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Environmental Microbiology Reports (2011) 3(1), 54–58
doi:10.1111/j.1758-2229.2010.00187.x
Quantification of nitrogenase in Trichodesmium IMS
101: implications for iron limitation of nitrogen fixation
in the ocean
emi4_187
54..58
Sherrie Whittaker,1 Kay D. Bidle,1 Adam B. Kustka1,2
and Paul G. Falkowski1,3*
1
Environmental Biophysics and Molecular Ecology, Institute of Marine and Coastal Sciences, Rutgers, The State
University of New Jersey, New Brunswick, NJ, USA.
2
Department of Earth and Environmental Sciences,
Rutgers, The State University of New Jersey – Newark,
Newark, NJ, USA.
3
Department of Earth and Planetary Sciences, Rutgers,
The State University of New Jersey, New Brunswick, NJ,
USA.
Summary
Iron is widely thought to limit nitrogen fixation in the
open, oligotrophic ocean due to the low solubility of
Fe in oxic seawater and the high Fe demand for the
nitrogenase holozyme. However, empirical evidence
for Fe limitation of field populations of Trichodesmium
based on either incubation experiments or molecular
and physiological indicators has not quantitatively
related Fe supply to the cellular Fe quotas for nitrogenase. Rather, the Fe required for N2 fixation has been
inferred from in vivo catalytic activity. Using a pet14b
expression vector, we cloned the nif H gene (encoding
the Fe-protein, which contains 4Fe atoms per subunit)
from Trichodesmium IMS 101, and purified the Histagged apoprotein with which we derived a primary
standard based on quantitative Western blots. Using a
standard curve derived from the cloned Trichodesmium Fe apoprotein, we measured the absolute abundance of the Fe-protein in iron-replete cultures of this
marine diazotroph. At peak expression, we calculate
0.04 mg nitrogenase mg-1 C. Assuming a conservative
stoichiometry of two Fe-protein subunits per MoFe
protein (which contains 15 Fe atoms per subunit, or a
total of 38 atoms of Fe per holozyme), we estimate
236 mmol Fe is bound to nitrogenase per mol cellular C.
This estimate is about 10 times greater than the Fe
previously calculated to support diazotrophic growth
under these conditions. Our results suggest that
Received 13 December, 2009; accepted 26 April, 2010. *For correspondence. E-mail [email protected]; Tel. (+1) 732 932 6555
x. 370; Fax (+1) 732 932 4083.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd
under bloom conditions in the subtropical North
Atlantic and North Pacific, as much as ~2.22 and
0.06 mmol m-3 of Fe is bound to Trichodesmium nitrogenase respectively. Such a high quota represents
between ~50% and > 100% summer-time average
particulate Fe in surface waters, suggesting the importance of this taxon for the retention and biogeochemical cycling of Fe. Moderate growth (0.10 day-1) towards
the end of these blooms would require a vertical flux
as high as ~23 mmol Fe day-1 m-2 into the mixed layer.
Introduction
Trichodesmium spp., a filamentous, non-heterocystous
marine cyanobacterial genus widely distributed throughout the world’s tropical and subtropical oceans, fixes ~80–
200 Tg N per annum (Capone et al., 1997; Gruber and
Sarmiento, 1997; Karl et al., 2002), and contributes up to
90% of total pelagic marine nitrogen fixation (Bergman
et al., 1997; Capone et al., 1997; LaRoche and Breitbarth,
2005). Despite this metabolic capacity, the central gyres
throughout most of the world’s oceans are extremely
deficient in fixed inorganic nitrogen. Hence, the fundamental question remains – why is fixed inorganic nitrogen
a major factor limiting primary productivity in the ocean
(Falkowski, 1997)? One possibility is that the expression
of nitrogenase, the enzyme responsible for nitrogen fixation in all diazotrophs, is itself limited by another element.
The nitrogenase complex is composed of two ~38 kDa
Fe-proteins each containing one 4Fe–4S cluster, and a
~220 kDa dimeric MoFe protein, which contains 30 Fe
atoms. Hence the holoenzyme contains 38 atoms of Fe,
making it one of the most Fe-rich enzymes in nature.
Because Fe is extremely rare in the soluble or colloidal
pools of the subtropical gyres, it has been hypothesized
that nitrogenase synthesis may be limited by Fe availability (Reuter et al., 1990; Falkowski, 1997; Karl et al., 1997).
While increasing Fe availability stimulates Trichodesmium rates of photosynthesis and N2 fixation in both cultured and natural populations (Reuter, 1988; 1990; Paerl
et al., 1994; Mills et al., 2004) few studies have attempted
to quantify the relationships among Fe quota and physiological rate processes. Berman-Frank and colleagues
(2001) and Kustka and colleagues (2003) evaluated the
Quantifying nitrogenase iron quotas in Trichodesmium 55
relationships between cellular Fe quota and N2-supported
growth or N2 fixation rates in cultures of Trichodesmium
IMS 101. Both studies suggest that ⱕ 50 mmol : mol
Fe : C is required for diazotrophic growth at 0.1 day-1.
Semi-empirical calculations based on maximum catalytic
rates from in vitro preparations of nitrogenase from Azotobacter, Klebsiella and other heterotrophic diazotrophs
suggest a similar quantity of Fe is required. These calculations, however, rely on several assumptions regarding
the quantities of Trichodesmium nitrogenase. Here we
specifically evaluate the nitrogenase quota in Trichodesmium IMS 101 using a quantitative immunoblot approach
to assess the iron allocated to the enzyme. In conjunction
with our results, we estimate an upper boundary for
Fe requirements of nitrogen fixation in the tropical and
subtropical gyres.
Results and discussion
Purification of the Fe-protein and standardization of
Western blots
A purified 6XHis-tagged, recombinant nif H protein
(Fig. S1), constructed in the pET® expression system
(Novagen), was used in conjunction with a universal nitrogenase antibody (derived from purified dinitrogenase
reductase from Azotobacter vinelandii and Rhodospirillum
rubrum and provided courtesy of Paul Ludden) to generate a quantitative measure of the total enzyme per unit
algal carbon. The antibody reacted with a single ~38 kDa
protein that corresponded to the 6XHis-tagged, recombinant construct. The resulting purified protein and concurrent immunoblot of the nif H protein standards were linear
over 2.5 orders of magnitude (Fig. 1).
Total nitrogenase proteins and Fe quotas from
Trichodesmium cultures
Total nitrogenase protein was extracted from cultured
Trichodesmium IMS 101 and assayed by quantitative
Western blot analysis (Fig. 2) using a concurrent standard
curve (Fig. 1). The analyses were performed on cells
grown over a diel cycle (12:12 light/dark) with illumination
beginning at 0700 local time. The Western blots revealed
a monomodal curve with peak intracellular concentrations
of nitrogenase between 1600 and 1900 h. The concentration of nitrogenase was normalized per unit cell carbon,
with maximum values of ~40 mg nitrogenase mg C-1.
Assuming a ratio of two nif H gene products per holozyme, we calculated a maximum Fe quota per unit C in
the nitrogenase holozyme of ~240 mmol : mol (Fig. 2). Our
results differ from previous calculations of iron quotas for
Trichodesmium in that they strictly focus on the amount
of iron bound in the structural component of the nitrogenase complex. Our estimates of iron quotas are generally
Fig. 1. Western blot quantification of nitrogenase concentration
(ng) versus pixel density. A strong linear relationship was observed
for nitrogenase values spanning three orders of magnitude
(y = 11.939x = 258.09; r2 = 0.99). Inset: Western blot showing the
relative detection signal of serially diluted, purified recombinant
nitrogenase. Lane numbers refer to the amount of purified
nitrogenase loaded (in ng). The Trichodesmium IMS 101 nif H gene
(NCBI Accession No. U90952) was cloned and overexpressed in a
BL21(DE3)pLysS expression host under an IPTG-inducible T7
promoter using the pET® 14b vector system (Novagen) which
confers an N-terminal 6XHis tag. Transformants were grown in
Lauria Broth (LB) containing 50 mg ml-1 ampicillin and 34 mg ml-1
chloramphenicol to an optical density (OD) of 0.4, harvested via
centrifugation (10 000 g; 10 min; 4°C) and stored at -80°C until
processed. Induction of the target protein was confirmed on 10%
polyacrylamide gels with sodium-dodecyl-sulfate (SDS-PAGE) and
staining with Gel Code (Pierce; see Fig. S1). Recombinant
6X-tagged nif H proteins were found to be in inclusion bodies
(post-14 000 g pellets), which were solubilized in 100 mM
NaH2PO4, 10 mM Tris, 8 M urea, pH 8, 0.2 mm filtered, and used
as source material for protein purification. Purification of the
overexpressed, recombinant 6xHis-tagged nif H protein was
achieved by Ni-NTA affinity chromatography (QIAexpress System,
Qiagen). A Bio-Scale MT2 column (2 ml; Bio-Rad) was packed
with 50% Ni-NTA superflow slurry and equilibrated with 100 mM
NaH2PO4, 10 mM Tris-Cl, 8 M urea, pH 8. Proteins were loaded,
washed and eluted in the above buffer at different pHs using
high-performance liquid chromatography (HPLC) (flow
rate = 1 ml min-1), according to manufacturer’s instructions. After
column loading, proteins were washed twice with buffer at pH 6.3
to remove non-specific proteins, followed by successive elutions at
pH 5.9 and 4.3, respectively. The effectiveness of purification was
verified with SDS-PAGE and Gel Code staining (Fig. S1). A standard
curve derived from a dilution series of the purified, recombinant nif H
protein was created using a universal antibody against dinitrogenase
reductase from Azotobacter vinelandii and Rhodospirillum rubrum
(titre = 1:30 000) and quantitative Western immunoblotting.
Membranes were subsequently probed with an anti-rabbit IgG
HRP conjugate (titre = 1:30 000) and visualized with SuperSignal
chemiluminescent substrate (Pierce). The resulting bands were
quantified by densitometry with Image J software (NIH).
higher than previous studies of estimates of total intracellular Fe : C from cultures and related more closely to
data derived from natural populations.
Extrapolating the results to the oceans
We calculated the amount of Fe bound in nitrogenase
for various ocean regions by extrapolating our peak
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 3, 54–58
56 S. Whittaker, K. D. Bidle, A. B. Kustka and P. G. Falkowski
Fig. 2. Representative, quantitative analysis of nif H expression
over a diel cycle using a concurrently generated standard curve
(see Fig. 1) and analysis of pixel density at each time point.
Upper panel: Western blot of Trichodesmium IMS 101 cell extracts
showing the relative expression of nif H over the diel cycle.
Lower panel: Nitrogenase : C and Fe : C ratios for the nitrogenase
complex over the diel time-course. Replicate Trichodesmium IMS
101 cultures were grown in YBCII media (Chen et al., 1996) under
a 12:12 h light : dark (L : D) cycle at 26°C under 80 mmol
quanta m-2 s-1 with constant bubbling. Exponentially growing cells
were harvested over one diel cycle onto 5-mm-pore-size
polycarbonate filters and stored at -80°C until further processing.
Sampling started 1 h before the photoperiod began (0700 h) and
continued every 3 h. Frozen cells were resuspended in buffer
containing 4% SDS/0.1 M NaCO3 and sonicated for 30 s (twice) on
ice, followed by addition of 1 mM phenylmethanesulfonylfluoride
(PMSF) and protein quantification (BCA protein assay; Pierce).
Loading buffer (15% glycerol, 0.05% bromothymol blue and 100 mM
DTT) was added to the remaining cell extracts prior to SDS-PAGE
and Western blot analysis (as described in Fig. 1). Total protein
loaded per lane was between 17.25 and 18.00 mg. Elemental
carbon and nitrogen analysis was performed on a Carlo Erba
NA1500 Series 2 Elemental Analyser. Fifteen millilitres of samples
were collected on pre-combusted GF/F filters, placed in tin foil
capsules and stored at -80°C until analysed. Fe : C quotas were
calculated using known iron requirements for the nitrogenase
enzyme structure; two Fe-proteins (each containing one 4Fe–4S
cluster) and one MoFe protein (containing 30 Fe atoms), yielding
a holoenzyme with 38 total atoms of Fe. Assuming a 2:1 ratio
between the Fe : MoFe proteins and the molecular weight of
the Fe-protein, we converted the nitrogenase : carbon (mg : mg)
to Fe : C (mmol : mol) with the following equation: Fe : C
(mmol : mol) = nitrogenase ¥ carbon (mg)/carbon
(mol) ¥ (1/molecular weight of MoFe) ¥ ratio of nitrogenase
complex to Fe-protein ¥ total Fe per complex.
nitrogenase expression data from laboratory cultures
(Fig. 2) to estimates of in situ Trichodesmium biomass. To
provide a comparison between varying locations in which
Trichodesmium is found, we reported from three different
regions: the Subtropical North Pacific, Tropical North
Atlantic and a bloom in the Arabian Sea (Table 1). The
value of 50 ng C trichome-1 is based on measurements of
particulate carbon (PC) from single filaments collected off
Hawaii at station ALOHA in the subtropical North Pacific
(Letelier and Karl, 1996). The lowest value of nitrogenase
was 0.11 mg m-3 at station ALOHA in the subtropical
North Pacific; the highest value reflected a bloom in
the Arabian Sea, 19 mg m-3. The tropical North Atlantic
samples fell between these two values but, at 4.28 mg
nitrogenase m-3, were also very high compared with
samples from station ALOHA.
Based on the estimated nitrogenase concentrations,
the calculated concentration of Fe bound in nitrogenase
in Trichodesmium over various ocean regions spanned
a range of 0.06 to 9.86 mmol m-3 (Table 1). The lowest
values were found in the subtropical North Pacific, while
the highest values were derived from a bloom located
in the Arabian Sea. In the North Atlantic, nitrogenase
was 4.28 mg m-3 and the iron bound in nitrogenase was
2.22 mmol m-3.
The relationship between iron and Trichodesmium is not
easily defined. In the open ocean, iron is primarily delivered through Aeolian dust transport originating from the
world’s deserts (Jickells et al., 2005), and from vertical
fluxes from the ocean interior. Once in seawater, iron is
dominated by the particulate form because of its low solubility; and how much iron is available to Trichodesmium
is further confounded by the amount and types of iron
species that are bioavailable. In addition to the soluble
forms, Trichodemsium has been demonstrated to uptake
some ligand bound iron and iron made bioavailable
through siderophores produced from associated bacteria
(Achilles et al., 2003). As the primary goal of this study is
to estimate the amount of iron bound to nitrogenase in
Trichodesmium, a comprehensive look at the relationship
of iron and Trichodesmium is beyond the scope of this
article. Comparing these data solely with particulate Fe
data from surface waters of the Sargasso Sea (Sherrell
Table 1. Iron bound in Trichodesmium biomass, based on published field data.
Location
Trichomes
m-3
ng C
trichome-1
ng C m-3
mg C m-3
mg nitrogenase
mg C-1
mg nitrogenase
m-3
mmol Fe in
nitrogenase m-3
Subtropical North Pacifica
Arabian Sea bloomb
Tropical North Atlanticc
5.67E + 04
1.00E + 07
2.25E + 06
50
50
50
2.84E + 06
5.00E + 08
1.13E + 08
2.84
500
112.5
0.038
0.038
0.038
0.11
19.00
4.28
0.06
9.86
2.22
a. Letelier and Karl (1996).
b. Capone and colleagues (1998).
c. Carpenter and colleagues (2004), based on data from May–June 1994 cruise.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 3, 54–58
Quantifying nitrogenase iron quotas in Trichodesmium 57
-3
and Boyle, 1992; ~0.32 mmol m ) and the central North
Pacific (Bruland et al. 1994; ~0.15 mmol m-3) suggests
that blooms of Trichodesmium represent at least 50% of
the standing stock of particulate Fe in the upper mixed
layer. A population growing at bloom concentrations, with
an intrinsic growth rate of 0.1 day-1, would require an
Fe flux into the upper water column as high as
23 mmol m-2 day-1.
These data provide one more piece to the ever-growing
body of work related to the iron requirements of Trichodemium by representing an upper limit for the Fe bound in
Trichodesmium nitrogenase in each ocean region. These
data reveal the difference in Trichodesmium biomass
between the three regions, highlighting the increased iron
quantities that are bound in nitrogenase in the subtropical
North Atlantic versus the North Pacific. It also points to
the importance of dense blooms with values that more
than quadruple those from the North Atlantic. Historically,
it has been speculated that estimated rates of fixed
nitrogen would significantly increase if Trichodesmium
blooms were included in global nitrogen fixation estimates
(Carpenter, 1983; Capone et al., 1997).
The above calculations are based on regions characterized by considerable Trichodesmium populations. To
broaden this perspective to an entire ocean basin, we
explored the question of iron limitation in the North Atlantic. An area of the North Atlantic Ocean was outlined with
a spring global sea surface temperature map generated
from the NOAA Satellites and Information website. The
sole requirement was sea surface temperatures 20°C and
above, the lower limit for physiologically active Trichodesmium. This region spanned the equator to approximately
35°N, measuring 2 ¥ 1013 m2. Assuming average euphotic
zone depth of ~100 m (Davis and McGillicuddy, 2006), the
total volume obtained is 2 ¥ 1015 m3. Further, assuming
the average chlorophyll concentration in this region to
be 0.2 mg m-3 (Falkowski et al., 1998), the calculated total
chlorophyll in the basin is ~4 ¥ 1014 mg, representing the
average for the total amount of phytoplankton within the
entire outlined basin.
To present a range of possible Trichodesmium biomass
estimates in the chosen area, we examined two possible
scenarios in which 1% and 10% of total biomass is
Trichodesmium. In both cases the cells have an average
C : Chl ratio of 100 (by mass), and we apply a ratio
of 0.038 mg nitrogenase mg C-1 (this study). For 1%
Trichodesmium to total biomass, ~400 ¥ 1012 mg C is contained in the diazotroph, which translates to an estimate
of the standing stock of nitrogenase to be ~15 ¥ 109 g
(i.e. 15 Gg). This is approximately six orders of magnitude
lower than the global standing stock of ribulose 1,5 bisphosphate oxygenase/carboxylase in the world oceans.
Using the same assumptions and calculations for 10%
Trichodesmium to total biomass, we find a standing stock
of nitrogenase of 15 ¥ 1011 g. Using the calculations
described previously, this translates to 7.8 ¥ 106 mol Fe
and 7.8 ¥ 107 mol Fe, respectively, bound in Trichodesmium nitrogenase protein pools.
Conclusion
To date, there has not been direct testing of the hypothesis that iron limits nitrogen fixation in the contemporary
open ocean via large-scale iron fertilization experiments
in a central oligotrophic gyre. Until such time, all data
supporting or refuting the hypothesis are based on
indirect analyses. Only through a solid understanding
of the physiological requirements and capabilities of
Trichodesmium and other nitrogen-fixing organisms will
a global picture of the relationship between iron and
nitrogen fixation emerge.
Acknowledgements
We thank Kevin Wyman for technical assistance and two
anonymous reviewers for their comments. This work was
supported by the Agouron Foundation and NASA (to P.G.F.)
by NSF Grant IOS-0717494 (to K.D.B.).
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Supporting information
Additional Supporting Information may be found in the online
version of this article:
Fig. S1. Gel Code-stained, SDS-PAGE of the purified 6XHistagged, recombinant Hif H protein from Trichodesmium IMS
101. Lane designations are as follows: lane 1, total protein
extract from IPTG-induced BL21(DE3)pLysS Escherichia coli
cells containing the pET 14b nif H construct; lane 2, Ni-NTA
flow-through after pH 6.3 wash; lane 3, Ni-NTA flow-through
after pH 5.9 wash; lane 4, purified protein eluted at pH of 4.3;
mw, Precision Plus protein standard (Bio-Rad).
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 3, 54–58