Although the microcrustacean Daphnia is becoming an organism of choice for proteomic studies, protein expression across its life cycle have not been fully characterized. Proteomes of adult females, juveniles, asexually produced embryos, and the ephippia-resting stages containing sexually produced diapausing freezing- and desiccation-resistant embryos are analyzed. Overall, proteins with known molecular functions are more likely to be detected than proteins with no detectable orthology. Similarly, proteins with stronger gene model support in two independent genome assemblies can be detected, than those without such support. This suggests that the proteomics pipeline can be applied to verify hypothesized proteins, even given questionable reference gene models. In particular, upregulation of vitellogenins and downregulation of actins and myosins in embryos of both types, relative to juveniles and adults, and overrepresentation of cell-cycle related proteins in the developing embryos, relative to diapausing embryos and adults, are observed. Upregulation of small heat-shock proteins and peroxidases, as well as overrepresentation of stress-response proteins in the ephippium relative to the asexually produced non-diapausing embryos, is found. The ephippium also shows upregulation of three trehalose-synthesis proteins and downregulation of a trehalose hydrolase, consistent with the role of trehalose in protection against freezing and desiccation.

Pathway optimization is, in general, a very demanding task due to the complex, nonlinear and largely unknown interactions of enzymes, regulators and metabolites. While in vitro reconstruction and pathway analysis is a viable alternative, a major limitation of this approach is the availability of the pathway enzymes for reliable pathway reconstruction. Here, we report the application of RNA display methods for the construction of fusion (chimeric) molecules, comprising mRNA and the protein they express, that can be used for the above purpose. The chimeric molecule is immobilized via hybridization of its mRNA end with homologous capture DNA spotted on a substrate surface. We show that the protein (enzyme) end of the fusion molecule retains its function under immobilized conditions and that the enzymatic activity is proportional to the amount of capture DNA spotted on the surface of a microarray or 96-well microplate. The relative amounts of all pathway enzymes can thus be changed at will by changing the amount of the corresponding capture DNA. Hence, entire pathways can be reconstructed and optimized in vitro from genomic information alone by generating chimeric molecules for all pathway enzymes in a single in vitro translation step and hybridizing on 96-well microplates where each well contains a different combination of capture DNA. We provide validation of this concept with the sequential reactions catalyzed by luciferase and nucleoside diphosphate kinase and further illustrate this method with the optimization of the five-step pathway for trehalose synthesis. Multi-enzyme pathways leading to the synthesis of specialty molecules can thus be optimized from genomic information about the pathway enzymes, provided the latter retain their activity under the in vitro immobilized conditions. / Singapore-MIT Alliance (SMA)

Background: Biological systems adapt to changing environments by reorganizing their cellula r and physiological program with metabolites representing one important response level. Different stresses lead to both conserved and specific responses on the metabolite level which should be reflected in the underl ying metabolic network. Methodology/Principal Findings: Starting from experimental data obtained by a GC-MS based high-throughput metabolic profiling technology we here develop an approach that: (1) extracts network representations from metabolic conditiondependent data by using pairwise correlations, (2) determines the sets of stable and condition-dependent correlations based on a combination of statistical significance and homogeneity tests, and (3) can identify metabolites related to the stress response, which goes beyond simple ob servation s about the changes of metabolic concentrations. The approach was tested with Escherichia colias a model organism observed under four different environmental stress conditions (cold stress, heat stress, oxidative stress, lactose diau xie) and control unperturbed conditions. By constructing the stable network component, which displays a scale free topology and small-world characteristics, we demonstrated that: (1) metabolite hubs in this reconstructed correlation networks are significantly enriched for those contained in biochemical networks such as EcoCyc, (2) particular components of the stable network are enriched for functionally related biochemical path ways, and (3) ind ependently of the response scale, based on their importance in the reorganization of the cor relation network a set of metabolites can be identified which represent hypothetical candidates for adjusting to a stress-specific response. Conclusions/Significance: Network-based tools allowed the identification of stress-dependent and general metabolic correlation networks. This correlation-network-ba sed approach does not rely on major changes in concentration to identify metabolites important for st ress adaptation, but rather on the changes in network properties with respect to metabolites. This should represent a useful complementary technique in addition to more classical approaches.