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1.  Quantitative Genome-Wide Genetic Interaction Screens Reveal Global Epistatic Relationships of Protein Complexes in Escherichia coli 
PLoS Genetics  2014;10(2):e1004120.
Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.
Author Summary
Genome-wide genetic interaction (GI) screens have been performed in yeast, but no analogous large-scale studies have yet been reported for bacteria. Here, we have used E. coli synthetic genetic array (eSGA) technology developed by our group to quantitatively map GIs to reveal epistatic dependencies and functional cross-talk among ∼600,000 digenic mutant combinations. By combining this epistasis information with functional modules derived by our group's earlier efforts from proteomic and genomic context (GC)-based methods, we identify several unexpected pathway-level dependencies, functional links between protein complexes, and biological roles of uncharacterized bacterial gene products. As part of the study, two of our pathway predictions from GI screens were validated experimentally, where we confirmed the role of these new components in iron-sulphur biogenesis and ribosome integrity. We also extrapolated the epistatic connectivity diagram of E. coli to 233 distantly related γ-proteobacterial species lacking GI information, and identified co-conserved genes and functional modules important for bacterial pathogenesis. Overall, this study describes the first genome-scale map of GIs in gram-negative bacterium, and through integrative analysis with previously derived protein-protein and GC-based interaction networks presents a number of novel insights into the architecture of bacterial pathways that could not have been discerned through either network alone.
PMCID: PMC3930520  PMID: 24586182
2.  Integrative Network Analysis of Signaling in Human CD34+ Hematopoietic Progenitor Cells by Global Phosphoproteomic Profiling Using TiO2 Enrichment Combined with 2D LC-MS/MS and Pathway Mapping 
Proteomics  2013;13(8):10.1002/pmic.201200369.
Protein kinase signaling regulates human hematopoietic stem/progenitor cell (HSPC) fate, yet little is known about critical pathway substrates. To address this, we have developed and applied a large-scale, empirically-optimized phosphopeptide affinity enrichment strategy with high-throughput 2D LC-MS/MS screening to evaluate the phosphoproteome of an isolated human CD34+ HSPC population. We first used hydrophilic interaction chromatography (HILIC) as a first dimension separation to separate and simplify protein digest mixtures into discrete fractions. Phosphopeptides were then enriched offline using TiO2-coated magnetic beads and subsequently detected online by C18 reverse phase nanoflow HPLC using data-dependent MS/MS High-Energy Collision-activated Dissociation (HCD) fragmentation on a high performance Orbitrap hybrid tandem mass spectrometer. We identified 15533 unique phosphopeptides in 3574 putative phosphoproteins. Systematic computational analysis revealed biological pathways and phosphopeptides motifs enriched in CD34+ HSPC that are markedly different from those observed in an analogous parallel analysis of isolated human T cells, pointing to the possible involvement of specific kinase-substrate relationships within activated cascades driving hematopoietic renewal, commitment and differentiation.
PMCID: PMC3883140  PMID: 23401153
human; hematopoietic; stem cell; signaling; phosphoprotein; phosphopeptide; chromatography; enrichment; tandem mass spectrometry
3.  A Census of Human Soluble Protein Complexes 
Cell  2012;150(5):1068-1081.
Cellular processes often depend on stable physical associations between proteins. Despite recent progress, knowledge of the composition of human protein complexes remains limited. To close this gap, we applied an integrative global proteomic profiling approach, based on chromatographic separation of cultured human cell extracts into more than one thousand biochemical fractions which were subsequently analyzed by quantitative tandem mass spectrometry, to systematically identify a network of 13,993 high-confidence physical interactions among 3,006 stably-associated soluble human proteins. Most of the 622 putative protein complexes we report are linked to core biological processes, and encompass both candidate disease genes and unnanotated proteins to inform on mechanism. Strikingly, whereas larger multi-protein assemblies tend to be more extensively annotated and evolutionarily conserved, human protein complexes with 5 or fewer subunits are far more likely to be functionally un-annotated or restricted to vertebrates, suggesting more recent functional innovations.
PMCID: PMC3477804  PMID: 22939629
4.  A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair 
Molecular microbiology  2010;79(2):484-502.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks, and 5′-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.
PMCID: PMC3071548  PMID: 21219465
Cas1; CRISPR; DNA recombination; DNA repair; nuclease; YgbT
5.  Genetic Interaction Maps in Escherichia coli Reveal Functional Crosstalk among Cell Envelope Biogenesis Pathways 
PLoS Genetics  2011;7(11):e1002377.
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.
Author Summary
Proper assembly of the cell envelope is essential for bacterial growth, environmental adaptation, and drug resistance. Yet, while the biological roles of the many genes and pathways involved in biosynthesis of the cell envelope have been studied extensively in isolation, how the myriad components intersect functionally to maintain envelope integrity under different growth conditions has not been explored systematically. Genome-scale genetic interaction screens have increasingly been performed to great impact in yeast; no analogous comprehensive studies have yet been reported for bacteria despite their prominence in human health and disease. We addressed this by using a synthetic genetic array technology to generate quantitative maps of genetic interactions encompassing virtually all the components of the cell envelope biosynthetic machinery of the classic model bacterium E. coli in two common laboratory growth conditions (rich and minimal medium). From the resulting networks of high-confidence genetic interactions, we identify condition-specific functional dependencies underlying envelope assembly and global remodeling of genetic backup mechanisms that ensure envelope integrity under environmental challenge.
PMCID: PMC3219608  PMID: 22125496
6.  Ribosome-Dependent ATPase Interacts with Conserved Membrane Protein in Escherichia coli to Modulate Protein Synthesis and Oxidative Phosphorylation 
PLoS ONE  2011;6(4):e18510.
Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.
PMCID: PMC3083400  PMID: 21556145
7.  Global Functional Atlas of Escherichia coli Encompassing Previously Uncharacterized Proteins 
PLoS Biology  2009;7(4):e1000096.
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.
Author Summary
One goal of modern biology is to chart groups of proteins that act together to perform biological processes via direct and indirect interactions. Such groupings are sometimes called functional modules. The types of protein interactions within modules include physical interactions that generate protein complexes and biochemical associations that make up metabolic pathways. We have combined proteomic and bioinformatic tools, and used them to decipher a large number of protein interactions, complexes, and functional modules with high confidence. In addition, exploring the topology of the resulting interaction networks, we successfully predicted specific biological roles for a number of proteins with previously unknown functions, and identified some potential drug targets. Although our work is focused on E. coli, our phylogenetic projections suggest that a considerable fraction of our observations and predictions can be extrapolated to many other bacterial taxa. As all the data derived from this study are publicly available, others may build on our work for further hypothesis-driven studies of gene function discovery.
A novel resource integrating proteomic and genome context-based tools provides a "systems-wide" functional blueprint ofE. coli, with insights into the biological and evolutionary significance of previously uncharacterized proteins.
PMCID: PMC2672614  PMID: 19402753
8.—an integrated protein interaction database for E. coli 
Nucleic Acids Research  2007;36(Database issue):D632-D636.
High throughput methods are increasingly being used to examine the functions and interactions of gene products on a genome-scale. These include systematic large-scale proteomic studies of protein complexes and protein–protein interaction networks, functional genomic studies examining patterns of gene expression and comparative genomics studies examining patterns of conservation. Since these datasets offer different yet highly complementary perspectives on cell behavior it is expected that integration of these datasets will lead to conceptual advances in our understanding of the fundamental design and evolutionary principles that underlie the organization and function of proteins within biochemical pathways. Here we present, a resource that combines locally generated interaction and evolutionary datasets with a previously generated knowledgebase, to provide an integrated view of the Escherichia coli interactome. Tools are provided which allow the user to select and visualize functional, evolutionary and structural relationships between groups of interacting proteins and to focus on genes of interest. Currently the database contains three interaction datasets: a functional dataset consisting of 3989 interactions between 1927 proteins; a ‘core’ high quality experimental dataset of 4863 interactions between 1100 proteins and an ‘extended’ experimental dataset of 9860 interactions between 2131 proteins. is available online at
PMCID: PMC2238847  PMID: 17942431

Results 1-8 (8)