Oxana Pogoutse

Global Functional Atlas of Escherichia coli Encompassing Previously Uncharacterized Proteins

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.

eSGA: E. coli Synthetic Genetic Array analysis

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.NOTE: In the version of this article initially published online two author names (Gabriel Moreno-Hagelseib and Constantine Christopolous) were spelled incorrectly. The correct author names are Gabriel Moreno-Hagelsieb and Constantine Christopoulos. The error has been corrected for the print, PDF and HTML versions of this article.

A dual function of the CRISPR–Cas system in bacterial antivirus immunity and DNA repair

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.

Impaired tRNA Nuclear Export Links DNA Damage and Cell-Cycle Checkpoint

In response to genotoxic stress, cells evoke a plethora of physiological responses collectively aimed at enhancing viability and maintaining the integrity of the genome. Here, we report that unspliced tRNA rapidly accumulates in the nuclei of yeast Saccharomyces cerevisiae after DNA damage. This response requires an intact MEC1- and RAD53-dependent signaling pathway that impedes the nuclear export of intron-containing tRNA via differential relocalization of the karyopherin Los1 to the cytoplasm. The accumulation of unspliced tRNA in the nucleus signals the activation of Gcn4 transcription factor, which, in turn, contributes to cell-cycle arrest in G1 in part by delaying accumulation of the cyclin Cln2. The regulated nucleocytoplasmic tRNA trafficking thus constitutes an integral physiological adaptation to DNA damage. These data further illustrate how signal-mediated crosstalk between distinct functional modules, namely, tRNA nucleocytoplasmic trafficking, protein synthesis, and checkpoint execution, allows for functional coupling of tRNA biogenesis and cell-cycle progression.

Genetic Interaction Maps in Escherichia coli Reveal Functional Crosstalk among Cell Envelope Biogenesis Pathways

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.

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p

BackgroundHistone modifications have been implicated in the regulation of transcription and, more recently, in DNA replication and repair. In yeast, a major conserved histone acetyltransferase, Hat1p, preferentially acetylates lysine residues 5 and 12 on histone H4.ResultsHere, we report that a nuclear sub-complex consisting of Hat1p and its partner Hat2p interacts physically and functionally with the origin recognition complex (ORC). While mutational inactivation of the histone acetyltransferase (HAT) gene HAT1 alone does not compromise origin firing or initiation of DNA replication, a deletion in HAT1 (or HAT2) exacerbates the growth defects of conditional orc-ts mutants. Thus, the ORC-associated Hat1p-dependent histone acetyltransferase activity suggests a novel linkage between histone modification and DNA replication. Additional genetic and biochemical evidence points to the existence of partly overlapping histone H3 acetyltransferase activities in addition to Hat1p/Hat2p for proper DNA replication efficiency. Furthermore, we demonstrated a dynamic association of Hat1p with chromatin during S-phase that suggests a role of this enzyme at the replication fork.ConclusionWe have found an intriguing new association of the Hat1p-dependent histone acetyltransferase in addition to its previously known role in nuclear chromatin assembly (Hat1p/Hat2p-Hif1p). The participation of a distinct Hat1p/Hat2p sub-complex suggests a linkage of histone H4 modification with ORC-dependent DNA replication.

Target Identification by Chromatographic Co-elution: Monitoring of Drug-Protein Interactions without Immobilization or Chemical Derivatization

Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates. Correlative proteomic analysis of drug-bound protein fractions by shotgun sequencing is then performed to identify candidate target(s). The method is highly reproducible, does not require immobilization or derivatization of drug or protein, and is applicable to diverse natural products and synthetic compounds. The capability of TICC to detect known drug-protein target physical interactions (Kd range: micromolar to nanomolar) is demonstrated both qualitatively and quantitatively. We subsequently used TICC to uncover the sterol biosynthetic enzyme Erg6p as a novel putative anti-fungal target. Furthermore, TICC identified Asc1 and Dak1, a core 40 S ribosomal protein that represses gene expression, and dihydroxyacetone kinase involved in stress adaptation, respectively, as novel yeast targets of a dopamine receptor agonist.

Sequential Peptide Affinity Purification System for the Systematic Isolation and Identification of Protein Complexes from Escherichia coli

Biochemical purification of affinity-tagged proteins in combination with mass spectrometry methods is increasingly seen as a cornerstone of systems biology, as it allows for the systematic genome-scale characterization of macromolecular protein complexes, representing demarcated sets of stably interacting protein partners. Accurate and sensitive identification of both the specific and shared polypeptide components of distinct complexes requires purification to near homogeneity. To this end, a sequential peptide affinity (SPA) purification system was developed to enable the rapid and efficient isolation of native Escherichia coli protein complexes (J Proteome Res 3:463-468, 2004). SPA purification makes use of a dual-affinity tag, consisting of three modified FLAG sequences (3X FLAG) and a calmodulin binding peptide (CBP), spaced by a cleavage site for tobacco etch virus (TEV) protease (J Proteome Res 3:463-468, 2004). Using the lambda-phage Red homologous recombination system (PNAS 97:5978-5983, 2000), a DNA cassette, encoding the SPA-tag and a selectable marker flanked by gene-specific targeting sequences, is introduced into a selected locus in the E. coli chromosome so as to create a C-terminal fusion with the protein of interest. This procedure aims for near-endogenous levels of tagged protein production in the recombinant bacteria to avoid spurious, non-specific protein associations (J Proteome Res 3:463-468, 2004). In this chapter, we describe a detailed, optimized protocol for the tagging, purification, and subsequent mass spectrometry-based identification of the subunits of even low-abundance bacterial protein complexes isolated as part of an ongoing large-scale proteomic study in E. coli (Nature 433:531-537, 2005).

Hsp110 is required for spindle length control

The Hsp70–Hsp110 chaperone complex antagonizes Cin8 plus-end motility and prevents premature spindle elongation in S phase.

Synthetic Peptide Arrays for Pathway-Level Protein Monitoring by Liquid Chromatography-Tandem Mass Spectrometry

Effective methods to detect and quantify functionally linked regulatory proteins in complex biological samples are essential for investigating mammalian signaling pathways. Traditional immunoassays depend on proprietary reagents that are difficult to generate and multiplex, whereas global proteomic profiling can be tedious and can miss low abundance proteins. Here, we report a target-driven liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy for selectively examining the levels of multiple low abundance components of signaling pathways which are refractory to standard shotgun screening procedures and hence appear limited in current MS/MS repositories. Our stepwise approach consists of: (i) synthesizing microscale peptide arrays, including heavy isotope-labeled internal standards, for use as high quality references to (ii) build empirically validated high density LC-MS/MS detection assays with a retention time scheduling system that can be used to (iii) identify and quantify endogenous low abundance protein targets in complex biological mixtures with high accuracy by correlation to a spectral database using new software tools. The method offers a flexible, rapid, and cost-effective means for routine proteomic exploration of biological systems including “label-free” quantification, while minimizing spurious interferences. As proof-of-concept, we have examined the abundance of transcription factors and protein kinases mediating pluripotency and self-renewal in embryonic stem cell populations.