Life-long epigenetic programming of cortical architecture by maternal 'Western' diet during pregnancy.

The evolution of human diets led to preferences toward polyunsaturated fatty acid (PUFA) content with 'Western' diets enriched in ω-6 PUFAs. Mounting evidence points to ω-6 PUFA excess limiting metabolic and cognitive processes that define longevity in humans. When chosen during pregnancy, ω-6 PUFA-enriched 'Western' diets can reprogram maternal bodily metabolism with maternal nutrient supply precipitating the body-wide imprinting of molecular and cellular adaptations at the level of long-range intercellular signaling networks in the unborn fetus. Even though unfavorable neurological outcomes are amongst the most common complications of intrauterine ω-6 PUFA excess, cellular underpinnings of life-long modifications to brain architecture remain unknown. Here, we show that nutritional ω-6 PUFA-derived endocannabinoids desensitize CB1 cannabinoid receptors, thus inducing epigenetic repression of transcriptional regulatory networks controlling neuronal differentiation. We found that cortical neurons lose their positional identity and axonal selectivity when mouse fetuses are exposed to excess ω-6 PUFAs in utero. Conversion of ω-6 PUFAs into endocannabinoids disrupted the temporal precision of signaling at neuronal CB1 cannabinoid receptors, chiefly deregulating Stat3-dependent transcriptional cascades otherwise required to execute neuronal differentiation programs. Global proteomics identified the immunoglobulin family of cell adhesion molecules (IgCAMs) as direct substrates, with DNA methylation and chromatin accessibility profiling uncovering epigenetic reprogramming at >1400 sites in neurons after prolonged cannabinoid exposure. We found anxiety and depression-like behavioral traits to manifest in adult offspring, which is consistent with genetic models of reduced IgCAM expression, to suggest causality for cortical wiring defects. Overall, our data uncover a regulatory mechanism whose disruption by maternal food choices could limit an offspring's brain function for life.

Impact of DICER1 and DROSHA on the Angiogenic Capacity of Human Endothelial Cells.

RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients.

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

A secretagogin locus of the mammalian hypothalamus controls stress hormone release.

A hierarchical hormonal cascade along the hypothalamic-pituitary-adrenal axis orchestrates bodily responses to stress. Although corticotropin-releasing hormone (CRH), produced by parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and released into the portal circulation at the median eminence, is known to prime downstream hormone release, the molecular mechanism regulating phasic CRH release remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single-cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH-releasing neuron population reliant on secretagogin's Ca(2+) sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. Pharmacological tools combined with RNA interference demonstrate that secretagogin's loss of function occludes adrenocorticotropic hormone release from the pituitary and lowers peripheral corticosterone levels in response to acute stress. Cumulatively, these data define a novel secretagogin neuronal locus and molecular axis underpinning stress responsiveness.

Stathmin is enriched in the developing corticospinal tract.

Understanding the intra- and extracellular proteins involved in the development of the corticospinal tract (CST) may offer insights into how the pathway could be regenerated following traumatic spinal cord injury. Currently, however, little is known about the proteome of the developing corticospinal system. The present study, therefore, has used quantitative proteomics and bioinformatics to detail the protein profile of the rat CST during its formation in the spinal cord. This analysis identified increased expression of 65 proteins during the early ingrowth of corticospinal axons into the spinal cord, and 36 proteins at the period of heightened CST growth. A majority of these proteins were involved in cellular assembly and organization, with annotations being most highly associated with cytoskeletal organization, microtubule dynamics, neurite outgrowth, and the formation, polymerization and quantity of microtubules. In addition, 22 proteins were more highly expressed within the developing CST in comparison to other developing white matter tracts of the spinal cord of age-matched animals. Of these differentially expressed proteins, only one, stathmin 1 (a protein known to be involved in microtubule dynamics), was both highly enriched in the developing CST and relatively sparse in other developing descending and ascending spinal tracts. Immunohistochemical analyses of the developing rat spinal cord and fetal human brain stem confirmed the enriched pattern of stathmin expression along the developing CST, and in vitro growth assays of rat corticospinal neurons showed a reduced length of neurite processes in response to pharmacological perturbation of stathmin activity. Combined, these findings suggest that stathmin activity may modulate axonal growth during development of the corticospinal projection, and reinforces the notion that microtubule dynamics could play an important role in the generation and regeneration of the CST.

ND3, ND1 and 39kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I.

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I+III2+IV (S1) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.

An investigation into membrane bound redox carriers involved in energy transduction mechanism in Brevibacterium linens DSM 20158 with unsequenced genome.

Brevibacterium linens (B. linens) DSM 20158 with an unsequenced genome can be used as a non-pathogenic model to study features it has in common with other unsequenced pathogens of the same genus on the basis of comparative proteome analysis. The most efficient way to kill a pathogen is to target its energy transduction mechanism. In the present study, we have identified the redox protein complexes involved in the electron transport chain of B. linens DSM 20158 from their clear homology with the shot-gun genome sequenced strain BL2 of B. linens by using the SDS-Polyacrylamide gel electrophoresis coupled with nano LC-MS/MS mass spectrometry. B. linens is found to have a branched electron transport chain (Respiratory chain), in which electrons can enter the respiratory chain either at NADH (Complex I) or at Complex II level or at the cytochrome level. Moreover, we are able to isolate, purify, and characterize the membrane bound Complex II (succinate dehydrogenase), Complex III (menaquinone cytochrome c reductase cytochrome c subunit, Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) of B. linens strain DSM 20158.

An enzymatic route to selenazolines.

Ringing the changes: Selenazolines have applications in medicinal chemistry, but their synthesis is challenging. We report a new convenient and less toxic route to these heterocycles that starts from commercially available selenocysteine. The new route depends on a heterocyclase enzyme that creates oxazolines and thiazolines from serines/threonines and cysteines.

A functional approach to uncover the low-temperature adaptation strategies of the archaeon Methanosarcina barkeri.

Low-temperature anaerobic digestion (LTAD) technology is underpinned by a diverse microbial community. The methanogenic archaea represent a key functional group in these consortia, undertaking CO2 reduction as well as acetate and methylated C1 metabolism with subsequent biogas (40 to 60% CH4 and 30 to 50% CO2) formation. However, the cold adaptation strategies, which allow methanogens to function efficiently in LTAD, remain unclear. Here, a pure-culture proteomic approach was employed to study the functional characteristics of Methanosarcina barkeri (optimum growth temperature, 37°C), which has been detected in LTAD bioreactors. Two experimental approaches were undertaken. The first approach aimed to characterize a low-temperature shock response (LTSR) of M. barkeri DSMZ 800(T) grown at 37°C with a temperature drop to 15°C, while the second experimental approach aimed to examine the low-temperature adaptation strategies (LTAS) of the same strain when it was grown at 15°C. The latter experiment employed cell viability and growth measurements (optical density at 600 nm [OD600]), which directly compared M. barkeri cells grown at 15°C with those grown at 37°C. During the LTSR experiment, a total of 127 proteins were detected in 37°C and 15°C samples, with 20 proteins differentially expressed with respect to temperature, while in the LTAS experiment 39% of proteins identified were differentially expressed between phases of growth. Functional categories included methanogenesis, cellular information processing, and chaperones. By applying a polyphasic approach (proteomics and growth studies), insights into the low-temperature adaptation capacity of this mesophilically characterized methanogen were obtained which suggest that the metabolically diverse Methanosarcinaceae could be functionally relevant for LTAD systems.

The pollen-specific class VIII-myosin ATM2 from Arabidopsis thaliana associates with the plasma membrane through a polybasic region binding anionic phospholipids.

Pollen tubes display polarized tip-growth and are a model to study the coordination of vesicular trafficking and cytoskeletal control. The molecular details of how dynamic actin filaments associate with the plasma membrane are currently unclear. In Arabidopsis thaliana, plasma membrane attachment of actin filaments may be mediated by four myosins representing the plant-specific myosin-subclass VIII, which localize to the plasma membrane and display only minor motor-activity. Here we explore the mode of membrane attachment of the pollen-expressed class VIII-myosins ATM2 and VIII-B through interaction with anionic membrane phospholipids. A fluorescent mCherry-ATM2-fusion decorated plasma membrane-peripheral actin filaments when expressed in tobacco pollen tubes, consistent with a role of class VIII-myosins at the membrane-cytoskeleton interface. As recombinant proteins, class VIII-myosins are prone to aggregation and to proteolysis, creating a challenge for their biochemical characterization. We describe a purification scheme for guanidinium chloride (GdmCl)-denatured recombinant proteins, followed by a renaturation protocol to obtain pure, soluble protein fragments of ATM2 and VIII-B. The fragments represent the C-terminal tail and coiled-coil-regions and lack the N-terminal actin-binding regions, IQ or motor domains. Based on lipid-overlays and liposome-sedimentation assays, the fragments of ATM2 and VIII-B bind anionic phospholipids. Small polybasic regions at the extreme C-termini were sufficient for lipid-binding of the respective protein fragments. When expressed in tobacco pollen tubes, a fluorescence-tagged variant of ATM2 lacking its lipid-binding region displayed substantially reduced plasma membrane association. The data indicate that class VIII-myosins may facilitate actin-plasma membrane attachment through interaction with anionic phospholipids, mediated by polybasic C-terminal lipid-binding domains.

miR-208b Reduces the Expression of Kcnj5 in a Cardiomyocyte Cell Line.

MicroRNAs (miRs) contribute to different aspects of cardiovascular pathology, among them cardiac hypertrophy and atrial fibrillation. Cardiac miR expression was analyzed in a mouse model with structural and electrical remodeling. Next-generation sequencing revealed that miR-208b-3p was ~25-fold upregulated. Therefore, the aim of our study was to evaluate the impact of miR-208b on cardiac protein expression. First, an undirected approach comparing whole RNA sequencing data to miR-walk 2.0 miR-208b 3'-UTR targets revealed 58 potential targets of miR-208b being regulated. We were able to show that miR-208b mimics bind to the 3' untranslated region (UTR) of voltage-gated calcium channel subunit alpha1 C and Kcnj5, two predicted targets of miR-208b. Additionally, we demonstrated that miR-208b mimics reduce GIRK1/4 channel-dependent thallium ion flux in HL-1 cells. In a second undirected approach we performed mass spectrometry to identify the potential targets of miR-208b. We identified 40 potential targets by comparison to miR-walk 2.0 3'-UTR, 5'-UTR and CDS targets. Among those targets, Rock2 and Ran were upregulated in Western blots of HL-1 cells by miR-208b mimics. In summary, miR-208b targets the mRNAs of proteins involved in the generation of cardiac excitation and propagation, as well as of proteins involved in RNA translocation (Ran) and cardiac hypertrophic response (Rock2).

The rise and fall of major royal jelly proteins during a honeybee (Apis mellifera) workers' life.

The genome of the western honeybee (Apis mellifera) harbors nine transcribed major royal jelly protein genes (mrjp1-9) which originate from a single-copy precursor via gene duplication. The first MRJP was identified in royal jelly, a secretion of the bees' hypopharyngeal glands that is used by young worker bees, called nurses, to feed developing larvae. Thus, MRJPs are frequently assumed to mainly have functions for developing bee larvae and to be expressed in the food glands of nurse bees. In-depth knowledge on caste- and age-specific role and abundance of MRJPs is missing. We here show, using combined quantitative real-time PCR with quantitative mass spectrometry, that expression and protein amount of mrjp1-5 and mrjp7 show an age-dependent pattern in worker's hypopharyngeal glands as well as in brains, albeit lower relative abundance in brains than in glands. Expression increases after hatching until the nurse bee period and is followed by a decrease in older workers that forage for plant products. Mrjp6 expression deviates considerably from the expression profiles of the other mrjps, does not significantly vary in the brain, and shows its highest expression in the hypopharyngeal glands during the forager period. Furthermore, it is the only mrjp of which transcript abundance does not correlate with protein amount. Mrjp8 and mrjp9 show, compared to the other mrjps, a very low expression in both tissues. Albeit mrjp8 mRNA was detected via qPCR, the protein was not quantified in any of the tissues. Due to the occurrence of MRJP8 and MRJP9 in other body parts of the bees, for example, the venom gland, they might not have a hypopharyngeal gland- or brain-specific function but rather functions in other tissues. Thus, mrjp1-7 but not mrjp8 and mrjp9 might be involved in the regulation of phenotypic plasticity and age polyethism in worker honeybees.

The Effect of Diet on the Composition and Stability of Proteins Secreted by Honey Bees in Honey.

Honey proteins are essential bee nutrients and antimicrobials that protect honey from microbial spoilage. The majority of the honey proteome includes bee-secreted peptides and proteins, produced in specialised glands; however, bees need to forage actively for nitrogen sources and other basic elements of protein synthesis. Nectar and pollen of different origins can vary significantly in their nutritional composition and other compounds such as plant secondary metabolites. Worker bees producing and ripening honey from nectar might therefore need to adjust protein secretions depending on the quality and specific contents of the starting material. Here, we assessed the impact of different food sources (sugar solutions with different additives) on honey proteome composition and stability, using controlled cage experiments. Honey-like products generated from sugar solution with or without additional protein, or plant secondary metabolites, differed neither in protein quality nor in protein quantity among samples. Storage for 4 weeks prevented protein degradation in most cases, without differences between food sources. The honey-like product proteome included several major royal jelly proteins, alpha-glucosidase and glucose oxidase. As none of the feeding regimes resulted in different protein profiles, we can conclude that worker bees may secrete a constant amount of each bee-specific protein into honey to preserve this highly valuable hive product.

Crystal structures of the mitochondrial deacylase Sirtuin 4 reveal isoform-specific acyl recognition and regulation features.

Sirtuins are evolutionary conserved NAD+-dependent protein lysine deacylases. The seven human isoforms, Sirt1-7, regulate metabolism and stress responses and are considered therapeutic targets for aging-related diseases. Sirt4 locates to mitochondria and regulates fatty acid metabolism and apoptosis. In contrast to the mitochondrial deacetylase Sirt3 and desuccinylase Sirt5, no prominent deacylase activity and structural information are available for Sirt4. Here we describe acyl substrates and crystal structures for Sirt4. The enzyme shows isoform-specific acyl selectivity, with significant activity against hydroxymethylglutarylation. Crystal structures of Sirt4 from Xenopus tropicalis reveal a particular acyl binding site with an additional access channel, rationalizing its activities. The structures further identify a conserved, isoform-specific Sirt4 loop that folds into the active site to potentially regulate catalysis. Using these results, we further establish efficient Sirt4 activity assays, an unusual Sirt4 regulation by NADH, and Sirt4 effects of pharmacological modulators.

Low-temperature anaerobic digestion is associated with differential methanogenic protein expression.

Anaerobic digestion (AD) is an attractive wastewater treatment technology, leading to the generation of recoverable biofuel (methane). Most industrial AD applications, carry excessive heating costs, however, as AD reactors are commonly operated at mesophilic temperatures while handling waste streams discharged at ambient or cold temperatures. Consequently, low-temperature AD represents a cost-effective strategy for wastewater treatment. The comparative investigation of key microbial groups underpinning laboratory-scale AD bioreactors operated at 37, 15 and 7°C was carried out. Community structure was monitored using 16S rRNA clone libraries, while abundance of the most prominent methanogens was investigated using qPCR. In addition, metaproteomics was employed to access the microbial functions carried out in situ. While δ-Proteobacteria were prevalent at 37°C, their abundance decreased dramatically at lower temperatures with inverse trends observed for Bacteroidetes and Firmicutes. Methanobacteriales and Methanosaeta were predominant at all temperatures investigated while Methanomicrobiales abundance increased at 15°C compared to 37 and 7°C. Changes in operating temperature resulted in the differential expression of proteins involved in methanogenesis, which was found to occur in all bioreactors, as corroborated by bioreactors' performance. This study demonstrated the value of employing a polyphasic approach to address microbial community dynamics and highlighted the functional redundancy of AD microbiomes.

Functional responses and adaptation of mesophilic microbial communities to psychrophilic anaerobic digestion.

Psychrophilic (<20°C) anaerobic digestion (AD) represents an attractive alternative to mesophilic wastewater treatment. In order to investigate the AD microbiome response to temperature change, with particular emphasis on methanogenic archaea, duplicate laboratory-scale AD bioreactors were operated at 37°C followed by a temperature drop to 15°C. A volatile fatty acid-based wastewater (composed of propionic acid, butyric acid, acetic acid and ethanol) was used to provide substrates representing the later stages of AD. Community structure was monitored using 16S rRNA gene clone libraries, as well as DNA and cDNA-based DGGE analysis, while the abundance of relevant methanogens was followed using qPCR. In addition, metaproteomics, microautoradiography-fluorescence in situ hybridization, and methanogenic activity measurements were employed to investigate microbial activities and functions. Methanomicrobiales abundance increased at low temperature, which correlated with an increased contribution of CH4 production from hydrogenotrophic methanogenesis at 15°C. Methanosarcinales utilized acetate and H2/CO2 as CH4 precursors at both temperatures and a partial shift from acetoclastic to hydrogenotrophic methanogenesis was observed for this archaeal population at 15°C. An upregulation of protein expression was reported at low temperature as well as the detection of chaperones indicating that mesophilic communities experienced stress during long-term exposure to 15°C. Overall, changes in microbial community structure and function were found to underpin the adaptation of mesophilic sludge to psychrophilic AD.

Charting the cellular and extracellular proteome analysis of Brevibacterium linens DSM 20158 with unsequenced genome by mass spectrometry-driven sequence similarity searches.

Brevibacterium linens DSM 20158 is an industrially important actinobacterium which is well-known for the production of amino acids and enzymes. However, as this strain has an unsequenced genome, there is no detailed information regarding its proteome although another strain of this microbe, BL2, has a shotgun genome sequence. However, this still does not cover the entire scope of its proteome. The present study is carried out by first identifying proteins by homology matches using the Mascot search algorithm followed by an advanced approach using de novo sequencing and MS BLAST to expand the B. linens proteome. The proteins identified in the secretome and cellular portion appear to be involved in various metabolic and physiological processes of this unsequenced organism. This study will help to enhance the usability of this strain of B. linens in different areas of research in the future rather than mainly in the food industries. BIOLOGICAL SIGNIFICANCE: The present study describes the construction of the first detailed proteomic reference map of B. linens DSM 20158 with unsequenced genome by comparative proteome research analysis. This opens new horizons in proteomics to understand the role of proteins involved in the metabolism and physiology of other organisms with unsequenced genomes.

Conformation-specific crosslinking of mitochondrial complex I.

Complex I is the only component of the eukaryotic respiratory chain of which no high-resolution structure is yet available. A notable feature of mitochondrial complex I is the so-called active/de-active conformational transition of the idle enzyme from the active (A) to the de-active, (D) form. Using an amine- and sulfhydryl-reactive crosslinker of 6.8Å length (SPDP) we found that in the D-form of complex I the ND3 subunit crosslinked to the 39 kDa (NDUFA9) subunit. These proteins could not be crosslinked in the A-form. Most likely, both subunits are closely located in the critical junction region connecting the peripheral hydrophilic domain to the membrane arm of the enzyme where the entrance path for substrate ubiquinone is and where energy transduction takes place.

The quantitative proteomic response of Synechocystis sp. PCC6803 to phosphate acclimation.

BACKGROUND: Inorganic phosphate (Pi) is a critical nutrient for all life and is periodically limiting in marine and freshwater provinces, yet little is understood how organisms acclimate to fluctuations in Pi within their environment. To investigate whole cell adaptation, we grew Synechocystis sp. PCC6803, a model freshwater cyanobacterium, in 3%, and 0.3% inorganic phosphate (Pi) media. The cells were allowed to acclimate over 60 days, and cells were harvested for quantitative high throughput mass spectrometry-based proteomics using the iTRAQ™ labelling technology. RESULTS: In total, 120 proteins were identified, and 52 proteins were considered differentially abundant compared to the control. Alkaline phosphatase (APase) activities correlated significantly (p < 0.05) with observed relative PhoA abundances. PstS1 and PstS2 were both observed, yet PstS1 was not differentially more abundant than the control. Phycobilisome protein abundances appeared to be coordinated, and are significantly less abundant in 0.3% Pi than 3% Pi cultures. Also, the central metabolic cell function appears to have shifted towards the production of (NADPH) reducing energy and nucleotide sugars. CONCLUSIONS: This acclimation response bears strong similarity to the previously reported response to nitrogen deprivation within Synechocystis sp. PCC 6803. However, it also demonstrates some characteristics of desiccation stress, such as the regulation of fatty acids and increased abundance of rehydrin in the 3% Pi culture.

Comparative quantitative proteomics of prochlorococcus ecotypes to a decrease in environmental phosphate concentrations.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.