Marisol Fernández

Contribution of crenarchaeal autotrophic ammonia oxidizers to the dark primary production in Tyrrhenian deep waters (Central Mediterranean Sea)

Mesophilic Crenarchaeota have recently been thought to be significant contributors to nitrogen (N) and carbon (C) cycling. In this study, we examined the vertical distribution of ammonia-oxidizing Crenarchaeota at offshore site in Southern Tyrrhenian Sea. The median value of the crenachaeal cell to amoA gene ratio was close to one suggesting that virtually all deep-sea Crenarchaeota possess the capacity to oxidize ammonia. Crenarchaea-specific genes, nirK and ureC, for nitrite reductase and urease were identified and their affiliation demonstrated the presence of ‘deep-sea’ clades distinct from ‘shallow’ representatives. Measured deep-sea dark CO2 fixation estimates were comparable to the median value of photosynthetic biomass production calculated for this area of Tyrrhenian Sea, pointing to the significance of this process in the C cycle of aphotic marine ecosystems. To elucidate the pivotal organisms in this process, we targeted known marine crenarchaeal autotrophy-related genes, coding for acetyl-CoA carboxylase (accA) and 4-hydroxybutyryl-CoA dehydratase (4-hbd). As in case of nirK and ureC, these genes are grouped with deep-sea sequences being distantly related to those retrieved from the epipelagic zone. To pair the molecular data with specific functional attributes we performed [14C]HCO3 incorporation experiments followed by analyses of radiolabeled proteins using shotgun proteomics approach. More than 100 oligopeptides were attributed to 40 marine crenarchaeal-specific proteins that are involved in 10 different metabolic processes, including autotrophy. Obtained results provided a clear proof of chemolithoautotrophic physiology of bathypelagic crenarchaeota and indicated that this numerically predominant group of microorganisms facilitate a hitherto unrecognized sink for inorganic C of a global importance.

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis—the paradigm of mesophilic hydrocarbonoclastic bacteria—O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.

Nuclease activities and cell death processes associated with the development of surface cultures of Streptomyces antibioticus ETH 7451

The presence and significance of developmentally regulated nucleases in Streptomyces antibioticus ETH 7451 has been studied in relation to the lytic processes occurring during differentiation. The cell-death processes have been followed in surface cultures by a propidium iodide viability assay. This has allowed the visualization of dead (membrane-damaged, red fluorescent) and live (membrane-intact, green fluorescent) mycelium during development, and has facilitated the analysis of the role of nucleases in these processes. A parallel activity-gel analysis showed the appearance of 20-22 kDa, 34 kDa and 44 kDa nucleases, the latter appearing only when aerial mycelium is formed. The appearance of these nucleases shows a remarkable correlation with the death process of the mycelium during differentiation and with chromosomal DNA degradation. The 20-22 kDa enzymes are possibly related to the lytic phenomena taking place in the vegetative substrate mycelium before the emergence of the reproductive aerial mycelium, whereas the function of the 44 kDa nuclease seems to be related to the sporulation step. The 20-22 kDa nucleases require Ca2+ for activity and are inhibited by Zn2+. The nucleases are loosely bound to the cell wall from where they can be liberated by simple washing. Conceivably, these enzymes work together and co-ordinate to achieve an efficient hydrolysis of DNA from dying cells. The results show that the biochemical reactions related with the lytic DNA degradation during the programmed cell death are notably conserved in Streptomyces. Some of the features of the process and the biochemical characteristics of the enzymes involved are analogous to those taking place during the DNA fragmentation processes in eukaryotic apoptotic cells.

SPARC Promotes Cathepsin B-Mediated Melanoma Invasiveness through a Collagen I/α2β1 Integrin Axis

In melanoma, the extracellular protein SPARC (secreted protein acidic and rich in cysteine) is related to tumor progression. Some of the evidence that links SPARC to melanoma progression indicates that SPARC may be involved in the acquisition of mesenchymal traits that favor metastatic dissemination. However, no molecular pathways that link extracellular SPARC to a mesenchymal phenotype have been described. In this study, global protein expression analysis of the melanoma secretome following enforced downregulation of SPARC expression led us to elucidate a new molecular mechanism by which SPARC promotes cathepsin B-mediated melanoma invasiveness using collagen I and α2β1 integrins as mediators. Interestingly, we also found that the transforming growth factor (TGF)-β1 contribution to cathepsin B-mediated invasion is highly SPARC dependent. In addition, induction of the E-cadherin to N-cadherin switch by SPARC enabled melanoma cells to transmigrate across an endothelial layer through a mechanism independent to that of enhancing invasion. Finally, SPARC also enhanced the extracellular expression of other proteins involved in epithelial-mesenchymal transformation, such as family with sequence similarity 3, member C/interleukin-like EMT-inducer. Our findings demonstrate a previously unreported molecular pathway for SPARC activity on invasion and support an active role of SPARC in the mesenchymal transformation that contributes to melanoma dissemination.

Evaluation of isotope-coded protein labeling (ICPL) in the quantitative analysis of complex proteomes

An evaluation of the ICPL (isotope-coded protein labeling) non-isobaric labeling technique was performed using two different biological models. Two samples containing phage T4 capsids were mixed in a 1:1 ratio after being labeled with the light or heavy versions of the ICPL reagent. The analysis of this proteome demonstrated the feasibility of this approach for differential quantitative proteomics and was employed to optimize the experimental parameters of the ICPL workflow. ICPL-mediated analysis of two more complex proteomes, those of a Salmonella enterica serovar Typhimurium virulent strain and an isogenic attenuated mutant, and its comparison with the results obtained in a 2D-PAGE classical approach confirmed that ICPL is a valuable alternative to other labeling techniques currently in use. In addition, our results suggest that labeling at the peptide level instead of following the standard ICPL workflow should increase both the number of proteins quantified and the reliability of the quantification.

Mycelium development in Streptomyces antibioticus ATCC11891 occurs in an orderly pattern which determines multiphase growth curves

BackgroundThe current model for the developmental cycle of Streptomyces confluent cultures on agar surface is based on the assumption that the only differentiation takes place along the transverse axis (bottom-up): a vegetative (substrate) mycelium grows completely live and viable on the surface and inside the agar until it undergoes a death process and differentiates to a reproductive (aerial) mycelium which grows into the air. Hence, this vertical description assumes that the development in the pre-sporulating phases is more or less homogeneous in all zones of the plate surface.ResultsThe work presents a detailed analysis of the differentiation cycle in Streptomyces antibioticus ATCC11891 considering a different spatial dimension: the longitudinal axes, represented by the plate surface. A previously unsuspected complexity during the substrate mycelial phase was detected. We have demonstrated that the young substrate hyphae suffer an early death round that has not been previously described. Subsequently, the remaining mycelium grows in successive waves which vary according to the density of the spore inoculum. In the presence of dense inocula (1.5 × 106 spores per plate), the hyphae develop in regular circles, approximately 0.5 cm in diameter. By contrast, with highly diluted inocula (6 × 103 spores per plate), aerial mycelium develops initially in the form of islands measuring 0.9 mm in diameter. Further mycelial development occurs between the circles or islands until the plate surface is totally covered. This pattern persists throughout the entire developmental cycle including the sporulation phases.ConclusionAn early death round during the substrate mycelial phase of Streptomyces antibioticus ATCC11891 takes place prior to successive growth periods in surface cultures. These developmental periods in turn, determine the shape of the complex multiphase growth curves observed. As shown here, these results also apply to other Streptomyces strains and species. Understanding these peculiarities of the Streptomyces developmental cycle is essential in order to properly interpret the morphological/biochemical data obtained from solid cultures and will expand the number of potential phenotypes subject to study.

Viability staining and terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling of the mycelium in submerged cultures of Streptomyces antibioticus ETH7451.

Viability stain and terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling (TUNEL) have been applied to submerged cultures of Streptomyces antibioticus ETH7451, the last technique after a suitable permeabilization treatment. Areas of dead mycelium can be clearly delineated by the viability stain within the network of hyphae which forms the mycelial masses characteristic of the submerged cultures. In addition, the TUNEL reaction shows that DNA fragmentation accompanies the death processes in the mycelium. These techniques permit the investigation of the influence of the medium and nutritional conditions on the viability of the cells. This has relevant biotechnological implications for the study of these important filamentous bacteria in the industrial fermentation processes. These techniques also allow a straight forward analysis of the physical and chemical reagents which provoke damage in Streptomyces DNA.

Proteomics identifies ubiquitin–proteasome targets and new roles for chromatin-remodeling in the Arabidopsis response to phosphate starvation

UNLABELLEDnIn order to identify new regulators of the phosphate (Pi) starvation signaling pathway in plants, we analyzed variation in the abundance of nuclear-enriched proteins isolated from Arabidopsis roots that depends on Pi supply. We used 2-D fluorescence difference gel electrophoresis and MALDI-TOF/TOF techniques for proteome separation, visualization and relative protein abundance quantification and identification. Pi-controlled proteins identified in our analysis included components of the chromatin remodeling, DNA replication, and mRNA splicing machineries. In addition, by combining Pi starvation conditions with proteasome inhibitor treatments, we characterized the role of the ubiquitin-proteasome system, a major mechanism for targeted protein degradation in eukaryotes, in the control of the stability of Pi-responsive proteins. Among Pi-responsive proteins, the histone chaperone NAP1;2 was selected for further characterization, and was shown to display differential nucleo-cytoplasmic accumulation in response to Pi deprivation. We also found that mutants affecting three members of the NAP1 family accumulate lower Pi levels and display reduced expression of Pi starvation-inducible genes, reflecting a potential regulatory role for these chromatin-remodeling proteins in Pi homeostasis.nnnBIOLOGICAL SIGNIFICANCEnIn this study, we explore the feasibility of nuclear proteomics to identify regulatory proteins and ubiquitin-proteasome targets within a specific stress signaling pathway in plants, in our case phosphate starvation signaling in Arabidopsis. It will be of interest for researchers involved in the dissection of any signaling pathway in plants, in particular those with an interest in the ubiquitin-proteasome functions, and for the plant nutrition community.

Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera

JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori.

The functional characterization of Open Reading Frames (ORFs) from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem. We present here the biochemical analysis of six proteins from Helicobacter pylori ATCC 26695, a focus organism in COMBREX. Initial hypotheses were based upon affinity capture of proteins from total cellular lysate using derivatized nano-particles, and subsequent identification by mass spectrometry. Candidate genes encoding these proteins were cloned and expressed in Escherichia coli, and the recombinant proteins were purified and characterized biochemically and their biochemical parameters compared with the native ones. These proteins include a guanosine triphosphate (GTP) cyclohydrolase (HP0959), an ATPase (HP1079), an adenosine deaminase (HP0267), a phosphodiesterase (HP1042), an aminopeptidase (HP1037), and new substrates were characterized for a peptidoglycan deacetylase (HP0310). Generally, characterized enzymes were active at acidic to neutral pH (4.0–7.5) with temperature optima ranging from 35 to 55°C, although some exhibited outstanding characteristics.