
medium without added N, biosynthetic GS activity increased before
the onset of the light period and then again in the late afternoon (M.R.
Mulholland et al. unpublished), consistent with the pattern of glnA
mRNA abundance in natural populations45.
The absence of a strong diel cycle of GS activity might reflect the
observation that Trichodesmium spp. colonies and filaments take up
regenerated N sources in addition to fixing N2 during the day. It
might also reflect the observation that only a fraction of cells along
a filament or within a colony may contain nitrogenase and are,
therefore, capable of N2 fixation. If two pools of GS are present in
Trichodesmium spp., the small changes in total filament or colony
GS activity over a diel cycle might be significant in terms of the
capacity for N assimilation in cells fixing N2. The ratio of GS transferase:biosynthetic activity in natural and cultured populations of
Trichodesmium spp. varies by ~20 to 30% over a diel cycle13,14. The
magnitude of this change is consistent with the suggestion that
nitrogenase is confined to ,10–15% of cells along a filament or
within a colony29,31, that GS is twice as abundant in cells containing
nitrogenase35, and that a portion of the GS pool is regulated in conjunction with nitrogenase.
Based on observations of the distribution and activity of GS during N2 fixation, it has been suggested that GS plays an important
regulatory role in N2 fixation, either directly or indirectly, by
preventing feedback inhibition from accumulated metabolites46.
Glutamine and glutamate are primary products of N metabolism
via the GS/GOGAT metabolic pathway. Intracolonial Glu and Gln
concentrations parallel the daily cycle of N2 fixation35,36 and might
be factors in subcellular regulation of nitrogenase. However, in
cultures of Trichodesmium NIBB1067 growing with and without
combined N, the Gln:Glu ratio reflects the pattern of total N uptake
(positive relationship)13. In natural populations, the magnitude and
timing of the daily peak in Gln:Glu ratios varies among sites and
populations. However, the highest Gln:Glu ratios are observed
during the period when rates of N2 fixation are highest14,36.
Light might be an important determinant for N2 fixation and the
N status of Trichodesmium spp. cells. Higher Gln:Glu ratios (0.7)
were observed in Trichodesmium spp. collected in the surface
waters compared with colonies collected from deep waters14. The
decrease in the intracellular Gln:Glu ratios in colonies collected
from deeper in the euphotic zone might be due to light or energy
limitation of N2 fixation rates. Higher rates of photosynthesis by
populations growing in surface waters might result in increases
in the supply of energy to support higher rates of N2 fixation.
In cultures of Trichodesmium IMS101 and NIBB1067, grown
on medium without added N substrates, the intracellular ratios of
Gln:Glu are lower (a maximum of ~0.3) and this might reflect the
low light levels at which these isolates are maintained13,14.
Nitrogen regulation
Investigators have suggested that the nitrogenase of Trichodesmium
NIBB1067 is regulated at different levels. An endogenous rhythm
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April 2000, Vol. 5, No. 4
has been identified for nitrogenase transcription, translation and
activity27. Alternatively, it has been suggested that the modification
and expression of nitrogenase is also regulated by the availability of
combined N (Ref. 39). Both regulatory mechanisms appear to be
important. Recently, a global N-regulating gene, ntcA, has been
identified in a Trichodesmium spp. isolate from the Red Sea (D.
Lindell pers. commun.), but the conditions under which it is
expressed have not been ascertained.
Community nitrogen dynamics and future directions
Although we are beginning to understand the physiology of N2
fixation by Trichodesmium spp., there remain many uncertainties
regarding the mechanisms that support the growth of Trichodesmium spp. populations in nature and the growth of organisms
living in association with Trichodesmium spp. colonies. For example, Trichodesmium spp. might have a larger role in the marine N
cycle than previously suspected. Although net growth of populations might depend on inputs of new N from N2 fixation,
Trichodesmium spp. also contribute to overall N turnover in oceanic
systems. They contribute both directly through the release of amino
acids, DON and NH41 (Refs 13,36,37), and indirectly through the
regeneration of dissolved inorganic and organic N by bacteria and
grazers living in association with Trichodesmium spp. colonies47–49.
Pathways of N cycling among Trichodesmium spp. filaments and
colonies and associated organisms remain to be directly quantified.
Although Trichodesmium spp. alleviate N limitation of growth by
fixing N2, the effects of deficits of other nutrients on Trichodesmium
spp. growth and N2 fixation are poorly understood. In particular, Fe,
an essential component of the nitrogenase enzyme complex, might
limit N2 fixation in oceanic regions with low aeolian dust inputs.
Phosphorous is also in short supply in oceanic regions where
Trichodesmium spp. grow and form blooms. It is unclear what the
nutrient demands are for Trichodesmium spp. growth and how they
acquire sufficient P to support observed growth rates. High N:P
ratios have been observed in populations from the Pacific Ocean10.
Although we are gaining in our understanding of how these species
affect the marine N cycle where they occur, much needs to be done
to integrate N2 fixation into global C, N and P budgets.
Acknowledgements
We would like to thank three anonymous reviewers for their helpful comments and suggestions. This work was supported by grants
from the National Science Foundation Division of Ocean Sciences.
References
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2 Carpenter, E.J. and Romans, K. (1991) Major role of the cyanobacteria
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3 Michaels, A. et al. (1996) Excess nitrate and the rate of nitrogen fixation in the
Sargasso Sea. Biogeochemistry 35, 181–226
4 Karl, D.M. et al. (1992) Trichodesmium blooms and new nitrogen in the North
Pacific gyre. In Marine Pelagic Cyanobacteria: Trichodesmium and Other
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14 Mulholland, M.R. and Capone, D.G. (1999) N2 fixation, N uptake and N
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16 Prufert-Bebout, L. et al. (1993) Growth, nitrogen fixation, and spectral
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17 Ben-Porath, J. et al. (1993) Genotype relationships in the genus
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18 Janson, S. et al. (1995) Cytomorphological characterization of the planktonic
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20 Carpenter, E.J. et al. (1993) The tropical diazotrophic phytoplankter
Trichodesmium: biological characteristics of two common species. Mar. Ecol.
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21 Bergman, B. et al. (1997) N2 fixation by non-heterocystous cyanobacteria.
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22 Chen, Y-B. et al. (1996) Growth and nitrogen fixation of the diazotrophic
filamentous nonheterocystous cyanobacterium Trichodesmium sp. IMS101 in
defined media: evidence for a circadian rhythm. J. Phycol. 32, 916–923
23 Capone, D.G. et al. (1990) Basis for diel variation in nitrogenase activity in
the marine planktonic cyanobacterium Trichodesmium thiebautii. Appl.
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24 Zehr, J.P. et al. (1993) Modification of the Fe protein of nitrogenase in natural
populations of Trichodesmium thiebautii. Appl. Environ. Microbiol. 59, 669–676
25 Ohki, K. and Fujita, Y. (1988) Aerobic nitrogenase activity measured as
acetylene reduction in the marine non-heterocystous cyanobacterium
Trichodesmium spp. grown under artificial conditions. Mar. Biol. 98, 111–114
26 Wyman, M. et al. (1996) Temporal variability in nitrogenase gene expression
in a natural population of the marine cyanobacterium Trichodesmium
thiebautii. Appl. Environ. Microbiol. 62, 1073–1075
27 Chen, Y-B. et al. (1998) Circadian rhythm of nitrogenase gene expression in
the diazotrophic filamentous nonheterocystous cyanobacterium
Trichodesmium sp. strain IMS101. J. Bacteriol. 180, 3598–3605
28 Janson, S. et al. (1994) Compartmentalization of nitrogenase in a nonheterocystous cyanobacterium: Trichodesmium contortum. FEMS Microbiol.
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29 Fredriksson, C. and Bergman, B. (1995) Nitrogenase quantity varies diurnally
in a subset of cells within colonies of the non-heterocystous cyanobacteria
Trichodesmium spp. Microbiology 141, 2471–2478
30 Fredriksson, C. and Bergman, B. (1997) Ultrastructural characterization of
cells specialised for nitrogen fixation in a non-heterocystous cyanobacterium,
Trichodesmium spp. Protoplasma 197, 76–86
31 Lin, S. et al. (1998) Whole-cell immunolocalization of nitrogenase in marine
diazotrophic cyanobacteria, Trichodesmium spp. Appl. Environ. Microbiol. 64,
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32 Bronk, D.A. et al. (1994) Nitrogen uptake, dissolved organic nitrogen release,
and new production. Science 265, 1843–1846
33 Glibert, P.M. (1993) The interdependence of uptake and release of NH41
and organic nitrogen. Mar. Microbial Food Webs 7, 53–67
34 Devassy, V.P. (1987) Trichodesmium red tides in the Arabian Sea. In
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(Qasim, S.Z., ed.), pp. 61–66, India
35 Carpenter, E.J. et al. (1992) Glutamine synthetase and nitrogen cycling in
colonies of the marine diazotrophic cyanobacteria Trichodesmium spp. Appl.
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36 Capone, D.G. et al. (1994) Amino acid cycling in colonies of the planktonic marine
cyanobacterium, Trichodesmium thiebautii. Appl. Environ. Microbiol. 60, 3989–3995
37 Glibert, P.M. and Bronk, D.A. (1994) Release of dissolved organic nitrogen
by marine diazotrophic cyanobacteria, Trichodesmium spp. Appl. Environ.
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38 Ohki, K. et al. (1991) Regulation of nitrogen fixation by different nitrogen
sources in the marine non-heterocystous cyanobacterium Trichodesmium sp.
NIBB1067. Arch. Microbiol. 156, 335–337
39 Ohki, K. et al. (1992) Regulation of nitrogenase activity in relation to the
light–dark regime in the filamentous non-heterocystous cyanobacterium
Trichodesmium sp. NIBB 1067. J. Gen. Microbiol. 138, 2679–2685
40 Tumer, N.E. et al. (1983) Different promoters for the Anabaena glutamine synthetase
gene during growth using molecular or fixed nitrogen. Nature 306, 337–342
41 Bergman, B. et al. (1985) Immuno-gold localization of glutamine synthetase
in a nitrogen-fixing cyanobacterium (Anabaena cylindrica). Planta 166, 329–334
42 Renstrom-Kellner, E. et al. (1990) Correlation between nitrogenase and
glutamine synthetase expression in the cyanobacterium Anabaena cylindrica.
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45 Kramer, J.G. et al. (1996) Diel variability in transcription of the structural gene
for glutamine synthetase (glnA) in natural populations of the marine diazotrophic
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46 Flores, E. and Herrero, A. (1994) Assimilatory nitrogen metabolism and its
regulation. In The Molecular Biology of Cyanobacteria (Bryant, D.A., ed.),
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47 Mulholland, M.R. et al. (1998) Extracellular amino acid oxidation by
phytoplankton and cyanobacteria: a cross-ecosystem comparison. Aquat.
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48 O’Neil, J.M. et al. (1996) Ingestion of 15N2-labelled Trichodesmium spp. and
ammonium regeneration by the harpacticoid copepod Macrosetella gracilis.
Mar. Biol. 125, 89–96
49 Paerl, H.W. et al. (1989) Bacterial associations with marine Oscillatoria sp.
(Trichodesmium sp.) populations: ecophysiological implications. J. Phycol.
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50 Mague, T.H. et al. (1977) Physiology and chemical composition of nitrogenfixing phytoplankton in the central North Pacific Ocean. Mar. Biol. 41, 213–227
51 Carpenter, E.J. and Price, C.C. (1977) Nitrogen fixation, distribution, and
production of Oscillatoria (Trichodesmium) spp. in the western Sargasso and
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Margaret Mulholland* is at the Marine Sciences Research Center,
SUNY Stony Brook, Stony Brook, NY 11794-5000, USA; Douglas
Capone is at the Wrigley Institute for Environmental Studies
and Dept of Biological Sciences, University of Southern California,
Los Angeles, CA 90089, USA (e-mail capone@wrigley.usc.edu).
*Author for correspondence (tel 11 631 632 3163;
fax 11 631 632 8820; e-mail mmulholland@notes.cc.sunysb.edu).
April 2000, Vol. 5, No. 4
153
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The nitrogen physiology of the
marine N2-fixing cyanobacteria
Trichodesmium spp.
Margaret R. Mulholland and Douglas G. Capone
Trichodesmium spp. have proved to be enigmatic organisms, and their ecology and physiology
are unusual among diazotrophs. Recent research shows that they can simultaneously fix N2
and take up combined nitrogen. The co-occurrence of these two processes is thought to be
incompatible, but they could be obligatory in Trichodesmium spp. if only a small fraction of
cells within a colony or along a filament are capable of N2 fixation. Combined nitrogen is
released from cells during periods of active growth and N2 fixation, and concomitantly taken up
by Trichodesmium spp. or cells living in association with colonies. Although the nitrogenase of
Trichodesmium spp. is affected by high concentrations of combined nitrogen, it might be relatively less sensitive to low concentrations of combined nitrogen typical of the oligotrophic
ocean and culture conditions. Nitrogenase activity and synthesis exhibits an endogenous
rhythm in Trichodesmium spp. cultures, which is affected by the addition of nitrogen.
I
n spite of the observation that nitrogen (N) is often the nutrient
that limits primary productivity in many oceanic environments,
marine N2 fixation has been largely overlooked as a source of
fixed N in these systems. This is probably because reports in the
early 1980s suggested that integrated rates of marine N2 fixation
were small compared with overall phytoplankton N demand.
However, a range of new evidence indicates that marine N2 fixation
does provide a significant source of new N to tropical marine
ecosystems. This evidence includes:
• Recognition of a more widespread occurrence and higher densities of the cyanobacteria Trichodesmium spp. than previously
reported1,2.
• Deficits and imbalances in the marine N budget indicating
unquantified N sources3–5.
• Particulate organic N and NO32 in surface waters with N isotopic signatures close to that of atmospheric N2 (low d15N)6,7.
• Measurements of rates of N2 fixation comparable to the flux and
uptake of fixed N (primarily NO32) into the euphotic zone1,2,11.
Furthermore, evidence is accumulating for other N2 fixers, which
could contribute substantially to the ocean’s fixed N budget8.
N2 fixation by Trichodesmium spp. is a significant source of new
N to tropical and subtropical marine systems where they occur1,9–11.
Species of the non-heterocystous, diazotrophic, cyanobacterial
genus Trichodesmium spp., are common throughout the N-depleted
tropical and subtropical oligotrophic ocean, and rates of N2 fixation
by Trichodesmium spp. have been measured in a variety of locations1. Although it is often assumed that Trichodesmium spp.
depend primarily on N2 fixation to meet their nutritional needs12,
they are also capable of taking up other nitrogenous compounds
(e.g. NH41, NO32 and some forms of organic N)13,14.
Much of what we know about the physiology and ecology of
Trichodesmium spp. derives from work on field populations because attempts to culture these species were unsuccessful for many
years. However, at least two Trichodesmium clones (IMS101 and
NIBB1067) have now been isolated from natural populations and
maintained for several years15,16. Both clones have been tentatively
identified as T. erythraeum based on their nifH gene sequence17–19.
Species-specific differences in N metabolism have not been
broadly investigated. In natural assemblages, T. thiebautii fixed
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April 2000, Vol. 5, No. 4
N2 at about twice the rate of T. erythraeum at light intensities
.500 mmol quanta m22 s21 and fixed N2 at ~1.6 times higher rates at
300 mmol quanta m22 s21 (Ref. 20). More rapid rates of N2 fixation
under high light intensities might explain the greater abundance of
T. thiebautii relative to T. erythraeum under such conditions. Low
light intensities were used for cultures during the isolation of strains
from natural populations, and might have selected for T. erythraeum.
Ecology of marine N2 fixation
Epiphytic, endosymbiotic and free-living unicellular and colonial
cyanobacterial species, as well as bacteria, have been shown to fix
N in pelagic marine systems21. However, to date, Trichodesmium
spp. appear to be the most quantitatively significant pelagic N2
fixers in marine systems. They are broadly distributed throughout
the marine tropics and sub-tropics, and surface aggregations can
occur over large expanses of ocean1,12. Besides providing important N inputs, these species can dominate primary productivity
and N cycling in the upper water column2,9,10, particularly when
they occur as episodic ‘blooms’1,4.
In the ocean, multi-cellular trichomes of Trichodesmium spp.
often form spherical aggregates called ‘puffs’ and fusiform bundles
called ‘tufts’, each containing up to several hundred trichomes20.
Free trichomes are also dispersed throughout the upper water column in tropical and subtropical seas11. Cultures of Trichodesmium
IMS101 and NIBB1067 occur largely as free filaments, forming
colonies only during stationary-phase growth13,15,16,22. The precise
causes of bundle formation, aggregation and the formation of
so-called ‘blooms’ are unknown, but many investigators have
suggested that oxygen protection of oxygen-labile nitrogenase, Fe
or other nutrient limitation might be important. Rates of N2 fixation
by Trichodesmium spp. colonies vary widely between sample
sites1,9,10,14 and this might be attributed to environmental and physiological factors affecting natural populations. Highly variable rates
of combined N uptake have also been measured14.
Nitrogenase synthesis and activity in natural23,24 and cultured22,25
populations of Trichodesmium spp. exhibits a daily cycle, and
nitrogenase activity is confined to the light portion of the day. This
daily cycle of N2 fixation corresponds with the pattern of photosynthetic C fixation. In natural populations of T. thiebautii, new
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01576-4
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Table 1. N and C-based growth rates for natural and cultured populations of Trichodesmium spp.
Location
Subtropical oceans
N. Pacific
Sargasso
N. Atlantic
Tropical oceans
Caribbean
Bahamas
N. Atlantic
Cultures
IMS101
Cell specific
N-based
N-based
Cell specific
C-based
C-based
Notes Ref.
N2 fixation rate turnover time doubling time CO2 fixation rate turnover time doubling time
(fmol cell21 h21)
(days)
(days)
(fmol cell21 h21)
(days)
(days)
33
5
2
21.0
133.0
350.0
14.7
92.0
243.0
54
85
18
93.0
59.0
286.0
64.0
41.0
198.0
13
50
14
56.0
14.0
52.0
39.0
9.7
36.0
165
530
935
30.0
9.0
5.5
21.0
6.5
3.8
32
6.1
2.8
515
1.3
2.2
a,b
a,b,c
a,b
50
51
52
a,b,c
51
a,b
d
a,b
e
f
Chlorophyll-based turnover time (days)
IMS101
IMS101
NIBB1067
NIBB1067
4.0–5.0
2.0–4.0
12.5–14.5
3.0–4.0
Assumed C biomass of 1 mmol C col21.
Assumed N biomass of 0.14 mmol N col21.
c
Data recalculated using 3:1 ratio of C2H2 reduction:N2 fixation.
d
D.G. Capone, unpublished.
e
E.J. Carpenter and D.G. Capone, unpublished.
f
M.R. Mulholland and D.G. Capone, unpublished.
a
b
nitrogenase is synthesized each morning23,26, the enzyme is modified and thereby inactivated during the afternoon and subsequently degraded through the night24. Peak N2 fixation occurs
around midday23, suggesting that nitrogenase activity might be
regulated by light. However, nifH (the gene encoding the nitrogenase protein) mRNA abundance in cells collected from natural
populations is highest before dawn, indicating that light itself does
not directly promote nitrogenase synthesis26. The metabolic signals
causing the modification and inactivation of nitrogenase have not
been identified23,24. An endogenous rhythm for N2 fixation and
the transcription, translation and activity of nitrogenase in
Trichodesmium IMS101 has been confirmed22,27.
Recently, it has been observed that not all cells within a colony
or along a filament of Trichodesmium spp. contain nitrogenase.
Immunological studies suggest that ,10–15% of cells clustered
along regions of filaments contain nitrogenase and are therefore
capable of fixing N2 (Refs 21,28–31). Cells within these clusters have
been named diazocytes. Although they have some ultrastructural similarities to heterocysts, they do not have thickened cell walls and retain many of the photosynthetic characteristics of vegetative cells21,30.
However, what is unclear is whether particular cells permanently differentiate to perform N2 fixation or if all cells retain the capacity but
do not simultaneously differentiate and fix N2. It follows that cells that
do not fix N2 require fixed N for growth. The observation that all
Trichodesmium spp. cells do not simultaneously have the ability to
fix N2 has important implications with regard to N utilization and N
metabolism in these species: some of these will be discussed here.
Nitrogen nutrition of Trichodesmium spp.
Low rates of 15N incorporation of combined N, which have been
reported previously, and low d15N of Trichodesmium spp. led to
the general conclusion that natural populations of Trichodesmium
spp. growing in N-depleted oligotrophic waters use N2 to meet the
majority of their cellular N requirements7. In this regard, estimates
of Trichodesmium spp. growth rates for natural populations
vary widely among and even within studies depending on whether
they are based on CO2 fixation (Table 1) or on N2 fixation
(Table 1; M.R. Mulholland and D.G. Capone, unpublished). Interestingly, doubling times are ~2–14 days for cultures of
Trichodesmium NIBB1067 and IMS101 growing exponentially
on seawater or on artificial media13–16,22, which is close to the Cbased doubling times for field populations (Table 1). The higher
growth rates estimated from C relative to N in natural populations
suggests that there are other sources of N. Alternatively, C-based
growth rates might be artifactually high (M.R. Mulholland and
D.G. Capone, unpublished).
Although surface waters in the tropical oligotrophic ocean
gyres are generally depleted in combined N – NH41 and dissolved
organic N (DON) are rapidly recycled to support much of the
apparent N demand of primary production by the phytoplankton
in these systems32,33. Dissolved pools of N (NH41, NO32 1 NO22
and DON) and PO423 can become enriched in and around blooms,
presumably because of N inputs from cells fixing N2 (Refs 4,34).
Trichodesmium spp. appear to simultaneously fix N2 and take
up combined N when it is available at low concentrations13,14.
Although low rates of NH41, NO32 and urea-uptake have been
reported in some studies using populations from the N Atlantic
Ocean and from the Caribbean Sea, there is considerable evidence
that Trichodesmium spp. are capable of taking up combined nitrogen (e.g. NH41, NO32 and DON)14. Natural populations of Trichodesmium spp. from the NW Pacific, the tropical N Atlantic, the
Caribbean Sea and the tropical Atlantic, as well as cultured populations, all have a capacity to take up NH41 at high rates14. High
rates of NH41 uptake have been correlated with NH41 concentrations
in exponentially growing cultured populations of Trichodesmium
NIBB1067 and IMS101 (Refs 13,14). A capacity for glutamate
(Glu) and glutamine (Gln) uptake has also been demonstrated in
natural and cultured populations of Trichodesmium spp.13,14,35,36.
April 2000, Vol. 5, No. 4
149
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3000
2500
2000
1500
1000
500
0
00.00
06.00
N Utilization (nmol N l–1 h–1)
(b) Exponential
12.00
Time (h)
18.00
24.00
18.00
24.00
18.00
24.00
3000
2500
2000
1500
1000
500
N2 fixation
0
00.00
06.00
(c) Stationary
N Utilization (nmol N l–1 h–1)
Trends in Plant Science
N Utilization (nmol N l–1 h–1)
(a) Early exponential
12.00
Time (h)
3000
2500
2000
1500
1000
NH4+ uptake
500
0
00.00
06.00
12.00
Time (h)
Fig. 1. Pattern of NH41 uptake (light gray) and N2 fixation (dark gray)
over diel cycles during (a) early exponential or lag phase growth, (b)
exponential phase growth and (c) stationary phase growth. Note that
the total N uptake varies with the growth stage as does the proportion
of the total N turnover as a result of N2 fixation. N2 fixation accounts
for most of the N turnover during early exponential growth, less than
half the total N turnover during exponential growth and less than a
There are several possible explanations for the lack of N uptake
in some of the earlier studies. For example, when natural populations of Trichodesmium spp. are encountered in the field, there
is no way of knowing the prior nutritional history or of knowing
what stage of growth they are in at the time of sampling. In culture
systems, NH41 can accumulate to several mM in the growth
medium over the growth cycle and consequently cells take up this
recycled N (which has been measured using 15NH41 tracer additions) at rates comparable to rates of N2 fixation13,14. The relative
proportion of N utilization, derived from N2 fixation and N uptake,
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April 2000, Vol. 5, No. 4
changes over the course of the growth cycle as more N is fixed into the
system and becomes available through recycling33 (M.R. Mulholland
and D.G. Capone, unpublished). Because Trichodesmium spp. must
first fix N2 into the system to grow, rates of N regeneration might be
lower during the early growth stages. If natural populations are
encountered early in the growth cycle, when the proportion of N2
fixation is a higher fraction of the total N uptake, then measurements of NH41 uptake might be low. Alternatively, higher measured rates of N uptake and lower rates of N2 fixation might be
expected in populations sampled during late stationary phase
growth, when new biomass production is low (Fig. 1).
Another possible explanation for the variability in reported
NH41-uptake rates is that NH41 concentrations in the water in
interfilamental spaces of colonies can be higher relative to concentrations measured in surrounding waters. In field studies, although
ambient water column NH41 concentrations have always been at
the limits of analytical detection, Glu, Gln, NH41 and urea concentrations are enriched in the interfilamental spaces of colonies14,36.
Consequently, cells within or adjacent to these microenvironments
are exposed to higher ambient nutrient concentrations. Calculations of NH41 uptake using stable isotopes are sensitive to the
ambient concentrations of nutrients used in the calculations. If, in
actuality, cells are exposed to higher concentrations of N substrates
because of elevated N within the intrafilamental spaces, 15Nlabelled substrates diffusing in would be diluted relative to the
enrichment factor used in the calculation based on ambient seawater
concentrations. Using the extracolonial or ‘ambient’ concentrations
in making uptake-rate calculations might thereby result in an
underestimation of the true rate of uptake.
With respect to the natural abundance data, although low d15N signatures in aquatic communities typically indicate a N2 N source, this
does not preclude the utilization of other isotopically light N sources.
For instance, the NH41 and DON pools derived directly from the
release of recently fixed N2 would be isotopically lighter than
NH41 or DON derived from recycled material derived from nonatmospheric N sources. Rapid reassimilation of this isotopically light
pool of regenerated N would allow Trichodesmium spp. to retain
their light isotopic signature while using regenerated N sources.
If only a small fraction of Trichodesmium spp. cells contain nitrogenase, a high capacity for NH41 uptake and an ability to concurrently take up nitrogenous substrates and fix N2 during the light
period might be an important adaptation for the extracellular distribution of N among cells and filaments13,14,35. Examination of Trichodesmium spp. filaments has not produced evidence of structural
mechanisms for along-filament transport of fixed N (Ref. 30). Consequently, it is unlikely that fixed N is transferred between cells
along filaments. An extracellular mechanism for the transfer of fixed
N among Trichodesmium spp. cells that are fixing N2 and among
those that are not has been proposed. It is suggested that fixed N, primarily amino acids, are released by cells fixing N2 and these are
available for uptake by cells that are not fixing N2 (Ref. 35). High
rates of amino acid36, DON (Ref. 37) and NH41 (Ref. 13) release,
and a simultaneous uptake capacity for amino acids and NH41 by
Trichodesmium spp. colonies supports this contention (Fig. 2).
In cultures of Trichodesmium NIBB1067 and IMS101, growing exponentially on a defined seawater medium without added N
sources, N2 fixation accounts for ~15% whereas the sum of NH41
and glu uptake accounts for ~85% of the total-measured daily
N uptake13,14. This is also consistent with the idea that 85% of
Trichodesmium spp. cells along a filament or within a colony do
not fix N2, but instead must rely on the uptake of combined N.
Trichodesmium NIBB1067, an isolate from the Kuroshio current
near Japan, can grow on NH41, NO32 and urea-enriched media in
the laboratory38,39. However, nitrogenase synthesis and activity are
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affected by the N source. Cultures grown on
NO32, NH41 and urea do not exhibit nitrogeNH4+
Glu
N2
nase activity or reduced nitrogenase activity
13,22,38
during light periods
. In a study using
Trichodesmium NIBB1067, the modified,
inactive form of the nitrogenase Fe-protein
was found in cells grown on NO32 or NH41,
NH4+
but nitrogenase was absent entirely from cells
+
NH
4
Glu
grown on urea39. In Trichodesmium IMS101,
N2
the Fe-protein was absent from cells treated
N2
with 20 mM of either NH41 or urea. In another
NH4+
study of Trichodesmium NIBB1067, cells
cultured on medium containing excess NO32
Gln
or urea retained nitrogenase and a capacity to
fix N2 during at least a portion of the light
NH4+
period. However, they did so at rates lower
+
NH4 + Keto acid
N2
than those measured in cultures maintained
13
on medium without added N substrates .
Cultures growing in medium with excess
Trends in Plant Science
urea turned over their cell N much more
Fig. 2. Conceptual diagram of nitrogen cycling within a colony of Trichodesmium. Four filarapidly than cultures growing on NO32ments are shown, with green cells representing vegetative or ‘non-N2-fixing’ cells. Blue cells
enriched medium or on medium without
represent N2-fixing cells clustered in regions along filaments. Dinitrogen diffuses into N2-fixing
added N sources13. However, growth rates
cells, where it is fixed in the form of NH41, Gln and Glu. Some of these compounds are released
were similar among cultures grown with or
directly into the growth medium where they are available for uptake by Trichodesmium cells or
without combined N sources13,38. The variother organisms and other non-assimilatory reactions (pink ovals). Organisms mediating other
ation in N turnover rates between cultures
possible fates of fixed N are depicted in red. Associated organisms with cell-surface amino acid
growing on different N sources could occur
oxidases might oxidize the amino acids, thereby liberating NH41 (depicted as red circles, lower
left). Additionally, NH41 might be released during grazing (red copepod in upper left).
because N2 fixation is confined to the light
period regardless of the N regime in natural
and cultured populations of Trichodesmium
spp. By contrast urea and NH41 are taken up
at comparable rates during both the dark and the light periods. Uptake might be satisfied by NH41 uptake supplied by N recycled within the
of NO32 occurs primarily during the light period13, and probably upper water column community13,14. Because Trichodesmium spp. are
competes with N2 fixation for photosynthetic energy and reductant. able to take up combined N, C-based productivity might not be a meaThe absolute growth rate (e.g. doubling of biomass) might be limited sure of new production in systems where allochthonous N is imported.
intrinsically.
Clearly, additional studies investigating the effect of combined N Nitrogen metabolism
on growth and the competitive advantage of Trichodesmium spp. Nitrogen assimilation in natural populations of Trichodesmium
need to be pursued. Although Trichodesmium NIBB1067 and spp. proceeds via the glutamine synthetase/glutamate synthase
IMS101 appear to be able to take up combined N and fix N2 simul- (GS/GOGAT) pathway35. GS is necessary for NH41 assimilation
taneously or alternately in cultures, little is known about the growth regardless of the primary form of N being used. High rates of GS
characteristics of Trichodesmium spp. under different N regimes in transferase activity (a reverse reaction assay that provides an
nature. Trichodesmium spp. can alleviate N limitation and therefore approximation of total GS active sites) relative to rates of total N
out-compete other species in N-limiting environments in nature, uptake have been observed in natural and cultured populations of
although blooms persist even after N concentrations become Trichodesmium spp. (Refs 13,14). Rates of both GS transferase
elevated. It is unclear how increasing availability of combined and GS biosynthetic activity (which approximates in vivo forward
N species affects the growth of Trichodesmium spp. and their reaction activity) in Trichodesmium spp. increase in the afternoon
during the period when rates of N2 fixation are highest. The ratio of
competitive success in natural systems.
The modification and expression of nitrogenase might be at least GS transferase:biosynthetic activity decreases during the period of
partially regulated by the N regime. NH41, Glu and Gln additions maximum N2 fixation, indicating that the proportion of the GS pool
have been shown to reduce N2 fixation over time, probably because that is biosynthetically active increases during the day.
The biosynthetic capacity of GS is sufficient to allow Triof feedback inhibition of nitrogenase by metabolite pools23 (M.R.
Mulholland et al. unpublished). From studies using metabolic chodesmium spp. colonies to turnover their cell N at least three times
inhibitors, it appears that metabolites that accumulate during N per day, suggesting that N assimilation does not limit the rate of N utiassimilation, are important in regulating nitrogenase activity and lization by cells, even during midday when N2 fixation rates are highsynthesis in natural and cultured populations of Trichodesmium est13,14. Cells appear to have sufficient capacity to assimilate all of the
spp.39. However, the presence of low concentrations of NH41 and intracellular N substrates derived from N2 fixation and N uptake in
amino acids in the culture medium during growth do not inhibit N2 cultures growing on media with or without added N (Ref. 13).
Excess GS activity is characteristic of cells limited by N or
fixation. Additions of combined N of 5–10 mM or more might be
necessary before nitrogenase synthesis or activity is affected. Such using N2 as their N source40. A positive correlation between GS and
high concentrations are not typical of the oligotrophic regions in nitrogenase enzyme abundance and distribution has been observed
which these species occur.
in a variety of heterocystous41,42 and non-heterocystous43,44 cyanoAlthough N2 fixation might supply the ‘new’ N necessary for alle- bacteria including Trichodesmium spp35. The GS enzyme is twice
viating N limitation of net growth, much of the gross N turnover as abundant in Trichodesmium spp. cells that contain nitrogenase
April 2000, Vol. 5, No. 4
151
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than in those that do not35. Although different types of GS might
be present in cells, different promoters for transcribing the gene
encoding GS during growth on fixed N and molecular N2 have
also been observed40. Thus, there might be both a constitutive pool
of GS, regulated for the general assimilation of N derived from
various N sources, and a nitrogenase-linked pool co-regulated
specifically with nitrogenase under low N conditions.
In support of two separately regulated GS pools in Trichodesmium
spp., glnA transcript abundance is elevated both during the pre-dawn
period and again in the late afternoon45. The early morning peak in
glnA transcript abundance corresponds with peak nifH (a gene encoding the Fe-protein of nitrogenase) transcript abundance26, whereas the
afternoon peak does not. In Trichodesmium NIBB1067 grown on
medium without added N, biosynthetic GS activity increased before
the onset of the light period and then again in the late afternoon (M.R.
Mulholland et al. unpublished), consistent with the pattern of glnA
mRNA abundance in natural populations45.
The absence of a strong diel cycle of GS activity might reflect the
observation that Trichodesmium spp. colonies and filaments take up
regenerated N sources in addition to fixing N2 during the day. It
might also reflect the observation that only a fraction of cells along
a filament or within a colony may contain nitrogenase and are,
therefore, capable of N2 fixation. If two pools of GS are present in
Trichodesmium spp., the small changes in total filament or colony
GS activity over a diel cycle might be significant in terms of the
capacity for N assimilation in cells fixing N2. The ratio of GS transferase:biosynthetic activity in natural and cultured populations of
Trichodesmium spp. varies by ~20 to 30% over a diel cycle13,14. The
magnitude of this change is consistent with the suggestion that
nitrogenase is confined to ,10–15% of cells along a filament or
within a colony29,31, that GS is twice as abundant in cells containing
nitrogenase35, and that a portion of the GS pool is regulated in conjunction with nitrogenase.
Based on observations of the distribution and activity of GS during N2 fixation, it has been suggested that GS plays an important
regulatory role in N2 fixation, either directly or indirectly, by
preventing feedback inhibition from accumulated metabolites46.
Glutamine and glutamate are primary products of N metabolism
via the GS/GOGAT metabolic pathway. Intracolonial Glu and Gln
concentrations parallel the daily cycle of N2 fixation35,36 and might
be factors in subcellular regulation of nitrogenase. However, in
cultures of Trichodesmium NIBB1067 growing with and without
combined N, the Gln:Glu ratio reflects the pattern of total N uptake
(positive relationship)13. In natural populations, the magnitude and
timing of the daily peak in Gln:Glu ratios varies among sites and
populations. However, the highest Gln:Glu ratios are observed
during the period when rates of N2 fixation are highest14,36.
Light might be an important determinant for N2 fixation and the
N status of Trichodesmium spp. cells. Higher Gln:Glu ratios (0.7)
were observed in Trichodesmium spp. collected in the surface
waters compared with colonies collected from deep waters14. The
decrease in the intracellular Gln:Glu ratios in colonies collected
from deeper in the euphotic zone might be due to light or energy
limitation of N2 fixation rates. Higher rates of photosynthesis by
populations growing in surface waters might result in increases
in the supply of energy to support higher rates of N2 fixation.
In cultures of Trichodesmium IMS101 and NIBB1067, grown
on medium without added N substrates, the intracellular ratios of
Gln:Glu are lower (a maximum of ~0.3) and this might reflect the
low light levels at which these isolates are maintained13,14.
Nitrogen regulation
Investigators have suggested that the nitrogenase of Trichodesmium
NIBB1067 is regulated at different levels. An endogenous rhythm
152
April 2000, Vol. 5, No. 4
has been identified for nitrogenase transcription, translation and
activity27. Alternatively, it has been suggested that the modification
and expression of nitrogenase is also regulated by the availability of
combined N (Ref. 39). Both regulatory mechanisms appear to be
important. Recently, a global N-regulating gene, ntcA, has been
identified in a Trichodesmium spp. isolate from the Red Sea (D.
Lindell pers. commun.), but the conditions under which it is
expressed have not been ascertained.
Community nitrogen dynamics and future directions
Although we are beginning to understand the physiology of N2
fixation by Trichodesmium spp., there remain many uncertainties
regarding the mechanisms that support the growth of Trichodesmium spp. populations in nature and the growth of organisms
living in association with Trichodesmium spp. colonies. For example, Trichodesmium spp. might have a larger role in the marine N
cycle than previously suspected. Although net growth of populations might depend on inputs of new N from N2 fixation,
Trichodesmium spp. also contribute to overall N turnover in oceanic
systems. They contribute both directly through the release of amino
acids, DON and NH41 (Refs 13,36,37), and indirectly through the
regeneration of dissolved inorganic and organic N by bacteria and
grazers living in association with Trichodesmium spp. colonies47–49.
Pathways of N cycling among Trichodesmium spp. filaments and
colonies and associated organisms remain to be directly quantified.
Although Trichodesmium spp. alleviate N limitation of growth by
fixing N2, the effects of deficits of other nutrients on Trichodesmium
spp. growth and N2 fixation are poorly understood. In particular, Fe,
an essential component of the nitrogenase enzyme complex, might
limit N2 fixation in oceanic regions with low aeolian dust inputs.
Phosphorous is also in short supply in oceanic regions where
Trichodesmium spp. grow and form blooms. It is unclear what the
nutrient demands are for Trichodesmium spp. growth and how they
acquire sufficient P to support observed growth rates. High N:P
ratios have been observed in populations from the Pacific Ocean10.
Although we are gaining in our understanding of how these species
affect the marine N cycle where they occur, much needs to be done
to integrate N2 fixation into global C, N and P budgets.
Acknowledgements
We would like to thank three anonymous reviewers for their helpful comments and suggestions. This work was supported by grants
from the National Science Foundation Division of Ocean Sciences.
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Margaret Mulholland* is at the Marine Sciences Research Center,
SUNY Stony Brook, Stony Brook, NY 11794-5000, USA; Douglas
Capone is at the Wrigley Institute for Environmental Studies
and Dept of Biological Sciences, University of Southern California,
Los Angeles, CA 90089, USA (e-mail capone@wrigley.usc.edu).
*Author for correspondence (tel 11 631 632 3163;
fax 11 631 632 8820; e-mail mmulholland@notes.cc.sunysb.edu).
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