Cellular acclimation strategies of a minimal picocyanobacterium to phosphate stress

R E S E A R C H L E T T E R

Cellularacclimation strategiesofaminimal picocyanobacteriumtophosphate stressMatthew A. Fuszard, Phillip C. Wright & Catherine A. Biggs

Department of Chemical and Process Engineering, ChELSI Institute, University of Sheffield, UK

Correspondence: Catherine A. Biggs,

Department of Chemical and Process

Engineering, University of Sheffield, Mappin

Street, Sheffield S1 3JD, UK. Tel.: 144 114

222 7510; fax: 144 114 222 7501; e-mail:

[email protected]

Received 6 December 2009; revised 24

February 2010; accepted 24 February 2010.

Final version published online 30 March 2010.

DOI:10.1111/j.1574-6968.2010.01942.x

Editor: Aharon Oren

Keywords

Prochlorococcus; cyanobacteria; proteomics;

phosphate starvation; MED4.

Abstract

The proteomic response of Prochlorococcus marinus MED4, subjected to extended

phosphate (P) starvation, was measured utilizing the quantitative technique

isobaric tags for relative and absolute quantitation. Seventeen proteins were

identified as significantly more abundant in MED4 cultures grown under P-

stressed conditions than the nonstressed cultures, while 14 proteins were observed

to be significantly less abundant. Proteins involved in P acquisition, and mem-

brane-associated functions such as protein folding, export and recycling as well as

a protein putatively associated with maintaining DNA integrity were found to be

higher in abundance than the nonstressed cultures. The effect of P starvation was

also noticeable on the photosynthetic apparatus, whereby important proteins

involved with light harvesting were reduced in abundance directly affecting the

metabolism. This is expected, as the cell is starved of an essential nutrient; however,

proteins involved in maintaining structural integrity in the photosystems are more

abundant, which was not expected. We conclude that MED4 is capable of

acclimating to long periods of P deprivation through a suite of processes including

activating P transport and acquisition mechanisms, general stress responses,

reduction of energy-related metabolic processes and importantly maintaining

structural integrity in vital cell mechanisms.

Introduction

Prochlorococcus marinus are obligate oxygen-evolving

photoautotrophic marine picocyanobacteria of significant

importance to world biogeochemical cycles, and are con-

sidered the most abundant photosynthetic organism on

Earth (Partensky et al., 1999). They are widespread through-

out the photic regions of the world’s oceans between 401S

and 501N, with cell densities of up to 105 cells mL�1 in the

central oligotrophic gyres (Partensky et al., 1999). They are

principally distinguished into two taxonomic clades due to

physiological niche adaptation to light intensity: high light-

and low light-adapted ecotypes (Moore et al., 1998; West &

Scanlan, 1999; Rocap et al., 2003).

A great deal of interest has arisen around Prochlorococcus

due to its small size and specifically its near-minimal

genome. Indeed, the chromosomes of most Prochlorococcus

strains demonstrate significant genomic reduction, revealing

a central conserved core set of essential genes and a variable

shell, which is hypothesized to reflect each individual

strain’s evolutionary adaptation to a specific environmental

niche (Kettler et al., 2007; Shi & Falkowski, 2008). Closer

inspection of Prochlorococcus genomes reveals that the

majority of these strain-specific genes (74% in the case of

Prochlorococcus strain MED4) are located in highly variable

‘genomic islands’, suggesting a mosaic structure that con-

tinually undergoes genomic rearrangement (Coleman et al.,

2006).

A suggested source of pressure for these organisms to

reduce genome as well as cell size is thought to be reduced

nutrient availability (Raven, 1998), which is a characteristic

of subtropical oceans, particularly phosphate (P). Indeed, P

concentrations are hypothesized to have affected domain

shifts from a eukaryotic to a prokaryotic life in these

oligotrophic regions (Karl et al., 1995, 2001). Also, recent

studies have found that phytoplanktonic species within

nutrient-poor oceanic biomes substitute phospholipids with

sulpholipids in order to conserve ambient phosphorous for

FEMS Microbiol Lett 306 (2010) 127–134 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

MIC

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mailto:[email protected]

more essential metabolic use in the face of competition from

heterotrophic bacteria (Van Mooy et al., 2006, 2009).

A recent study of MED4 showed that a unique suite of

genes was upregulated under P stress (Martiny et al., 2006).

Most of these genes are orthologues of Escherichia coli genes

located in and around the phoB operon, but another set are

located within a variable genomic island, ‘Island 5’, and

unique to MED4. The function of these genes is as yet

uncharacterized; however, some putative annotations are

available at GenBank (http://www.ncbi.nlm.nih.gov/).

It is clear that the availability and ambient concentration

of inorganic P within oligotrophic regions is a crucial factor

determining the success of MED4 within those environ-

ments. Therefore, this study seeks to ascertain the global

quantitative proteomic response of MED4 to longer term P

starvation, and thereby providing further insight into how

this organism responds to P stress. For this proteomic study,

we will utilize a recently developed, but widely used,

proteomic technique: isobaric tags for relative and absolute

quantitation (iTRAQ) (Choe et al., 2007; Pandhal et al.,

2007). iTRAQ was chosen as this technique has a clear

advantage over more conventional proteomic methods

through conferring reproducibility and statistical confi-

dence to the measurements of protein abundance within a

cell at a fixed point in time (Ross et al., 2004; Gan et al.,

2007).

Materials and methods

For a complete description of the Materials and methods

refer to the Supporting Information Appendix S1; in brief,

however, P. marinus strain MED4 was grown in biological

triplicate under two separate conditions: P-deplete and P-

replete PCR-S11 media. The cells were harvested at the same

time in the late exponential phase (after 10 days), and

proteins were extracted (Meijer & Wijffels, 1998). Approxi-

mately 100 mg of protein from each replicate was then

reduced, alkylated, digested and labelled with 8-plex iTRAQ

reagents according to the manufacturer’s (Applied Biosys-

tems, Framingham, MA) protocol. The replicates were then

pooled before primary strong cation exchange (SCX) frac-

tionation (Pandhal et al., 2007). Mass spectrometeric analy-

sis of the SCX fractions was performed using both a

HCTUltra ESI TRAP MS/MS (Bruker Daltonics GmbH,

Bremen, Germany) and a QStar XL Hybrid ESI Quadrupole

time-of-flight tandem mass spectrometer, ESI-qQ-TOF-MS/

MS (Applied Biosystems; MDS-Sciex, Concord, Ontario,

Canada), coupled with an online capillary liquid chromato-

graphy system (Ultimate 3000, Dionex/LC Packings, the

Netherlands) (Pandhal et al., 2007). Preliminary data analy-

sis, peptide identification and quantification were carried

out using the PHENYX [Geneva Bioinformatics (GeneBio),

Geneva, Switzerland] software.

Results and discussion

Introduction

Ninety-eight proteins were identified by Z1 peptides [coef-

ficient of variation (CV) = 1.07] and eight false positives

were identified [false positive rate (FPR) = 0.016]. However,

for accurate determination of protein identification, Z2

peptides are required. With this restriction, 68 proteins were

identified (CV = 1.05), with three false positives

(FPR = 0.05), with quantification only possible for 62 of the

identified proteins. For a full list of identified proteins, see

Supporting Information, Table S1. This figure, while lower

than other iTRAQ experimental data of other cyanobacteria,

such as Synechocystis sp. PCC6803 (Gan et al., 2007) and

Nostoc sp. (Ow et al., 2009a), shows a broad coverage across

the chromosome for MED4 (Fig. 1). It is also similar to the

only other iTRAQ shotgun proteomic experiment con-

ducted on MED4, where 70 proteins were identified by Z2

peptides (Pandhal et al., 2007). Also, there was a significant

bias towards identification of particular proteins within the

results, where 75% of the peptides identified only mapped to

19% of the identified proteins (Table S1). This strongly

suggests that the cell’s proteome, particularly under P-

stressed conditions, is dominated by a small number of

these particular proteins.

Of the identified proteins, 17 were significantly more

abundant than the control, and 14 were less abundant. This

is a more balanced observation than the previous transcrip-

tomic study of P starvation of MED4 (Martiny et al., 2006)

that reported 30 upregulated genes and just four down-

regulated under P starvation conditions. This difference is

understandable as the earlier study monitored healthy cells

subjected to a P-depleted medium over a 2-day period,

Gene position

0 200 400 600 800 1000 1200 1400 1600 18000.1

1

10

100

+P

/–P

rel

ativ

e ab

unda

nce

(log

)

Fig. 1. Spread of identified proteins across the whole proteome. Fold

change is depicted on the y-axis. Solid circles represent increased

abundance, triangles represent reduced abundance and empty circles

depict proteins with no change in abundance compared with the

control. The central dotted region highlights the PhoB region, and the

upper dotted region highlights the genomically variable ‘Island 5’ region.

FEMS Microbiol Lett 306 (2010) 127–134c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

128 M.A. Fuszard et al.

http://www.ncbi.nlm.nih.gov/

whereas this study focused on the response of a longer term

(10 day) exposure to P depletion, and so can be regarded

more of an acclimation strategy rather than an immediate

stress response. This characteristic of stress against longer

term acclimation has been observed recently by comparing

the response to varying levels of salt-infused media of two

other cyanobacteria: Synechocystis sp. PCC6803 and Euha-

lothece sp. BAA001 (Pandhal et al., 2009). Moreover, as later

sections will show, the cell responds to prolonged P starva-

tion by regulating the abundance of proteins across the

proteome, and not just from limited specific areas (Fig. 1,

where all identified proteins are depicted with respect to

their chromosomal location), as opposed to an immediate

shock response (Martiny et al., 2006).

It is important to briefly consider the fundamental

methodological differences when introducing comparisons

between transcriptomic and proteomic data. The half-lives

of both mRNA and its encoded protein differ by up to an

order of magnitude, and so any direct quantitative correla-

tion between transcript levels and protein abundance is, at

the time of writing, very difficult to assert. There are issues

with the quantitative nature of both techniques; indeed,

microarray experiments have been observed to underestimate

the relative change in gene expression (Yuen et al., 2002),

and recently iTRAQ has also been shown to potentially

underestimate the relative changes in protein abundances

(Ow et al., 2009b). However, qualitative comparisons be-

tween the two methodologies are invaluable, and inferences

into the physiological state of the cell when stressed are

emphasized through the comparison of both transcriptomic

and proteomic data.

P-acquisition mechanisms

Here, only four proteins from those gene clusters identified

previously as responding to P starvation (Martiny et al.,

2006) were assessed as significantly more abundant than

the P-replete control: PhoA, the alkaline phosphatase; PhoE,

the putative orthophosphate membrane transporter; PstS,

the periplasmic P-binding protein; and one protein from the

genomic island operon, PMM1416 (Fig. 2a). The first three

are part of the phoB region with the pstABCS ortho-

phosphate transport system, and the last one is from the

genomic island group PMM1403-1416.

In agreement with the transcriptomic data (Martiny et al.,

2006), PhoE, PhoA and PMM1416 demonstrate the greatest

fold change in response to P deprivation (Fig. 2a), which

clearly shows that the cells respond quickly through the

production of these proteins and maintain their cellular

concentration throughout the period of P starvation. How-

ever, PMM1416 has been seen to be upregulated during both

P and light stress, indicating a general stress response role for

(a)

0.1

1

10

100(b)

0.1

1

10(c)

0.1

1

10

(d)

0.1

1

10(e)

0.1

1

10

100

+P

/−P

rel

ativ

e ab

unda

nce

(log 1

0)

Fig. 2. Relative abundance groups of identified proteins from the P-stressed phenotype in relation to the control. (a) Proteins involved in P acquisition

and stress, (b) photosynthesis, PETC and ATP synthase proteins, (c) differential abundance of glycolysis and carbon fixation proteins, (d) proteins

associated with transcription, translation protein folding and turnover, and (e) other uncategorized proteins.


129Adaptation of P. marinus MED4 to phosphate stress

this particular protein (Coleman et al., 2006). The levels of

alkaline phosphatase, PhoA, were c. 28-fold more abundant

in the stressed cultures, whereas the porin PhoE was c. 50-

fold more abundant (Fig. 2a). At the transcriptomic level

after 48 h, the regulated levels were almost at parity (Martiny

et al., 2006), suggesting the differential production of both

PhoE and PhoA over extended starvation periods. Increased

alkaline phosphatase activity has been measured previously

for oceanic picocyanobacteria under P stress (Moore et al.,

2005; Tetu et al., 2009) and in Synechocystis sp. PCC6803

(Gan, 2006), and so our results are in line with these

observations.

Photosynthesis and energy metabolism

The structure and functioning of the MED4 photosynthetic

apparatus is affected through extended P starvation (Fig. 3).

Seven proteins were recognized as differentially abundant

(Fig. 2b). Proteins that were less abundant than the control

were those associated with chlorophyll binding and light

harvesting (e.g. Pcb and CP43 within PSII). Interestingly,

this observation has also been identified recently at the

transcriptomic level in Synechococcus WH8102 when sub-

jected to extended P stress (Tetu et al., 2009). PsaA, which is

known to be an electron acceptor in PSI, is also less

abundant as well as the plastocyanin docking protein PsaF.

PsaA is also a vital part of the photosynthetic electron

transport chain (PETC), and binds almost 100 chlorophyll

molecules, making it an essential light-harvesting protein in

PSI (Barber, 2001), specifically as MED4 has only one copy

of the pcb gene, which is associated exclusively with PSII

(Fig. 3) (Rocap et al., 2003). From this, we conclude that the

cell reduced its photosynthetic capabilities. This would

directly reduce UV photodamage and oxidative stress from

reactive oxygen species produced as a byproduct of water

splitting at the oxygen-evolving complex at the base of PSII.

This conclusion is supported by the observation that the

known antioxidants, thioredoxin (TrxA) and thioredoxin

peroxidise (tpx), are not significantly differentially abundant

in the stressed phenotype (Fig. 2d). It is also clear that other

essential proteins in the PETC, besides PsaF, are less abun-

dant than the P-replete control. PsaF and ferredoxin-NADP

oxidoreductase are downregulated, which strongly suggests

that the cell is attempting to reduce certain reductive energy

production processes, specifically NADPH generation,

which in turn indicates a general metabolic slowdown. It is

interesting to note that essential protein subunits of the ATP

synthase complex are unaffected by long-term exposure to P

deprivation, which suggests that ATP was produced nor-

mally. This is in contrast to the only other known proteomic

assay of cyanobacterial response to P starvation, whereby

ATP synthase subunits were significantly upregulated in

Synechocystis sp. PCC6803 (Gan, 2006). The reason for this

is not clear, and warrants further research.

When considering the structural aspects of both photo-

systems, it appears that important proteins associated with

maintaining PSI and PSII structural integrity are more

abundant, notably the Mn-stabilizing protein (MSP) of PSII

Pcb

MSP

CP

43

P680

P700

D1 D2

Psb

28

PQH2

FeS

PsaF

PsaC

Cytochrom

e f

Cyt

ochr

ome b

FNR

PC

A0

A1

Fx

Fd

C ring

α αβ

PSII Cyt b6f PSIATP

synthase

PsaD

Fig. 3. The photosynthetic apparatus of MED4. Specific proteins are labelled. Proteins that exhibited increased abundance are represented with dotted

hatching, proteins that are less abundant than the control are represented with diagonal hatching and proteins that exhibit no change are represented

with brickwork hatching. The PETC is indicated by arrows.



and PsaD, which is responsible for docking ferredoxin as

well as stabilizing PSI (Barber, 2001). These findings suggest

that the photosystem, while protecting itself from photo-

induced damage, maintains structural integrity, possibly in

case ambient P concentrations return to normal. However,

when comparing this finding with WH8102, PsaD is upre-

gulated, but an MSP polypeptide is downregulated (Tetu

et al., 2009). The reason for this is not clear, and warrants

further investigation.

Three important proteins within glycolysis, the reductive

pentose phosphate (Calvin) cycle and carbon fixation are

significantly less abundant under P stress: rbcL, the large

subunit of Rubisco; rpe, ribulose-phosphate 3-epimerase,

both of which are vital enzymes in the Calvin cycle, as well as

gap2, glyceraldehyde 3-phosphate dehydrogenase, which is

the enzyme involved in the sixth step of the breakdown of

glucose (Fig. 2c). Both rbcL and rpe were also observably

downregulated within WH8102 (Tetu et al., 2009). This

result confirms that the cell metabolically slowed down

when exposed to long-term P starvation, coinciding with

the earlier observation of reduced photosynthetic capability

and energy production.

Protein turnover, stress response and generalcell processes

Of considerable interest is the possible increase in transla-

tion, where the ribosomal 30S subunit protein S6 and 50S

subunit L7/L12 were more abundant than the control;

however, transcription (measured by the concentration of

RpoA, the a subunit of RNA polymerase) seems to be

unaffected (Fig. 2d). This result has also been identified in

WH8102, whereby 10 out of the 17 ribosomal protein

transcripts quantified were significantly upregulated, and

RpoA was similarly unaffected during late P starvation (Tetu

et al., 2009). Interestingly, this may be an indication of

polysome usage in translating important proteins, and

coincidentally efficient usage of P expensive mRNA mole-

cules. This process would easily explain a higher proportion

of ribosomal proteins with regard to observed transcription.

However, in contrast to this, the elongation factor Tu (tuf),

which is involved in protein synthesis, specifically the

correct placement of aminoacyl tRNA into the ribosome, is

also not differentially abundant. This result has also been

found in P starvation of Synechocystis (Gan, 2006). An

explanation for this is not immediately available. Another

puzzling result affecting translation is the observation

that ivlH, an important regulatory subunit protein in de

novo synthesis of branched chain amino acids such as

valine, leucine and isoleucine, is less abundant in the

stressed cultures (Fig. 2e); however, this may be due to the

cells recycling amino acids from degraded misfolded

proteins.

In association with translation and amino acid synthesis,

the nitrogen metabolism regulator protein, P-II, is also more

abundant in P-starved cells (Fig. 2e). P-II is thought to

regulate the assimilation of nitrogen as well as carbon

sources on multiple levels (Osanai & Tanaka, 2007). P-II is

phosphorylated in cyanobacteria and as such interacts with

both a phosphatase and a kinase. However, P-II phosphatase

interaction is thought to control nitrate/nitrite assimilation,

and as MED4 is unable to grow on those particular nitrogen

sources (Moore et al., 2002), and that the kinase activity is

reduced when, in the presence of ammonia in another

cyanobacterium, Synechococcus elongatus PCC7942 (Lee

et al., 1999), this particular function of P-II may well be

redundant within MED4. With regard to amino acid synth-

esis, P-II has been shown to increase N-acetyl glutamate

kinase (NAGK) activity (Maheswaran et al., 2004), an

enzyme in the arginine biosynthetic pathway, and identified

in Synechococcus (Burillo et al., 2004; Heinrich et al., 2004).

As MED4 is known to have NAGK, it is safe to assume that

this cellular increase in P-II will have a constitutive affect on

arginine biosynthesis. In addition to this, P-II directly

influences nitrogen-related gene transcription (Paz-Yepes

et al., 2003), but this process is, as yet, unknown.

An intriguing result is the increased abundance of the

periplasmic protein, FKBP-type peptidyl-prolyl cis–trans

isomerase (PPIase) (Fig. 2d), which assists in the accelerated

and correct folding of proteins bound for extracellular use

(Lang et al., 1987; Lang & Schmid, 1988). This result is

interesting if considered in parallel with the significant

increase in a membrane-associated protease (PMM0516,

Fig. 2e), which would assist in recycling misfolded periplas-

mic proteins, and the significant increase in PhoA concen-

trations reported above. However, PPIase transcripts were

found to be downregulated in WH8102 (Tetu et al., 2009),

but this could indicate a strain-specific response to P

starvation, particularly when considering the increased

abundance of the MED4-specific protein PMM1416.

Fatty acid biosynthesis is also detrimentally affected by P

starvation. Two proteins essential in this process, acyl carrier

protein (acpP) and enoyl-(acyl carrier protein) reductase

(fabL), were less abundant than the control (Fig. 2e). Fatty

acids have multiple intracellular uses, notably fuel storage

and membrane manufacture. It could easily be deduced that

with a paucity of bioavailable P, phospholipid biosynthesis

and hence membrane manufacture, would be reduced.

However, it is known that o 1% of inducted Pi is incorpo-

rated into membranes, representing a small fraction of the

cellular quota for P, and there is no evidence, as yet, for P

regulation within the lipid membrane of MED4 (Van Mooy

et al., 2006). Hence, we conclude that this result reflects that

the process of fuel storage is reduced within the cells.

Finally, it is interesting to notice that of the five identified

stress-related heat shock proteins, GroES, GroEL1, GroEL2,



grpE and DnaK2, only GroES is differentially more abun-

dant (Fig. 2d). As GroES interacts with GroEL to form a

complex, which assists in correcting misfolded proteins, this

result is surprising, particularly when compared with MED4

subjected to high light Stress, whereby both GroES and

GroEL12 proteins were more abundant (Pandhal et al.,

2007). Another protein identified as being stress response

related, a histone-like DNA-binding protein (PMM1321),

was more abundant in the P-stressed phenotype (Fig. 2e).

These proteins are known to wrap DNA and stabilize it from

denaturation under extreme environmental conditions (Pet-

tijohn, 1988). Indeed, a homologue of this protein (HU) was

more abundant in Synechocystis sp. PCC6803 under P-

deplete conditions (Gan, 2006), but surprisingly, was not

observed in MED4 under light stress (Pandhal et al., 2007).

This observation suggests specificity in stress response for

this protein, possibly nutrient starvation; however, a more

detailed examination of the overall stress responses within

this organism is required.

Conclusions

It is clear that MED4 acclimates to long-term P starvation

through activating and also suppressing a wide range of

cellular processes. Important metabolic mechanisms such as

glycolysis are depressed, while other systems, most notably

P-acquisition mechanisms, are considerably elevated.

Photosynthesis and carbon fixation are reduced, while the

structures of the photosystems are reinforced. This, in

particular, is an indication of the stressed cell reducing its

metabolic activities while simultaneously maintaining cel-

lular integrity. Specific amino acid biosynthesis mechanisms

are either reinforced or reduced. This may be an indication

of individual amino acid requirements, which could well be

linked to intracellular recycling efficiency and/or specificity.

Indeed, translation, indicated through ribosome levels,

appears to be increased, indicating an active, ongoing

response. Specific chaperonins and protein-folding proteins,

particularly membrane-associated ones, are more abundant,

while DNA integrity is reinforced. Interestingly, there does

appear to be a specificity of the stress response to P

starvation, whereby under conditions of nitrogen depriva-

tion, ribosomal genes as well as the carboxysome shell

protein genes csoS12 and photosystem genes were all re-

pressed, whereas Rubisco is repressed under both N starva-

tion (Tolonen et al., 2006) and P starvation (this study).

However, the response to N deprivation was measured over

a 48-h period and may not reflect longer term acclimation.

The environmental conditions that MED4 are exposed to

in situ are considered to be consistent and unchanging;

however, these results appear to suggest that MED4 exhibits

a capability to withstand long periods of P starvation and

recover. This, in turn, implies that periodic fluctuations

within oligotrophic oceans are not uncommon. It has long

been known that MED4 can withstand short periods of P

starvation and recover (Moore et al., 2005; Martiny et al.,

2006), and these results suggest that the strain has the

capability to acclimate to and survive longer periods of P

stress.

Acknowledgements

We wish to acknowledge the provision of an EPSRC student-

ship, Advanced Research Fellowship for C.A.B. (EP/

E053556/01) and further EPSRC funding (GR/S84347/01

and EP/E036252/1). We also acknowledge the Roscoff

Culture Collection for the kind provision of cells. Finally,

we would like to acknowledge Dr Saw Yen Ow, Dr Jagroop

Pandhal and Dr Josselin Noirel for all assistance and instru-

ment help.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Appendix S1. Materials and methods.



Table S1. Proteins identified by two or more peptides and

quantitated.

Please note: Wiley-Blackwell is not responsible for the

content or functionality of any supporting materials sup-

plied by the authors. Any queries (other than missing

material) should be directed to the corresponding author

for the article.



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Cellular acclimation strategies of a minimal picocyanobacterium to phosphate stress