Comparative quantitative proteomics of prochlorococcus ecotypes to a decrease in environmental phosphate concentrations
© Fuszard et al; licensee BioMed Central Ltd. 2012
Received: 8 November 2011
Accepted: 19 March 2012
Published: 19 March 2012
The well-lit surface waters of oligotrophic gyres significantly contribute to global primary production. Marine cyanobacteria of the genus Prochlorococcus are a major fraction of photosynthetic organisms within these areas. Labile phosphate is considered a limiting nutrient in some oligotrophic regions such as the Caribbean Sea, and as such it is crucial to understand the physiological response of primary producers such as Prochlorococcus to fluctuations in the availability of this critical nutrient.
Prochlorococcus strains representing both high light (HL) (MIT9312) and low light (LL) (NATL2A and SS120) ecotypes were grown identically in phosphate depleted media (10 μM Pi). The three strains displayed marked differences in cellular protein expression, as determined by high throughput large scale quantitative proteomic analysis. The only strain to demonstrate a significantly different growth rate under reduced phosphate conditions was MIT9312. Additionally, there was a significant increase in phosphate-related proteins such as PhoE (> 15 fold increase) and a depression of the Rubisco protein RbcL abundance in this strain, whereas there appeared to be no significant change within the LL strain SS120.
This differential response between ecotypes highlights the relative importance of phosphate availability to each strain and from these results we draw the conclusion that the expression of phosphate acquisition mechanisms are activated at strain specific phosphate concentrations.
Within marine oligotrophic systems, such as central subtropical gyres, orthophosphate (Pi) is a crucial macronutrient governing microbial population densities, particularly within the well-lit surface waters of the euphotic zone [1–3]. The principal photosynthetic organism numerically dominating these areas is Prochlorococcus, which is estimated to represent about 50% of all photosynthetic activity within them [4, 5]. Prochlorococcus has been broadly delineated into two clades, or ecotypes, high light (HL) and low light (LL) based upon the ratios of divinylchlorophylla and b within their light harvesting apparatuses and as such their assumed depth within the water column [6, 7]. Further clade subdivisions have been implemented through phylogenetic analyses of 16S rRNA sequences . As a taxon, Prochlorococcus is characterised by its small size (~ 1 μm3), and significantly reduced genomes which ranges from 1.64 Mbps (the HL strain MIT9301) to 2.68 Mbps (the LL strain MIT9303) . This diminished volume and genome is hypothesised to be the result of an accelerated evolutionary process adapting to reduced phosphorus in its environment [10, 11]. Indeed, Prochlorococcus is known to replace phospholipids in its membranes with sulpholipids, which dramatically reduce its Pi requirements .
Given the importance of Pi to Prochlorococcus, perhaps it is surprising to find no significant correlation between ecotype distribution and Pi concentration . However, fluxes in Pi transport within these regions are important considerations, which could help to explain the discrepancy. Nevertheless the observation of a large number of known Pi acquisition genes in some LL ecotypes (i.e. MIT9313 and NATL2A), and not others (i.e. SS120) [14, 15] is confusing. Indeed, Pi acquisition genes are present in some HL strains (i.e. MED4) and not others (i.e. MIT9515) . However it was recently observed that the prevalence of Prochlorococcus genes involved in acquisition of phosphate substrates were correlated with areas of low Pi such as the Caribbean Sea and NW Mediterranean . This conflict is likely resolved due to the presence of hypervariable genomic islands within Prochlorococcus, allowing for evolutionarily rapid niche adaptation . Given this, it was hypothesised that the presence or absence of these genes could directly affect the protein content of cells when Pi stressed, and as such directly affect the ability of a strain to acclimate to environmental Pi fluctuations . So the question arises, how effective are cells with and without these genes at acclimating to a shift in environmental Pi? Indeed, the levels of mRNA transcripts of two strains, MED4 and MIT9313, which both contain the two component response regulation system phoBR, behaved quite differently to Pi starvation .
Details of the strains used in this study, as obtained from NCBI and CCMP.
Strain and genome details
P acquisition mechanisms
Isolation details and culture conditions
Culture temp (°C)
S. Frankel & L West-Johnsrud
Results and discussion
Using relative abundance cut-offs of 1.6 and 0.6 fold differences to represent increased or decreased relative abundances [21, 22], 4 proteins were more abundant in MIT9312 and 4 were less abundant than the replete cultures. Within NATL2A, 6 proteins were more abundant and 1 was less abundant than the replete cultures. In SS120, 4 were more abundant and none were lower than the replete cultures (Figure 2A).
Within NATL2A, PstS abundance is significantly greater within Pi-deplete conditions, though with greater uncertainty (Additional file 1: Table S1). However neither PhoA nor PhoE was observed with mass spectrometry here, which is surprising as we showed previously that both PhoA and PhoE are greater in abundance alongside PstS in the high light ecotype MED4 , as is true with MIT9312 in this study. However, considering that NATL2A cells are in mid-exponential phase as opposed to early stationary phase this may indicate a progressive strategy of protein expression within the cells, however more work is needed to clarify this.
What was also unexpected, was the absence of any Pi acquisition mechanisms (as reflected in observed peptide identifications) within SS120 cells (Additional file 1: Table S1), allied with no significant difference in growth rates between Pi-replete and Pi-deplete cultures (p > 0.05). SS120 is deficient in most Pi acquisition genes [14, 15], however it does have two copies of PstS, neither of which were present in our assay. At first glance, this result appears counter-intuitive, as a 'very' LL strain typically present in vivo within Pi-replete environments would be expected to be adversely affected by a substantial decrease in Pi. However, the absence of a phoBRregulon suggests that the strain is incapable of regulating a response to shifts in environmental concentrations of Pi that are not immediately starvation inducing . Curiously, this also infers that activation of the phoBR response mechanisms within MIT9312 and NATL2A were directly due to the mechanism's innate sensitivity to changing external Pi concentrations. This suggests that the intensity of response is directly proportional to external Pi concentration, coincidentally specific to each strain, and may be reflective of each strain's environmental niche and/or obligate cellular requirements.
Photosynthesis, biosynthesis and central metabolism
When considering NATL2A solely, there appear to be a few subtle discrepancies in protein abundances between stressed and non-stressed cultures. Fumerase (FumC) is an enzyme associated with both the tricarboxylic acid (TCA) cycle and arginine/proline biosynthesis, and appears to be more abundant within NATL2A cells when Pi-deplete (Additional file 1: Table S1). As NATL2A has an incomplete TCA cycle, it is safe to assume that its function within the cell is within arginine and proline metabolism. Also, the acyl carrier protein (AcpP) is an essential component of fatty acid biosynthesis, and is more abundant in Pi-deplete NATL2A cells (Additional file 1: Table S1). Fatty acids are for the most part used within either fuel storage or membrane manufacture. However it may be misleading to arrive at the conclusion that this is a specific cellular response to lower Pi concentrations. It is possibly a function of apparently slightly elevated (albeit not significant) growth within NATL2A Pi-deplete cultures, and as such could reflect comparatively greater metabolic activity. Nevertheless, this explanation cannot immediately address the lower abundance of CobJ, a Precorrin-3B C17-methyltransferase region-containing protein (Additional file 1: Table S1), part of the aerobic vitamin B12 biosynthesis pathway within Pi-stressed cells. However, B12 synthesis is a sub pathway offshoot from the main chlorophyll biosynthetic pathway, and as such may reflect a metabolic preference for chlorophyll production that, again, may be representative of faster growing populations.
An interesting observation is the abundance of CitT within Pi-stressed SS120 cells (Figure 3A). This protein functions as a di/tricarboxylate transporter, which implies that the cells are scavenging lysed cellular material from the environment. That stressed SS120 cells appear to be preferentially acquiring tricarboxylic acid intermediates when growing in Pi-deplete conditions, and not upregulatingPstS, is puzzling. However, it may indicate that this strain may be supplementing an affected glycolysis pathway through acquiring external carbon sources, and that this is more evidence that the cells response to an environmental stress is an iterative, evolving process. SS120 may simply have not initiated transcription of PstS in sufficiently detectable quantities. Indeed, even in starvation experiments pstSexperession is far from an immediate response [14, 23].
An interesting observation is the presence of LuxR, the response regulatory family protein involved in quorum sensing within bacteria, in NATL2A cells (Additional file 1: Table S1). To our knowledge, this is the first instance of observing proteins putatively indicated in quorum sensing capability in any marine cyanobacteria. However, we were unable to locate any LuxI homologues, an essential protein required for effective quorum sensing, within NATL2A (data not shown). However LuxR is known to be a transcriptional regulator activated when cell concentrations of a particular trigger compound (usually N-(3-oxohexanoyl)-L-homoserine lactone, which is generated through the enzymatic functioning of LuxI) reach particular levels. As such, we speculate that the protein acts as a density-dependant transcriptional regulator, but for an unknown function, and through another trigger compound.
Prochlorococcus are now widely considered to be evolutionarily adept at environmental niche domination, particularly within nutrient poor oligotrophic waters. The genus is typified by genomes characterised by hypervariable genomic islands , which are thought to contain genes obtained through phage-mediated horizontal gene transfer, and infer niche-specific advantages such as nutrient acquisition and phage resistance. Our results reinforce previous results concerning the importance of phosphate concentrations to specific strains, but also highlight the possibility of the cells employing a progressive acclimation strategy. It appears that Prochlorococcus strains evolutionarily adapted to life in a Pi-deplete environment respond to phosphate fluctuations through a succession of cellular processes, such as the upregulation of Pi acquisition mechanisms, a dissociation of photosynthesis from central metabolic pathways, and a staggered breakdown of the photosystems allowing prolonged photophosphorylated ATP generation. This progressive response allows the cell to react quickly to any subsequent increases in ambient Pi concentrations. It is our hypothesis that HL strains are also particularly sensitive to changes in Pi, and that ambient phosphate concentrations initiate a strong response regardless of being predominantly growth limited elsewhere.
We also note that our results strongly infer that the induction of Pi acquisition mechanisms are concentration specific between strains, particularly considering the absence of any stress response of the LL strain SS120 compared to MIT9312 when grown from identical initial concentration levels.
For a complete description of the Materials and Methods used please refer to the (Additional file 3: Material and Methods). In brief, however, biological triplicates of all three strains (MIT9312, NATL2A and SS120 (CCMP, Maine)) were grown under 2 separate conditions: Pi replete (Pro99 media with 50 μM NaH2PO4 ) and Pi deplete (Pro99 media with 10 μM NaH2PO4), and moderate white light intensities (30, 10 and 20 μE m-2 s-1 respectively), in a 13:11 h light:dark regime at 23°C.
For the proteomic analysis, the cells were harvested once measured optical densities reached 0.4 (after which populations had been observed to crash), and proteins were extracted from the three biological replicates for each phenotype . 100 μg of protein from each replicate was then reduced, alkylated, digested and labelled with 8-plex iTRAQ reagents according to the manufacturer's (ABSciex, Framingham, MA) protocol. The labelled replicates were then pooled before primary strong cation exchange (SCX) fractionation . Mass spectrometric analysis of the SCX fractions was performed with 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 chromatography system (Ultimate 3000, Dionex/LC Packings, The Netherlands) [21, 22]. Preliminary data analysis, protein identification and quantitation were carried out using the PHENYX [Geneva Bioinformatics (GeneBio), Geneva, Switzerland] software platform.
The authors wish to acknowledge the provision of an EPSRC studentship, Advanced Research Fellowship for CAB (EP/E053556/01) and further EPSRC funding (GR/S84347/01 and EP/E036252/1). We also acknowledge the Provasoli-Guillard National Center for Culture of Marine Phytoplankton for the kind provision of cells. Thanks also to Adam Martiny for additional input.
- Ammerman JW, Hood RR, Case DA, Cotner JB: Phosphorus deficiency in the Atlantic: An emerging paradigm in oceanography. Eos Trans Am Geophys Union. 2003, 84: 165-170.View ArticleGoogle Scholar
- Thingstad TF, Krom MD, Mantoura RF, et al: Nature of phosphorus limitation in the ultraoligotrophic eastern Mediterranean. Science. 2005, 309: 1068-1071. 10.1126/science.1112632.View ArticleGoogle Scholar
- Thingstad TF, Zweifel UL, Rassoulzadegan F: P limitation of heterotrophic bacteria and phytoplankton in the northwest Mediterranean. Limnol Oceanogr. 1998, 43: 88-94. 10.4319/lo.1998.43.1.0088.View ArticleGoogle Scholar
- Chisholm SW, Olson RJ, Zettler ER, Goericke R, Waterbury JB, Welschmeyer NA: A novel free-living prochlorophyte abundant in the oceanic euphotic zone. Nature. 1988, 334: 340-343. 10.1038/334340a0.View ArticleGoogle Scholar
- Partensky F, Hess WR, Vaulot D: Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiol Mol Biol Rev. 1999, 63: 106-127.Google Scholar
- Moore LR, Chisholm SW: Photophysiology of the marine cyanobacterium Prochlorococcus: ecotypic differences among cultured isolates. Limnol Oceanogr. 1999, 44: 628-638. 10.4319/lo.1999.44.3.0628.View ArticleGoogle Scholar
- Moore LR, Rocap G, Chisholm SW: Physiology and molecular phylogeny of coexisting Prochlorococcus ecotypes. Nature. 1998, 393: 464-467. 10.1038/30965.View ArticleGoogle Scholar
- Rocap G, Distel DL, Waterbury JB, Chisholm SW: Resolution of Prochlorococcus and Synechococcus ecotypes by using 16S-23S ribosomal DNA internal transcribed spacer sequences. Appl Environ Microbiol. 2002, 68: 1180-1191. 10.1128/AEM.68.3.1180-1191.2002.View ArticleGoogle Scholar
- Partensky F, Garczarek L: Prochlorococcus: Advantages and Limits of Minimalism. Ann Rev Marine Sci. 2009, 2: 305-331.View ArticleGoogle Scholar
- Coleman ML, Chisholm SW: Ecosystem-specific selection pressures revealed through comparative population genomics. ProcNatlAcadSci. 2010, 107: 18634-18639.View ArticleGoogle Scholar
- Dufresne A, Garczarek L, Partensky F: Accelerated evolution associated with genome reduction in a free-living prokaryote. Genome Biol. 2005, 6: R14-10.1186/gb-2005-6-2-r14.View ArticleGoogle Scholar
- Van Mooy BA, Rocap G, Fredricks HF, Evans CT, Devol AH: Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in oligotrophic marine environments. Proc Natl Acad Sci USA. 2006, 103: 8607-8612. 10.1073/pnas.0600540103.View ArticleGoogle Scholar
- Johnson ZI, Zinser ER, Coe A, McNulty NP, Woodward EM, Chisholm SW: Niche partitioning among Prochlorococcus ecotypes along ocean-scale environmental gradients. Science. 2006, 311: 1737-1740. 10.1126/science.1118052.View ArticleGoogle Scholar
- Martiny AC, Coleman ML, Chisholm SW: Phosphate acquisition genes in Prochlorococcus ecotypes: evidence for genome-wide adaptation. Proc Natl Acad Sci USA. 2006, 103: 12552-12557. 10.1073/pnas.0601301103.View ArticleGoogle Scholar
- Moore LR, Ostrowski M, Scanlan DJ, Feren K, Sweetsir T: Ecotypic variation in phosphorus-acquisition mechanisms within marine picocyanobacteria. AquatMicrobEcol. 2005, 39: 257-269.Google Scholar
- Martiny AC, Huang Y, Li W: Occurrence of phosphate acquisition genes in Prochlorococcus cells from different ocean regions. Environ Microbiol. 2009, 11: 1340-1347. 10.1111/j.1462-2920.2009.01860.x.View ArticleGoogle Scholar
- Coleman ML, Sullivan MB, Martiny AC, Steglich C, Barry K, Delong EF, Chisholm SW: Genomic islands and the ecology and evolution of Prochlorococcus. Science. 2006, 311: 1768-1770. 10.1126/science.1122050.View ArticleGoogle Scholar
- Copeland A, Lucas S, Lapidus A, Barry K, Detter JC, Hammon N, Israni S, Pitluck S, Thiel J, Schmutz J, Larimer F, Land M, Kyrpides N, Lykidis A, Richardson P: Complete sequence of Prochlorococcus marinus str. MIT. 2005, 9312-UnpublishedGoogle Scholar
- Kettler GC, Martiny AC, Huang K, Zucker J, Coleman ML, Rodrigue S, Chen F, Lapidus A, Ferriera S, Johnson J, et al: Patterns and implications of gene gain and loss in the evolution of Prochlorococcus. PLoS Genet. 2007, 3: e231-10.1371/journal.pgen.0030231.View ArticleGoogle Scholar
- Dufresne A, Salanoubat M, Partensky F, Artiguenave F, Axmann IM, Barbe V, Duprat S, Galperin MY, Koonin EV, Le Gall F, Makarova KS, Ostrowski M, Oztas S, Robert C, Rogozin IB, Scanlan DJ, Tandeau de Marsac N, Weissenbach J, Wincker P, Wolf YI, Hess WR: Genome sequence of the cyanobacterium Prochlorococcus marinus SS120, a nearly minimal oxyphototrophic genome. Proc Natl Acad Sci USA. 2003, 100 (17): 10020-10025. 10.1073/pnas.1733211100.View ArticleGoogle Scholar
- Fuszard MA, Wright PC, Biggs CA: Cellular acclimation strategies of a minimal picocyanobacterium to phosphate stress. FEMS MicrobiolLett. 2010, 306: 127-134. 10.1111/j.1574-6968.2010.01942.x.View ArticleGoogle Scholar
- Pandhal J, Wright PC, Biggs CA: A quantitative proteomic analysis of light adaptation in a globally significant marine cyanobacterium Prochlorococcus marinus MED4. J Proteome Res. 2007, 6: 996-1005. 10.1021/pr060460c.View ArticleGoogle Scholar
- Tetu SG, Brahamsha B, Johnson DA, Tai V, Phillippy K, Palenik B, Paulsen IT: Microarray analysis of phosphate regulation in the marine cyanobacterium Synechococcus sp. WH8102. ISME J. 2009, 3: 835-849. 10.1038/ismej.2009.31.View ArticleGoogle Scholar
- Huber AL, Hamel KS: Phosphatase activities in relation to phosphorus nutrition in Nodularia spumigen (Cyanobacteriaceae). Hydrobiologia. 1985, 123: 81-88. 10.1007/BF00006617.View ArticleGoogle Scholar
- Natesan R, Shanmugasundaram S: Extracellular phosphate solubilization by the cyanobacterium Anabaena ARM310. J Biosci. 1989, 14: 203-208. 10.1007/BF02716680.View ArticleGoogle Scholar
- Scanlan DJ, Mann NH, Carr NG: The response of the picoplanktonic marine cyanobacterium Synechococcus species WH7803 to phosphate starvation involves a protein homologous to the periplasmic phosphate-binding protein of Escherichia coli. Mol Microbiol. 1993, 10: 181-191. 10.1111/j.1365-2958.1993.tb00914.x.View ArticleGoogle Scholar
- Scanlan DJ, West NJ: Molecular ecology of the marine cyanobacterial genera Prochlorococcus and Synechococcus. FEMS Microbiol Ecol. 2002, 40: 1-12. 10.1111/j.1574-6941.2002.tb00930.x.View ArticleGoogle Scholar
- Adams MM, Gomez-Garcia MR, Grossman AR, Bhaya D: Phosphorus Deprivation Responses and Phosphonate Utilization in a Thermophilic Synechococcus sp. from Microbial Mats. J Bacteriol. 2008, 190: 8171-8184. 10.1128/JB.01011-08.View ArticleGoogle Scholar
- Collier JL, Grossman AR: Chlorosis induced by nutrient deprivation in Synechococcus sp. strain PCC 7942: not all bleaching is the same. J Bacteriol. 1992, 174: 4718-4726.Google Scholar
- Moore LR, Post AF, Rocap G, Chisholm SW: Utilization of Different Nitrogen Sources by the Marine Cyanobacteria Prochlorococcus and Synechococcus. LimnolOceanogr. 2002, 47: 989-996.Google Scholar
- Meijer EA, Wijffels RH: Development of a Fast, Reproducible and Effective Method for the Extraction and Quantification of Proteins of Micro-algae. Biotechnol Tech. 1998, 12: 353-358. 10.1023/A:1008814128995.View ArticleGoogle Scholar
- Moore LR, Goericke R, Chisholm SW: Comparative physiology of Synechococcus and Prochlorococcus: Influence of light and temperature on growth, pigments, fluorescence and absorptive properties. Mar Ecol Prog Ser. 1995, 116: 259-276.View ArticleGoogle Scholar
- Ow SY, Noirel J, Cardona T, Taton A, Lindblad P, Stensjo K, Wright PC: Quantitative Overview of N2 Fixation in Nostoc punctiforme ATCC 29133 through Cellular Enrichments and iTRAQ Shotgun Proteomics. Journal Proteome Research. 2009, 8: 187-198. 10.1021/pr800285v.View ArticleGoogle Scholar
- Team RDC: R: A Language and Environment for Statistical Computing. 2011, R Foundation for Statistical Computing: Vienna, AustriaGoogle Scholar
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