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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2015)

Living organisms are comprised of crowded and highly interconnected networks of biological molecules and metabolites. The scale and complexity of these networks makes them difficult to completely characterize, even for relatively streamlined organisms like the bacterium Escherichia coli K-12. After decades of work, many aspects of the growth and physiology of E. coli remain poorly understood. Its regulatory pathways remain only partially mapped. Functional-genomic approaches hold the promise of speeding discovery and characterization of the molecular networks underpinning life.

In this work, a chemical-genomic screen was conducted for two purposes. First, we were interested in the genetic-networks that make up an E. coli cell. Could we build on previous work to expand our knowledge of gene function in this model bacterium? Second, we were interested in pushing the resolution of functional genomics, not to characterize the relationship between genes in an organism but to characterize the relationships of amino acids and structural features of a single enzyme; RNA polymerase (RNAP).

To accomplish these tasks, we first used oligo-mediated recombineering to build a library of over 150 mutants in RNAP. We then screened this library of mutant strains, along with the KEIO deletion library of E. coli, for growth across more than 100 unique chemical stresses using colony array technology. This generated a map that expanded the chemical-sensitivity landscape of E. coli.

From features within this landscape, we identified a unique mechanism of illicit transport wherein the translation inhibitors kasugamycin and blasticidin S gain access to the cytoplasm of E. coli by hijacking the peptide ABC-importers Dpp and Opp. We used genetic analysis, in vivo translation assays, and in vitro binding assays to show that the peptide ABC-importers directly import the two drugs.

Next, we identified a new binding interface within RNAP that allows for the binding and function of the critical transcription regulator DksA. We used genetic analysis, in vitro site-specific crosslinking, molecular modeling, and functional assays of transcription both in vivo and in in vitro. We found that the lineage specific insertion SI1 directly binds DksA and is critical for the effects of DksA on transcription.

    Cover page: Insights into Illicit Transport and Transcription Regulation from a Chemical-Genomic Screen in Escherichia coli
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2012)
    The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr use a shared radical-mediated mechanism. In addition to the radical SAM CX(3)CX(2)C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function, putatively to form (cysteine 355 in RlmN) and resolve (cysteine 118 in RlmN) a covalent intermediate needed to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further clarify the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species between RlmN and its rRNA substrate and well as the roles of the conserved cysteine residues in catalysis.
      Cover page: Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis.
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2012)
      The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr use a shared radical-mediated mechanism. In addition to the radical SAM CX(3)CX(2)C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function, putatively to form (cysteine 355 in RlmN) and resolve (cysteine 118 in RlmN) a covalent intermediate needed to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further clarify the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species between RlmN and its rRNA substrate and well as the roles of the conserved cysteine residues in catalysis.
        Cover page: Covalent Intermediate in the Catalytic Mechanism of the Radical S‑Adenosyl‑l‑methionine Methyl Synthase RlmN Trapped by Mutagenesis
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2019)
        Abstract: The genomic catalogue of the human microbiota has expanded dramatically in recent years, and insights derived from human microbiota genomics has vast potential to generate treatments for human diseases. However, predictably harnessing the microbiota for beneficial outcomes is currently limited by our lack of understanding of the physiology of the constituent bacteria. For instance, the functions of most of their genes are not known. Here, we systematically measure mutant phenotypes for genes from the gut commensalBacteroides thetaiotaomicron. Using a barcoded transposon mutant library, we measured the fitness of 4,055B. thetaiotaomicrongenes across 492 experiments, including growth on 45 carbon substrates and in the presence of 57 stress-inducing compounds. Our data is in strong agreement with previous studies, and more importantly also uncovers the biological roles of poorly annotated genes. We identified 497 genes with a specific phenotype in only one or a handful of conditions, thus enabling informed predictions of gene function for a subset of these genes. For example, we identified a glycoside hydrolase important for growth on type I rhamnogalacturonan, a DUF4861 protein for glycosaminoglycan utilization, a DUF1080 protein for disaccharide utilization, and a tripartite multidrug resistance system specifically important for bile salt tolerance. Our approach can be applied to other members of the human microbiota to experimentally characterize their genes.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2021)
          Harnessing the microbiota for beneficial outcomes is limited by our poor understanding of the constituent bacteria, as the functions of most of their genes are unknown. Here, we measure the growth of a barcoded transposon mutant library of the gut commensal Bacteroides thetaiotaomicron on 48 carbon sources, in the presence of 56 stress-inducing compounds, and during mono-colonization of gnotobiotic mice. We identify 516 genes with a specific phenotype under only one or a few conditions, enabling informed predictions of gene function. For example, we identify a glycoside hydrolase important for growth on type I rhamnogalacturonan, a DUF4861 protein for glycosaminoglycan utilization, a 3-keto-glucoside hydrolase for disaccharide utilization, and a tripartite multidrug resistance system specifically for bile salt tolerance. Furthermore, we show that B. thetaiotaomicron uses alternative enzymes for synthesizing nitrogen-containing metabolic precursors based on ammonium availability and that these enzymes are used differentially in vivo in a diet-dependent manner.
            Cover page: Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2020)
            Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.
              Cover page: Genetic interaction mapping informs integrative structure determination of protein complexes
              • Article
              • Peer Reviewed
              LBL Publications (2024)
              Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes. We discuss the advantages and limitations of each approach, as well as factors to consider when selecting a strategy. In addition, we highlight experimental and computational advances in arraying condensed libraries from mutant pools. We focus on examples of successful library construction in gut bacteria and their application to gene function studies and drug discovery. Given the need for understanding gene function and organization in gut bacteria, we provide a comprehensive guide for researchers to construct randomly barcoded transposon mutant libraries.
                Cover page: Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library constructionCreative Commons 'BY-NC' version 4.0 license
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