Limnol. Oceanogr., **(*), ****, **** ****
E ****, by the American Society of Limnology and Oceanography, Inc.
doi:10.4319/lo.2010.55.5.2161
Polyphosphate in Trichodesmium from the low-phosphorus Sargasso Sea
Elizabeth D. Orchard,a Claudia R. Benitez-Nelson,b Perry J. Pellechia,c Michael W. Lomas,d and
Sonya T. Dyhrmane,*
a Massachusetts Institute of Technology Woods Hole Oceanographic Institution Joint Program in Oceanography/Applied Ocean Science
and Engineering, Woods Hole, Massachusetts
b Department of Earth and Ocean Sciences and Marine Sciences Program, University of South Carolina, Columbia, South Carolina
c Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina
d Bermuda Institute of Ocean Sciences, St. George, Bermuda
e Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts
Abstract
Polyphosphate (polyP) is often considered to be the product of luxury uptake in areas of excess phosphorus
(P), but can also accumulate in P-depleted cells in response to P resupply. To test the hypothesis that polyP is
present in phytoplankton from oligotrophic systems, the marine diazotroph Trichodesmium was collected from
the low-P surface waters of the Sargasso Sea and assayed with solid-state 31P nuclear magnetic resonance
spectroscopy. Up to 25% of Trichodesmium cellular P was characterized as polyP, despite physiological data that
indicated the colonies were P deplete. This was consistent with culture studies where there were high percentages
of polyP under P-deplete conditions. All Trichodesmium species examined had the genetic machinery to produce
and degrade polyP. Trends in the amount of Trichodesmium polyP along the cruise transect showed that
allocation of P to polyP was consistently high, and that the ratio of polyP : carbon varied with changes in
temperature and mixed-layer depth. It may be that Trichodesmium was taking advantage of pulses in P supply,
and that polyP is a physiological fingerprint of this variability. Additionally, if polyP formation is a common trait
in phytoplankton, polyP released from cells could be an additional bioavailable component of the dissolved
organic P pool. Taken together, this study highlights the importance of polyP to P cycling and cellular P
allocation even in oligotrophic regions.
and in marine (Diaz et al. 2008) and freshwater (Bertilsson
Inorganic polyphosphate (polyP) is a polymer of
et al. 2003) phytoplankton culture experiments. Luxury
phosphate ranging in length from three to thousands of
orthophosphate units. PolyP synthesis and catabolism are uptake could drive the accumulation of polyP in coastal
typically controlled by a gene (ppK) encoding a polyP systems or areas where P is in excess relative to nitrogen
kinase that reversibly adds phosphate to the end of the (N). Fundamentally distinct from luxury uptake is the
polyP chain (Tzeng and Kornberg 1998), and a gene (ppX) overplus response, wherein P-deplete cells accumulate
encoding an exopolyphosphatase that removes the terminal polyP in response to short-term pulses in P supply
phosphate from a polyP molecule (Akiyama et al. 1993). (Jacobson and Halmann 1982; Bolier et al. 1992). The
PolyP has been found in all major groups of life examined overplus response is not well studied in marine phyto-
to date, but its function is varied, and in many regards plankton, or in marine systems in general. However, it has
remains unclear (Kornberg et al. 1999). Accumulation of been hypothesized that overplus could drive the cellular
cellular polyP has been variously attributed to a stationary accumulation of polyP in low-P systems where phytoplank-
phase adaptation, an energy storage compound, an osmotic ton are P deficient, but may experience short-term
regulator, a buffer against alkali conditions, a factor in variations in their local P environment (Karl and Bjorkman
deoxyribonucleic acid (DNA) competency (as part of a 2002).
DNA channel), and in intracellular phosphorus (P) storage Several different taxa of marine phytoplankton are
(Kornberg 1995; Kornberg et al. 1999). It is the link known to accumulate polyP, including Skeletonema,
between polyP and phosphate storage that has driven Thalassiosira, Synechocystis, Nostoc, Calothrix (Mateo et
research on polyP dynamics as a function of microbial P al. 2006; Diaz et al. 2008), and Trichodesmium, an N-fixing
physiology. marine cyanobacteria (Romans et al. 1994). Genes puta-
There are two basic processes that have been studied tively involved in polyP synthesis and degradation also
involving polyP formation in bacteria in response to P: appear to be present, and expressed, in a number of both
luxury uptake and the overplus response. Luxury uptake is eukaryotic (Dyhrman et al. 2006b) and prokaryotic
the storage of P as polyP when P is consistently in excess phytoplankton (Gomez-Garcia et al. 2003; Martiny et al.
relative to other macronutrients (e.g., growth is not P 2006; Hewson et al. 2009). Taken together, these observa-
controlled) and uptake exceeds instantaneous growth tions suggest that many phytoplankton produce and
demands. Luxury uptake has been extensively studied in degrade polyP, but despite its potential significance to P
wastewater treatment applications (Crocetti et al. 2000), physiology and P cycling, there are only a few studies that
have assayed polyP in a marine setting (Romans et al. 1994;
Diaz et al. 2008). Furthermore, the presence of phyto-
* Corresponding author: ********@****.***
2161
2162 Orchard et al.
Fig. 1. Station locations for a May 2006 transect from north to south in the Sargasso Sea.
plankton polyP has never been examined in a chronically colonies were picked into 0.2-mm-filtered local surface
low-P environment, such as the Sargasso Sea. seawater, and then transferred into fresh 0.2-mm-filtered
water to reduce contamination of closely associated
In this study, Trichodesmium near-surface populations
organisms. Colonies were then separated for further
from the Sargasso Sea were assayed with solid-state 31P
analyses as indicated below.
nuclear magnetic resonance (NMR) spectroscopy to
examine cellular allocation of P as polyP in an oligotrophic
31P NMR spectroscopy Trichodesmium biomass was
system. Results are compared to culture data, and
filtered onto 47-mm 5-mm polycarbonate filters and dried at
covarying field parameters, to examine potential drivers
65uC prior to analysis, according to conditions used in
of intracellular polyP dynamics.
other work (Benitez-Nelson et al. 2004; Dyhrman et al.
2009). For each field station, approximately 100 colonies
Methods
were pooled onto a single filter. Solid-state 31P NMR
spectra were recorded on a Varian Inova 500 spectrometer
Culture conditions Trichodesmium cultures were grown
operating at 202.489 MHz using a Doty Scientific XC-4 mm
in RMP medium with Sargasso seawater as previously
magic angle spinning (MAS) probe. Bloch decays of 50 ms
described (Webb et al. 2001). Species examined include
were collected with a 200 d or parts per million (ppm, 1026)
Trichodesmium erythraeum IMS101, Trichodesmium tenue
window after 30u excitation pulses. The number of
Z-1, Trichodesmium thiebautii II-3, Trichodesmium spiralis
transients collected varied, depending on the amount of
KAT, and Trichodesmium spp. H9-4 obtained from the
material on the filter, and ranged from 50,000 to 100,000
culture collection of Dr. John Waterbury at the Woods
scans per sample (24 48 h). Two-pulse phase modulation
Hole Oceanographic Institution. All cultures were unialgal
but not axenic. Cultures were grown at 25uC on a shaker (McGeorge et al. 1999) proton dipolar decoupling with a
field strength of 45 kHz was applied during acquisition, and
table with daily cycles that consisted of 1 h at
a MAS speed of 10 kHz was used. The chemical shift (a
13.5 mE m22 s21, 10 h at 33.7 mE m22 s21, 1 h at
unit-less normalization of frequency) is reported as 1026
13.5 mE m22 s21, and 12 h of dark. Chlorophyll a
(ppm). Spectra were fit with five Lorentzian lines at a
fluorescence was monitored using an Aquafluor fluorom-
chemical shift of 18 to 20 (nominally phosphonate), 0 to 2
eter (Turner Designs). T. erythraeum IMS101 was grown
with (+P) and without (2P) 16 mmol L21 phosphoric acid (nominally orthophosphate), 212 (a sharp peak approxi-
mating a monospecific, but uncharacterized diester), 210
added to the media. Samples were taken for particulate
carbon (C), N, and P as well as for solid-state 31P NMR to 212 (a broader spectrum of diesters), and 222 to 224
spectroscopy (see below) throughout the experiments. (nominally polyP). The percentage of total P as polyP was
then determined (Fig. 2). This is likely to be a minimum
measurement given the assumption that all polyP has a
Field samples Samples were collected during a transect
of the Sargasso Sea (Sta. 1 7) in May 2006 (Fig. 1). peak signal within this 222 to 224 region. Results are
reported as a %polyP of total P or as the polyP : C. The
Trichodesmium colonies were collected from the near
surface (roughly within the top 20 m) using a handheld later value was determined as the proportion of polyP
130-mm net. Colonies were not sorted by morphology, but multiplied by the ratio of total P : C in the sample to
the puff morphology was typically most abundant. Single account for changes in the absolute amount of P in the
Trichodesmium polyphosphate dynamics 2163
to the persulfate oxidation method (Valderrama 1981).
Dissolved organic P (DOP) concentrations were calculated
as the difference between TDP and DIP. With the methods
used herein, polyP would be detected within the DOP pool
even though it may occur as an inorganic compound
(Monaghan and Ruttenberg 1999).
Statistics Pair-wise correlation coefficients (corr. coeff.)
and p values were calculated between all parameters:
Trichodesmium polyP : C, Trichodesmium APA, Trichodes-
mium N : P ratio, Trichodesmium C : P ratio, surface temper-
ature, mixed-layer depth, latitude, DOP concentration, and
DIP concentration at 20 m, using JMP software (SAS). The
mixed-layer depth was defined as the depth where the
temperature changed by 0.5uC relative to the surface. A two-
tailed t-test was used to compare %polyP among growth
phases of cultured Trichodesmium and between P-replete and
Fig. 2. A representative solid-state 31P NMR spectrum of
P-deplete culture treatments.
Trichodesmium from Sta. 2 during a May 2006 cruise transect in
the Sargasso Sea; the polyP peak is noted at a chemical shift of
222 to 224. Genome annotation The putative ppX and ppK genes
were identified using the Integrated Microbial Genomes
sample due to variations in P quota. The %polyP values portal at the Joint Genome Institute US Department of
reported have an error of 6 5%, similar to solid-state 31P Energy website (http://img.jgi.doe.gov/v1.1/main.cgi) and
NMR studies in other systems (Sannigrahi and Ingall 2005; the National Center for Biotechnology Information Gen-
Sannigrahi et al. 2006; Diaz et al. 2008). It is important to bank database (http://www.ncbi.nlm.nih.gov/), based on
note that Trichodesmium cultures and colonies likely homology.
contained epibionts. However, the particulate P was
dominated by Trichodesmium, and thus the epibionts DNA amplification and sequencing Cultures of Tricho-
should not have a major influence on the proportion of P desmium were collected by filtration onto a 5-mm polycar-
bonate filter and stored at 220uC until extraction. DNA
detected in a given bond class.
was extracted using the Instagene Matrix (Bio-Rad)
Alkaline phosphatase activity (APA) For Trichodes- according to the manufacturer s instructions or as de-
mium APA assays, 325 colonies were filtered onto a 5-mm scribed in Ehrenreich et al. (2005). Polymerase chain
polycarbonate filter. Samples were processed as described reaction (PCR) primers were designed to either amplify
elsewhere (Dyhrman and Ruttenberg 2006). Briefly, fragments of the putative ppX (F 59 GGAATGTCCGA
10 mmol L21 6,8-difluoro-4-methylumbelliferyl phosphate AAAGCGAGC 39 R 59 GCCCAAAAAGCAACCCC
GTTC 39) or ppK (F 59 CGCTTTATCAAACTGATT
(di-MUF-P; Invitrogen) was added to each sample in a
petri dish with artificial seawater containing no P, and CGTCGG 39 R 59 CGCAAACAACAAATACCACG
GAC 39) genes. Each PCR reaction consisted of a 5-min
fluorescence was measured on a Fluostar Optima plate
denaturation step at 95uC, followed by 35 cycles of 1 min at
reader (BMG Labtech) every 5 20 min for five time points,
95uC, 1 min at 60.7uC, 1 min at 72uC, and a final extension
within the linear range of the assay. Previous kinetics
of 10 min at 72uC, in a Bio-Rad iCycler. PCR amplification
experiments found the 10 mmol L21 substrate concentration
was done using 0.5 mL iTaq DNA polymerase (Bio-Rad),
to be saturating (data not shown). Standard fluorescence
2.5 mL of DNA template, 2.5 mL of 2 mmol L21
curves were generated for each assay using 6,8-difluoro-7-
deoxyribonucleotide triphosphate, 2.5 mL of 53 iTaq
hydroxy-4-methylcoumarin.
buffer, 50 pmol of each primer, and sterile water to a final
volume of 25 mL. The PCR products were gel extracted
Chemical analyses For C, N, and total P measurements
using the QIAquick gel extraction kit (Qiagen), and direct
20 Trichodesmium colonies from the field were collected
onto precombusted GF/F filters and dried at 65uC in sequenced at the MWG Biotech facility or at the University
of Maine, according to the facility s protocols. Sequences
precombusted foil. Each filter was split for analysis of C, N,
were edited using Sequencher (Gene Codes) and verified
and total particulate P. C and N concentrations were
manually. Alignments were done in MacVector. The
measured using a Perkin Elmer 2400 CHN Element
sequences have been deposited in Genbank (accession
Autoanalyzer with no acid fuming (Karl et al. 1991). Total
number GU299287 GU299292).
particulate P was measured as described in Benitez-Nelson
et al. (2004). Soluble reactive P (SRP) was measured
according to the magnesium-induced coprecipitation SRP Results
method (Karl and Tien 1992), with a detection limit of
0.5 nmol L21. SRP concentrations measured here are Genes T. erythraeum has gene homologs for ppK and
referred to as the dissolved inorganic P (DIP) concentra- for ppX. The genes are not contiguous, and do not appear
tion. The total dissolved P (TDP) was processed according to be downstream of a Pho box P regulatory sequence (Su
2164 Orchard et al.
mixed-layer depth (Table 2). There was no significant
Table 1. The %polyP detected in T. erythraeum IMS101
cultures grown on P-replete or P-deplete media in different correlation between Trichodesmium polyP : C and DOP,
growth phases. DIP, Trichodesmium N : P ratio, or Trichodesmium APA.
There was also no significant correlation between Tricho-
Log phase P- Stationary phase P- Log phase P-
desmium APA and DIP concentration, Trichodesmium C : P
replete* (n58) replete (n54) deplete (n53)
ratio, or Trichodesmium N : P ratio.
Mean 0.2{ 16 16
SD 0.5 9 13 Discussion
p{ 0.0003 0.006
Storage of cellular P as polyP is considered an important
* The average C : P ratio for the P-replete cultures was 130 6 35, and for
the P-deplete samples was 486 6 201. aspect of microbial P physiology, and could influence P
{ Each %polyP value has a 6 5% error.
cycling in aquatic systems. However, there are few studies
{ All samples were compared to the log phase P-replete condition using a
that have examined polyP in marine phytoplankton, and
two-tailed t-test; bold font denotes significance.
none have examined polyP accumulation in low-P oligo-
trophic systems. Others have hypothesized that polyP could
et al. 2007), although previous work has highlighted that
accumulate in phytoplankton from oligotrophic, low-P
there may be heterogeneity in the Trichodesmium Pho box
regions, as part of an overplus-type response (Karl and
sequence (Orchard et al. 2009). All species of Trichodes-
Bjorkman 2002). To address this hypothesis, polyP was
mium tested (T. erythraeum, T. spiralis, T. tenue, and T.
assayed in Trichodesmium cultures and from field popula-
thiebautii) had the ppK and ppX genes. Over the sequenced
tions from the Sargasso Sea.
156 base pair fragment, ppK is 88 93% identical at the
nucleotide level. The sequenced 223 base pair fragment of
PolyP dynamics in culture All of the Trichodesmium
ppX is 93 100% identical at the nucleotide level.
species tested in culture have the genetic machinery to
produce and degrade polyP. This is consistent with solid-
Culture experiments Multiple Trichodesmium species
state 31P NMR data that detected polyP in P-replete
had detectable polyP. The maximum %polyP that was
cultures of Trichodesmium, with maximal polyP percentag-
measured in P-replete cultures was 12% for T. tenue, 14%
es that were similar among species. These data suggest that
for T. spp. H9-4, 19% for T. thiebautii, and 22% for T.
the polyP dynamics observed in T. erythraeum IMS101 are
erythraeum. The %polyP in P-replete T. erythraeum
likely representative of the other species, although this
IMS101 cultures was significantly lower in log phase than
warrants further study. The solid-state 31P NMR method is
in stationary phase (Table 1). The %polyP in log phase P-
nondestructive and can provide data on P allocation
replete cultures (cultures with a C : P ratio less than 200)
patterns that are complementary to other approaches that
was significantly lower than log phase P-deplete cultures
have detected polyP accumulated in granules in Trichodes-
(grown with no added P and with a C : P ratio greater than
mium populations from the Caribbean Sea using electron
200; Table 1).
microscopy (Romans et al. 1994).
Under P-replete conditions, T. erythraeum IMS101 had
Field data The surface temperature increased with
the highest %polyP of total cellular P during stationary
decreasing latitude from north to south increasing from
24.6uC to 27.7uC (Table 2). The mixed-layer depth gener- phase. In fact, polyP was often below the detection limit in
P-replete log phase cultures. This is consistent with the
ally shoaled in the more southern latitudes decreasing from
hypothesis that polyP is important for survival in
a maximum of 32 m to a minimum of 13 m (Table 2). DIP
stationary phase or that polyP is accumulating in response
and DOP concentrations at 20 m were relatively constant
to general stress (Rao and Kornberg 1996; Kornberg et al.
along the cruise transect, ranging from 0.5 to 3.0 nmol L21
1999). Luxury uptake may also be occurring in these
and 22.9 to 46.8 nmol L21, respectively, and with no clear
cultures, but luxury P is either not being stored as polyP
trend from north to south (Table 2). The average Tricho-
during log phase growth or is below the detection limit for
desmium C : P ratio and N : P ratio were consistently above
this assay. These results contrast with those from P-deplete
the Redfield ratio of 106 : 1 and 16 : 1, respectively, and
cultures, where log phase P-deplete samples had signifi-
Trichodesmium APA ranged from 0.23 to 0.97 nmol P h21
cantly higher percentages of polyP relative to log phase P-
colony21 (Table 2). None of these potential metrics of P
replete samples. This could be the result of an overplus-like
physiology showed a consistent trend on the north to south
response (Fig. 3). Because DIP was not refed to P-deplete
transect (Table 2), or a significant correlation to other
cells, this is not entirely consistent with traditional
environmental parameters. However, these metrics of P
definitions of the overplus response. However, upon
physiology were consistently in the range of what would be
becoming P-deplete T. erythraeum up-regulates a suite of
considered evidence of P depletion from other studies
genes encoding enzymes to access P from DOP (Dyhrman
(Mulholland et al. 2002; Krauk et al. 2006; White et al.
et al. 2006a; Orchard et al. 2009). These cultures were
2006b).
grown on Sargasso Sea water containing DOP, and cells
In field-collected Trichodesmium colonies, %polyP
would have been able to access a new source of P upon DIP
ranged from 8% to 25% of the total P as detected with
depletion, the result of which might have been an overplus-
solid-state 31P NMR spectroscopy (Table 2). The polyP
like accumulation of polyP (Fig. 3). This could be
normalized between stations as the polyP : C ratio was
significantly correlated with temperature, latitude, and analogous to what could happen in P-deplete oligotrophic
Trichodesmium polyphosphate dynamics 2165
systems where cells experience variation in the P supply
Table 2. PolyP : C in Trichodesmium field samples compared to %polyP and indicators of Trichodesmium P physiology, including C : P, N : P, APA, and chemical and
Temperature Mixed-layer
through switching between growth on low-concentration
depth (m)
0.90
0.04
DIP to growth on comparatively higher concentrations of
17.5
5
32
19
30
13
24
17
DOP.
PolyP dynamics in the Sargasso Sea PolyP accumula-
tion in phytoplankton has previously been observed in the
20.92
0.03
coastal zone and eutrophic lakes, and in these regions
(uC)
24.6
25.7
25.3
26.2
26.8
27.9
27.7
5
polyP accumulation was generally attributed to luxury
uptake of P (Schelske and Sicko-Goad 1990; Diaz
et al. 2008). In the oligotrophic Sargasso Sea polyP was
between 8% and 25% of the total cellular P in Trichodes-
Latitude
27.67
26.66
25.66
24.66
23.66
22.66
21.66
0.87
0.05
mium despite the low DIP concentrations in this area
5
(, 5 nmol L21). These data are striking in that they
demonstrate that even in low-P regions some phytoplank-
{ Pairwise correlations reported as correlation coefficients (corr. coeff.) are compared to polyP : C at each station; bold font denotes significance.
ton sequester a significant proportion of their cellular P as
colony21 h21) (nmol L21) (nmol L21)
polyP. These field data are consistent with the culture
20.32
0.60
DIP
1.1
1.5
0.5
3.0
0.8
1.4
0.5
results that highlight the allocation of P to polyP can be
5
elevated in P-deplete Trichodesmium.
Unlike the other marine systems that have been
examined (e.g., Caribbean; Romans et al. 1994), the
0.20
0.75
DOP
43.1
22.9
46.8
28.9
45.2
45.6
presence of high percentages of polyP in Trichodesmium
5
nd
from the Sargasso Sea is unlikely to be due to luxury
uptake. Evidence from culture studies suggest Trichodes-
mium may be capable of luxury uptake (White et al. 2006b),
physical parameters from a north-to-south cruise transect in the Sargasso Sea. nd, not determined.
but elevated N : P, C : P, and APA (metrics of P deficiency)
(nmol P
APA
0.93
0.39
0.56
0.23
0.97
0.46
0.94
0.22
0.72
in this study and others from the region suggest that
5
Trichodesmium populations are P deficient (Dyhrman et al.