Physiological Regulation of Isocitrate Dehydrogenase and the Role of 2-Oxoglutarate in Prochlorococcus sp. Strain PCC 9511

The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2- oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus

Evaluación de métodos de extracción proteica en alevines de chame (Dormitator latifrons) para estudio proteómico

The chame (Dormitator latifrons) is a freshwater fish with aquaculture potential in Latin America. Its production is limited to the use of juveniles captured from the wild. It has been possible to reproduce this species in captivity from broodstock obtained from the natural environment, although in most cases, the process fails in the passage from prolarvae to larvae due to lack of food that contains the necessary nutritional requirements, especially proteins such as some of the main compounds needed by fish. The objective of the research was to evaluate two protein extraction protocols from Dormitator latifrons fingerlings. Fingerling samples were taken from the natural environment, sacrificed by immersion in liquid N2 and stored at -80°C until protein extraction. This work compares the protein concentration and the protein profile, obtained by using two extraction buffers: a traditional RIPA; and ammonium bicarbonate, a volatile salt compatible with mass spectrometry. As a result, a better protein profile is shown in the samples treated with ammonium bicarbonate, which is why this protocol is concluded and recommended for future investigations of the global proteome, by means of mass spectrometry.El chame (Dormitator latifrons), es un pez de agua dulce, con potencial acuícola en Latinoamérica. Su producción, está limitada a la utilización de juveniles capturados del medio silvestre. Se ha logrado reproducir esta especie en cautiverio a partir de reproductores obtenidos del medio natural, aunque en la mayoría de los casos, el proceso fracasa en el paso de prolarvas a larvas por falta de alimento que contenga los requerimientos nutricionales necesarios, especialmente proteínas como unos de los principales compuestos que necesitan los peces. El objetivo de la investigación fue evaluar dos protocolos de extracción de proteínas, a partir de alevines de Dormitator latifrons. Se tomaron muestras de alevines del medio natural, se sacrificaron por inmersión en N2 líquido y conservados a -80°C hasta la extracción de proteínas. Este trabajo compara la concentración de proteína y el perfil proteico, obtenidos mediante el uso de dos tampones de extracción: uno tradicional RIPA; y bicarbonato amónico, una sal volátil compatible con espectrometría de masas. Como resultado se muestra un mejor perfil proteico en las muestras tratadas con bicarbonato amónico, por lo que se concluye y recomienda este protocolo para futuras investigaciones del proteoma global, mediante espectrometría de masas

Glucose uptake in Prochlorococcus: diversity of kinetics and effects on the metabolism

We have previously shown that Prochlorococcus sp. SS120 strain takes up glucose by using a multiphasic transporter encoded by the Pro1404 gene. Here, we studied the glucose uptake kinetics in multiple Prochlorococcus strains from different ecotypes, observing diverse values for the Ks constants (15–126.60 nM) and the uptake rates (0.48–6.36 pmol min-1 mg prot-1). Multiphasic kinetics was observed in all studied strains, except for TAK9803-2. Pro1404 gene expression studies during the 21st Atlantic Meridional Transect cruise showed positive correlation with glucose concentrations in the ocean. This suggests that the Pro1404 transporter has been subjected to diversification along the Prochlorococcus evolution, in a process probably driven by the glucose availabilities at the different niches it inhabits. The glucose uptake mechanism seems to be a primary transporter. Glucose addition induced detectable transcriptomic and proteomic changes in Prochlorococcus SS120, but photosynthetic efficiency was unaffected. Our studies indicate that glucose is actively taken up by Prochlorococcus, but its uptake does not significantly alter the trophic ways of this cyanobacterium, which continues performing photosynthesis. Therefore Prochlorococcus seems to remain acting as a fundamentally phototrophic organism, capable of using glucose as an extra resource of carbon and energy when available in the environment

Molecular complexity of the major urinary protein system of the Norway rat, Rattus norvegicus

ABSTRACT Major urinary proteins (MUP) are the major component of the urinary protein fraction in house mice ( Mus spp.) and rats ( Rattus spp.). The structure, polymorphism and functions of these lipocalins have been well described in the western European house mouse ( Mus musculus domesticus ), clarifying their role in semiochemical communication. The complexity of these roles in the mouse raises the question of similar functions in other rodents, including the Norway rat, Rattus norvegicu s. Norway rats express MUPs in urine but information about specific MUP isoform sequences and functions is limited. In this study, we present a detailed molecular characterization of the MUP proteoforms expressed in the urine of two laboratory strains, Wistar Han and Brown Norway, and wild caught animals, using a combination of manual gene annotation, intact protein mass spectrometry and bottom-up mass spectrometry-based proteomic approaches. Detailed sequencing of the proteins reveals a less complex pattern of primary sequence polymorphism than the mouse. However, unlike the mouse, rat MUPs exhibit added complexity in the form of post-translational modifications including phosphorylation and exoproteolytic trimming of specific isoforms. The possibility that urinary MUPs may have different roles in rat chemical communication than those they play in the house mouse is also discussed

Molecular complexity of the major urinary protein system of the Norway rat, <i>Rattus norvegicus</i>

ABSTRACTMajor urinary proteins (MUP) are the major component of the urinary protein fraction in house mice (Mus spp.) and rats (Rattus spp.). The structure, polymorphism and functions of these lipocalins have been well described in the western European house mouse (Mus musculus domesticus), clarifying their role in semiochemical communication. The complexity of these roles in the mouse raises the question of similar functions in other rodents, including the Norway rat, Rattus norvegicus. Norway rats express MUPs in urine but information about specific MUP isoform sequences and functions is limited. In this study, we present a detailed molecular characterization of the MUP proteoforms expressed in the urine of two laboratory strains, Wistar Han and Brown Norway, and wild caught animals, using a combination of manual gene annotation, intact protein mass spectrometry and bottom-up mass spectrometry-based proteomic approaches. Detailed sequencing of the proteins reveals a less complex pattern of primary sequence polymorphism than the mouse. However, unlike the mouse, rat MUPs exhibit added complexity in the form of post-translational modifications including phosphorylation and exoproteolytic trimming of specific isoforms. The possibility that urinary MUPs may have different roles in rat chemical communication than those they play in the house mouse is also discussed.</jats:p

Glucose uptake and its effect on gene expression in prochlorococcus

11 pages, 6 figures.-- PMID: 18941506 [PubMed].-- PMCID: PMC2565063.-- Supplementary information (figs. S1-S2) available.The marine cyanobacteria Prochlorococcus have been considered photoautotrophic microorganisms, although the utilization of exogenous sugars has never been specifically addressed in them. We studied glucose uptake in different high irradiance- and low irradiance-adapted Prochlorococcus strains, as well as the effect of glucose addition on the expression of several glucose-related genes. Glucose uptake was measured by adding radiolabelled glucose to Prochlorococcus cultures, followed by flow cytometry coupled with cell sorting in order to separate Prochlorococcus cells from bacterial contaminants. Sorted cells were recovered by filtration and their radioactivity measured. The expression, after glucose addition, of several genes (involved in glucose metabolism, and in nitrogen assimilation and its regulation) was determined in the low irradiance-adapted Prochlorococcus SS120 strain by semi-quantitative real time RT-PCR, using the rnpB gene as internal control. Our results demonstrate for the first time that the Prochlorococcus strains studied in this work take up glucose at significant rates even at concentrations close to those found in the oceans, and also exclude the possibility of this uptake being carried out by eventual bacterial contaminants, since only Prochlorococcus cells were used for radioactivity measurements. Besides, we show that the expression of a number of genes involved in glucose utilization (namely zwf, gnd and dld, encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and lactate dehydrogenase, respectively) is strongly increased upon glucose addition to cultures of the SS120 strain. This fact, taken together with the magnitude of the glucose uptake, clearly indicates the physiological importance of the phenomenon. Given the significant contribution of Prochlorococcus to the global primary production, these findings have strong implications for the understanding of the phytoplankton role in the carbon cycle in nature. Besides, the ability of assimilating carbon molecules could provide additional hints to comprehend the ecological success of Prochlorococcus.This work was supported by grants BMC2003-09218-CO2-01, BMC2003-09218-CO2-02 and BFU2006-10011/BMC from Spanish Ministerio de Educación y Ciencia (MEC, co-funded by the European Social Fund from the European Union), Universidad de Córdoba and Junta de Andalucía (JA). G.G.-B., A.L.-L. and J.G.-M. were recipients of fellowships from JA and MEC and Spanish Consejo Superior de Investigaciones Científicas.Peer reviewe

Nitrogen starvation induces extensive changes in the redox proteome of Prochlorococcus sp. strain SS120

Very low nitrogen concentration is a critical limitation in the oligotrophic oceans inhabited by the cyanobacterium Prochlorococccus, one of the main primary producers on Earth. It is well known that nitrogen starvation affects redox homeostasis in cells. We have studied the effect of nitrogen starvation on the thiol redox proteome in the Prochlorococcus sp. SS120 strain, by using shotgun proteomic techniques to map the cysteine modified in each case and to quantify the ratio of reversibly oxidized/reduced species. We identified a number of proteins showing modified cysteines only under either control or N-starvation, including isocitrate dehydrogenase and ribulose phosphate 3-epimerase. We detected other key enzymes, such as glutamine synthetase, transporters and transaminases, showing that nitrogen-related pathways were deeply affected by nitrogen starvation. Reversibly oxidized cysteines were also detected in proteins of other important metabolic pathways, such as photosynthesis, phosphorus metabolism, ATP synthesis and nucleic acids metabolism. Our results demonstrate a wide effect of nitrogen limitation on the redox status of the Prochlorococcus proteome, suggesting that besides previously reported transcriptional changes, this cyanobacterium responds with post-translational redox changes to the lack of nitrogen in its environment

Regulatory and metabolic adaptations in the nitrogen assimilation of marine picocyanobacteria

Prochlorococcus and Synechococcus are the two most abundant photosynthetic organisms on Earth, with a strong influence on the biogeochemical carbon and nitrogen cycles. Early reports demonstrated the streamlining of regulatory mechanisms in nitrogen metabolism and the removal of genes not strictly essential. The availability of a large series of genomes, and the utilization of latest generation molecular techniques have allowed elucidating the main mechanisms developed by marine picocyanobacteria to adapt to the environments where they thrive, with a particular interest in the strains inhabiting oligotrophic oceans. Given that nitrogen is often limited in those environments, a series of studies have explored the strategies utilized by Prochlorococcus and Synechococcus to exploit the low concentrations of nitrogen-containing molecules available in large areas of the oceans. These strategies include the reduction in the GC and the cellular protein contents; the utilization of truncated proteins; a reduced average amount of N in the proteome; the development of metabolic mechanisms to perceive and utilize nanomolar nitrate concentrations; and the reduced responsiveness of key molecular regulatory systems such as NtcA to 2-oxoglutarate. These findings are in sharp contrast with the large body of knowledge obtained in freshwater cyanobacteria. We will outline the main discoveries, stressing their relevance to the ecological success of these important microorganisms

Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511.

Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed