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Open Access Publications from the University of California

Scholarly Works (14 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2020)

Bacteriophages (phages) are viruses that specifically infect bacteria including diverse, notable pathogens. They constitute the most abundant biological entities on earth, have driven the evolution of critical biotechnological tools, and are proposed as an emerging solution to treat the rise of antimicrobial resistant bacterial pathogens. Although these bacterial predators have been studied for nearly a century, our understanding of host-specificity, gene function, and the consequences of phage infection remain limited to a handful of model phage-host pairs. In this dissertation, we employ high-throughput genetic screening technologies to rapidly and economically assess genetic function for bacterial fitness against diverse phages. We rapidly identify phage-host interactions in competitive fitness experiments through the use of loss-of-function and gain-of-function screening technologies: RB-TnSeq (barcoded transposon insertion), CRISPRi (dCas9-mediated transcriptional repression), and Dub-seq (barcoded overexpression). We employ all 3 technologies to study phage-host interactions in E.coli and systematically elucidate the phage resistance landscape for this model organism and 14 of its phages, discovering new routes to phage resistance, and providing a comprehensive resource that can be mapped against hundreds of fitness conditions. We subsequently extended our investigations an additional model pathogen, Salmonella enterica serovar Typhimurium, one of the world’s most common sources of food poisoning and an emerging antibiotic resistance threat. We introduce two new barcoded libraries in Salmonella and investigate its interactions with 11 additional phages. In addition to the resource provided, we uncover an unanticipated phage cross-resistance network. This network includes multiple ties to Salmonella virulence and lifestyle associated with phage resistance, introducing new considerations for therapeutic avenues. In aggregate, we report the results of over hundreds of genome wide assays, corroborating known and discovering new modes of phage resistance. Here, we also further extend Dub-seq and genome engineering technologies for the study of phage gene function, which too remains limited. By employing Dub-seq as a co-expression platform, we re-tool Dub-seq overexpression assays to rapidly characterize the function of 8 single gene lysis systems from ssDNA and ssRNA phages. We discover new putative target inhibitors, introduce new factors in resistance acquisition, and put forward a target hypothesis for a general role of these proteins. The forementioned library screens were enabled by genome-engineering technologies. Finally, we report a genome-engineering chassis in E.coli based off of the integrative prophage, Lambda, that can be modified at enhanced rates and any nonessential position. We believe that the development of such chassis will be critical towards not only synthetic biology applications, but also the rapid development of strains to characterize phage-host interactions.

    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2022)
    Used widely for genome editing, CRISPR-Cas enzymes provide RNA-guided immunity to microbes by targeting foreign nucleic acids for cleavage. We show here that the native activity of CRISPR-Cas12c protects bacteria from phage infection by binding to DNA targets without cleaving them, revealing that antiviral interference can be accomplished without chemical attack on the invader or general metabolic disruption in the host. Biochemical experiments demonstrate that Cas12c is a site-specific ribonuclease capable of generating mature CRISPR RNAs (crRNAs) from precursor transcripts. Furthermore, we find that crRNA maturation is essential for Cas12c-mediated DNA targeting. These crRNAs direct double-stranded DNA binding by Cas12c using a mechanism that precludes DNA cutting. Nevertheless, Cas12c represses transcription and can defend bacteria against lytic bacteriophage infection when targeting an essential phage gene. Together, these results show that Cas12c employs targeted DNA binding to provide antiviral immunity in bacteria, providing a native DNase-free pathway for transient antiviral immunity.
      Cover page: A naturally DNase-free CRISPR-Cas12c enzyme silences gene expression
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2019)
      Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (i) a refactored λ-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (ii) genetic modifications that boost recombination efficiency, and (iii) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a 7-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context--neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.
        Cover page: A versatile platform strain for high-fidelity multiplex genome editing
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2023)
        Single-strand RNA (ssRNA) Fiersviridae phages cause host lysis with a product of single gene (sgl for single-gene lysis; product Sgl) that induces autolysis. Many different Sgls have been discovered, but the molecular targets of only a few have been identified. In this study, we used a high-throughput genetic screen to uncover genome-wide host suppressors of diverse Sgls. In addition to validating known molecular mechanisms, we discovered that the Sgl of PP7, an ssRNA phage of Pseudomonas aeruginosa, targets MurJ, the flippase responsible for lipid II export, previously shown to be the target of the Sgl of coliphage M. These two Sgls, which are unrelated and predicted to have opposite membrane topology, thus represent a case of convergent evolution. We extended the genetic screens to other uncharacterized Sgls and uncovered a common set of multicopy suppressors, suggesting that these Sgls act by the same or similar mechanism.
          Cover page: Multicopy suppressor screens reveal convergent evolution of single-gene lysis proteinsCreative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2022)
          CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.
            Cover page: Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing.Creative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2024)
            Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman is a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact double-stranded DNA (dsDNA). When the HamAB complex detects DNA damage, HamB helicase activity activates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating "phantom" cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and enzymes from eukaryotes and archaea suggest deep functional symmetries with other important helicases across domains of life.
              Cover page: Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complexCreative Commons 'BY' version 4.0 license
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2021)
              Though bacteriophages (phages) are known to play a crucial role in bacterial fitness and virulence, our knowledge about the genetic basis of their interaction, cross-resistance and host-range is sparse. Here, we employed genome-wide screens in Salmonella enterica serovar Typhimurium to discover host determinants involved in resistance to eleven diverse lytic phages including four new phages isolated from a therapeutic phage cocktail. We uncovered 301 diverse host factors essential in phage infection, many of which are shared between multiple phages demonstrating potential cross-resistance mechanisms. We validate many of these novel findings and uncover the intricate interplay between RpoS, the virulence-associated general stress response sigma factor and RpoN, the nitrogen starvation sigma factor in phage cross-resistance. Finally, the infectivity pattern of eleven phages across a panel of 23 genome sequenced Salmonella strains indicates that additional constraints and interactions beyond the host factors uncovered here define the phage host range.
                Cover page: The genetic basis of phage susceptibility, cross-resistance and host-range in Salmonella
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2020)

                Summary

                Though bacteriophages (phages) are known to play a crucial role in bacterial fitness and virulence, our knowledge about the genetic basis of their interaction, cross-resistance and host-range is sparse. Here, we employed genome-wide screens in Salmonella enterica serovar Typhimurium to discover host determinants involved in resistance to eleven diverse lytic phages including 4 new phages isolated from a therapeutic phage cocktail. We uncovered 301 diverse host factors essential in phage infection, many of which are shared between multiple phages demonstrating potential cross-resistance mechanisms. We validate many of these novel findings and uncover the intricate interplay between RpoS, the virulence-associated general stress response sigma factor and RpoN, the nitrogen starvation sigma factor in phage cross-resistance. Finally, the infectivity pattern of eleven phages across a panel of 23 genome sequenced Salmonella strains indicates that additional constraints and interactions beyond the host factors uncovered here define the phage host range.
                  Cover page: The Genetic Basis of phage susceptibility, cross-resistance and host-range in Salmonella
                  • Article
                  • Peer Reviewed
                  UC Berkeley Previously Published Works (2020)
                  Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.
                    Creative Commons 'BY-NC-ND' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UC Berkeley Previously Published Works (2020)
                    Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.
                      Cover page: High-throughput mapping of the phage resistance landscape in E. coli
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