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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.  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
3.  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
4.  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
5.  Recombination analysis of Soybean mosaic virus sequences reveals evidence of RNA recombination between distinct pathotypes 
Virology Journal  2008;5:143.
RNA recombination is one of the two major factors that create RNA genome variability. Assessing its incidence in plant RNA viruses helps understand the formation of new isolates and evaluate the effectiveness of crop protection strategies. To search for recombination in Soybean mosaic virus (SMV), the causal agent of a worldwide seed-borne, aphid-transmitted viral soybean disease, we obtained all full-length genome sequences of SMV as well as partial sequences encoding the N-terminal most (P1 protease) and the C-terminal most (capsid protein; CP) viral protein. The sequences were analyzed for possible recombination events using a variety of automatic and manual recombination detection and verification approaches. Automatic scanning identified 3, 10, and 17 recombination sites in the P1, CP, and full-length sequences, respectively. Manual analyses confirmed 10 recombination sites in three full-length SMV sequences. To our knowledge, this is the first report of recombination between distinct SMV pathotypes. These data imply that different SMV pathotypes can simultaneously infect a host cell and exchange genetic materials through recombination. The high incidence of SMV recombination suggests that recombination plays an important role in SMV evolution. Obtaining additional full-length sequences will help elucidate this role.
PMCID: PMC2627826  PMID: 19036160

Results 1-5 (5)