Depiction of metabolome changes in histidine-starved Escherichia coli by CE-TOFMS.

Metabolic changes in response to histidine starvation were observed in histidine-auxotrophic Escherichia coli using a capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolomics technique. Prior to the analysis, we prepared an E. coli metabolome list of 727 metabolites reported in the literature. An improved method for metabolite extraction was developed, which resulted in higher extraction efficiency in phosphate-rich metabolites, e.g., ATP and GTP. Based on the results, 375 charged, hydrophilic intermediates in primary metabolisms were analysed simultaneously, providing quantitative data of 198 metabolites. We confirmed that the intracellular levels of intermediates in histidine biosynthesis are rapidly accumulated in response to a drop in histidine level under histidine-starved conditions. Simultaneously, disciplined responses were observed in the glycolysis, tricarboxylic acid cycle, and amino acid and nucleotide biosynthesis pathways as regulated by amino acid starvation.

Comparison of metabolite production capability indices generated by network analysis methods.

UNLABELLED: A framework of constraint-based reconstruction and analysis (COBRA) is used for modeling large-scale metabolic networks. In COBRA, extreme pathway and optimization analyses are commonly used to study the properties of networks. While the results of both methods are completely consistent, extreme pathway analysis is considered to be better because of its wider representational ability. In this study, we assessed these two methods by computational knockout experiments. We examined a simple pathway model and found that the extreme pathway method led to misguided conclusions in specific cases, while optimization analysis calculated the correct knockout effects. We also investigated the Escherichia coli metabolic pathway model, and found that these methods result in inconsistent interpretations of the network properties. IN CONCLUSION: it has been claimed that these two methods result in the same producible metabolites, but we found a difference in individual results for a biological pathway. Our results could provide helpful guidance for when to use the methods, particularly extreme pathway analysis.

Large-scale identification of protein-protein interaction of Escherichia coli K-12.

Protein-protein interactions play key roles in protein function and the structural organization of a cell. A thorough description of these interactions should facilitate elucidation of cellular activities, targeted-drug design, and whole cell engineering. A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library. Of 4339 bait proteins tested, partners were found for 2667, including 779 of unknown function. Proteins copurifying with hexahistidine-tagged baits on a Ni2+-NTA column were identified by MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). An extended analysis of these interacting networks by bioinformatics and experimentation should provide new insights and novel strategies for E. coli systems biology.