One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a “systems-wide” functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.

Physical and functional interactions define the molecular organization of the cell. Genetic interactions, or epistasis, tend to occur between gene products involved in parallel pathways or interlinked biological processes. High-throughput experimental systems to examine genetic interactions on a genome-wide scale have been devised for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, but have not been reported previously for prokaryotes. Here we describe the development of a quantitative screening procedure for monitoring bacterial genetic interactions based on conjugation of Escherichia coli deletion or hypomorphic strains to create double mutants on a genome-wide scale. The patterns of synthetic sickness and synthetic lethality (aggravating genetic interactions) we observed for certain double mutant combinations provided information about functional relationships and redundancy between pathways and enabled us to group bacterial gene products into functional modules.

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among >235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target.

Exendin-4, a long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist, is a potential regulator of feeding behavior through its ability to inhibit gastric emptying, reduce food intake, and induce satiety. GLP-1R activation by exendin-4 induces anorexia; however, the specific populations of neuropeptidergic neurons activated by exendin-4 within the hypothalamus, the central regulator of energy homeostasis, remain unclear. This study determines whether exendin-4 regulates hypothalamic neuropeptide expression and explores the signaling mechanisms involved. The distribution and quantity of exendin-4-induced c-Fos immunoreactivity were evaluated to determine activation of α-melanocyte-stimulating hormone/proopiomelanocortin, neuropeptide Y, neurotensin (NT), and ghrelin neurons in hypothalamic nuclei during exendin-4-induced anorexia in mice. Additionally, exendin-4 action on NT and ghrelin transcript regulation was examined in immortalized hypothalamic neurons. With anorexia induced by intracerebroventricular exendin-4, α-melanocyte-stimulating hormone/proopiomelanocortin and neuropeptide Y neurons were activated in the arcuate nucleus, with simultaneous activation of NT-expressing neurons in the paraventricular nucleus, and ghrelin-expressing neurons in the arcuate nucleus, paraventricular nucleus, and periventricular hypothalamus, suggesting that neurons in one or more of these areas mediate the anorexic action of exendin-4. In the hypothalamic neuronal cell models, exendin-4 increased cAMP, cAMP response element-binding protein/activating transcription factor-1 and c-Fos activation, and via a protein kinase A-dependent mechanism regulated NT and ghrelin mRNA expression, indicating that these neuropeptides may serve as downstream mediators of exendin-4 action. These findings provide a previously unrecognized link between central GLP-1R activation by exendin-4 and the regulation of hypothalamic NT and ghrelin. Further understanding of this central GLP-1R activation may lead to safe and effective therapeutics for the treatment of metabolic disorders.

Central resistance to the actions of insulin and leptin is associated with the onset of obesity and type 2 diabetes mellitus, whereas leptin and insulin signaling is essential for both glucose and energy homeostasis. Although it is known that leptin resistance can lead to attenuated insulin signaling, whether insulin resistance can lead to or exacerbate leptin resistance is unknown. To investigate the molecular events underlying crosstalk between these signaling pathways, immortalized hypothalamic neuronal models, rHypoE-19 and mHypoA-2/10, were used. Prolonged insulin exposure was used to induce cellular insulin resistance, and thereafter leptin-mediated regulation of signal transduction and gene expression was assessed. Leptin directly repressed agouti-related peptide mRNA levels but induced urocortin-2, insulin receptor substrate (IRS)-1, IRS2, and IR transcription, through leptin-mediated phosphatidylinositol 3-kinase/Akt activation. Neuronal insulin resistance, as assessed by attenuated Akt phosphorylation, blocked leptin-mediated signal transduction and agouti-related peptide, urocortin-2, IRS1, IRS2, and insulin receptor synthesis. Insulin resistance caused a substantial decrease in insulin receptor protein levels, forkhead box protein 1 phosphorylation, and an increase in suppressor of cytokine signaling 3 protein levels. Cellular insulin resistance may cause or exacerbate neuronal leptin resistance and, by extension, obesity. It is essential to unravel the effects of neuronal insulin resistance given that both peripheral, as well as the less widely studied central insulin resistance, may contribute to the development of metabolic, reproductive, and cardiovascular disorders. This study provides improved understanding of the complex cellular crosstalk between insulin-leptin signal transduction that is disrupted during neuronal insulin resistance.