Anthony Shiver

All Publications

• Randomly barcoded transposon mutant libraries for gut commensals II: Applying libraries for functional genetics. Cell reports Voogdt, C. G., Tripathi, S., Bassler, S. O., McKeithen-Mead, S. A., Guiberson, E. R., Koumoutsi, A., Bravo, A. M., Buie, C., Zimmermann, M., Sonnenburg, J. L., Typas, A., Deutschbauer, A. M., Shiver, A. L., Huang, K. C. 2023; 43 (1): 113519

  Abstract

  The critical role of the intestinal microbiota in human health and disease is well recognized. Nevertheless, there are still large gaps in our understanding of the functions and mechanisms encoded in the genomes of most members of the gut microbiota. Genome-scale libraries of transposon mutants are a powerful tool to help us address this gap. Recent advances in barcoded transposon mutagenesis have dramatically lowered the cost of mutant fitness determination in hundreds of in vitro and in vivo experimental conditions. In an accompanying review, we discuss recent advances and caveats for the construction of pooled and arrayed barcoded transposon mutant libraries in human gut commensals. In this review, we discuss how these libraries can be used across a wide range of applications, the technical aspects involved, and expectations for such screens.

  View details for DOI 10.1016/j.celrep.2023.113519

  View details for PubMedID 38142398

• Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library construction. Cell reports Tripathi, S., Voogdt, C. G., Bassler, S. O., Anderson, M., Huang, P. H., Sakenova, N., Capraz, T., Jain, S., Koumoutsi, A., Bravo, A. M., Trotter, V., Zimmerman, M., Sonnenburg, J. L., Buie, C., Typas, A., Deutschbauer, A. M., Shiver, A. L., Huang, K. C. 2023; 43 (1): 113517

  Abstract

  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.

  View details for DOI 10.1016/j.celrep.2023.113517

  View details for PubMedID 38142397

• A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions. bioRxiv : the preprint server for biology Shiver, A. L., Sun, J., Culver, R., Violette, A., Wynter, C., Nieckarz, M., Mattiello, S. P., Sekhon, P. K., Friess, L., Carlson, H. K., Wong, D., Higginbottom, S., Weglarz, M., Wang, W., Knapp, B. D., Guiberson, E., Sanchez, J., Huang, P. H., Garcia, P. A., Buie, C. R., Good, B., DeFelice, B., Cava, F., Scaria, J., Sonnenburg, J., Sinderen, D. V., Deutschbauer, A. M., Huang, K. C. 2023

  Abstract

  Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in Bifidobacterium breve (Bb). We generated a high density, randomly barcoded transposon insertion pool in Bb, and used this pool to determine Bb fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. To enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1462 genes. We leveraged these tools to improve models of metabolic pathways, reveal unexpected host- and diet-specific requirements for colonization, and connect the production of immunomodulatory molecules to growth benefits. These resources will greatly reduce the barrier to future investigations of this important beneficial microbe.

  View details for DOI 10.1101/2023.08.29.555234

  View details for PubMedID 37693407

  View details for PubMedCentralID PMC10491234

• Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron. BMC biology Arjes, H. A., Sun, J., Liu, H., Nguyen, T. H., Culver, R. N., Celis, A. I., Walton, S. J., Vasquez, K. S., Yu, F. B., Xue, K. S., Newton, D., Zermeno, R., Weglarz, M., Deutschbauer, A., Huang, K. C., Shiver, A. L. 2022; 20 (1): 285

  Abstract

  Ordered transposon-insertion collections, in which specific transposon-insertion mutants are stored as monocultures in a genome-scale collection, represent a promising tool for genetic dissection of human gut microbiota members. However, publicly available collections are scarce and the construction methodology remains in early stages of development.Here, we describe the assembly of a genome-scale ordered collection of transposon-insertion mutants in the model gut anaerobe Bacteroides thetaiotaomicron VPI-5482 that we created as a resource for the research community. We used flow cytometry to sort single cells from a pooled library, located mutants within this initial progenitor collection by applying a pooling strategy with barcode sequencing, and re-arrayed specific mutants to create a condensed collection with single-insertion strains covering >2500 genes. To demonstrate the potential of the condensed collection for phenotypic screening, we analyzed growth dynamics and cell morphology. We identified both growth defects and altered cell shape in mutants disrupting sphingolipid synthesis and thiamine scavenging. Finally, we analyzed the process of assembling the B. theta condensed collection to identify inefficiencies that limited coverage. We demonstrate as part of this analysis that the process of assembling an ordered collection can be accurately modeled using barcode sequencing data.We expect that utilization of this ordered collection will accelerate research into B. theta physiology and that lessons learned while assembling the collection will inform future efforts to assemble ordered mutant collections for an increasing number of gut microbiota members.

  View details for DOI 10.1186/s12915-022-01481-2

  View details for PubMedID 36527020

• M-TUBE enables large-volume bacterial gene delivery using a high-throughput microfluidic electroporation platform. PLoS biology Huang, P., Chen, S., Shiver, A. L., Culver, R. N., Huang, K. C., Buie, C. R. 2022; 20 (9): e3001727

  Abstract

  Conventional cuvette-based and microfluidics-based electroporation approaches for bacterial gene delivery have distinct advantages, but they are typically limited to relatively small sample volumes, reducing their utility for applications requiring high throughput such as the generation of mutant libraries. Here, we present a scalable, large-scale bacterial gene delivery approach enabled by a disposable, user-friendly microfluidic electroporation device requiring minimal device fabrication and straightforward operation. We demonstrate that the proposed device can outperform conventional cuvettes in a range of situations, including across Escherichia coli strains with a range of electroporation efficiencies, and we use its large-volume bacterial electroporation capability to generate a library of transposon mutants in the anaerobic gut commensal Bifidobacterium longum.

  View details for DOI 10.1371/journal.pbio.3001727

  View details for PubMedID 36067229

• Design, construction, and invivo augmentation of a complex gut microbiome. Cell Cheng, A. G., Ho, P., Aranda-Diaz, A., Jain, S., Yu, F. B., Meng, X., Wang, M., Iakiviak, M., Nagashima, K., Zhao, A., Murugkar, P., Patil, A., Atabakhsh, K., Weakley, A., Yan, J., Brumbaugh, A. R., Higginbottom, S., Dimas, A., Shiver, A. L., Deutschbauer, A., Neff, N., Sonnenburg, J. L., Huang, K. C., Fischbach, M. A. 2022

  Abstract

  Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex, but not both, limiting their utility. Here, we construct and characterize invitro a defined community of 104 bacterial species composed of the most common taxa from the human gut microbiota (hCom1). We then used an iterative experimental process to fill open niches: germ-free mice were colonized with hCom1 and then challenged with a human fecal sample. We identified new species that engrafted following fecal challenge and added them to hCom1, yielding hCom2. In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by either hCom2 or a human fecal community are phenotypically similar, suggesting that this consortium will enable a mechanistic interrogation of species and genes on microbiome-associated phenotypes.

  View details for DOI 10.1016/j.cell.2022.08.003

  View details for PubMedID 36070752

• Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria. Nature protocols Shiver, A. L., Culver, R., Deutschbauer, A. M., Huang, K. C. 2021

  Abstract

  Commensal bacteria from the human intestinal microbiota play important roles in health and disease. Research into the mechanisms by which these bacteria exert their effects is hampered by the complexity of the microbiota, the strict growth requirements of the individual species and a lack of genetic tools and resources. The assembly of ordered transposon insertion libraries, in which nearly all nonessential genes have been disrupted and the strains stored as independent monocultures, would be a transformative resource for research into many microbiota members. However, assembly of these libraries must be fast and inexpensive in order to empower investigation of the large number of species that typically compose gut communities. The methods used to generate ordered libraries must also be adapted to the anaerobic growth requirements of most intestinal bacteria. We have developed a protocol to assemble ordered libraries of transposon insertion mutants that is fast, cheap and effective for even strict anaerobes. The protocol differs from currently available methods by making use of cell sorting to order the library and barcoded transposons to facilitate the localization of ordered mutations in the library. By tracking transposon insertions using barcode sequencing, our approach increases the accuracy and reduces the time and effort required to locate mutants in the library. Ordered libraries can be sorted and characterized over the course of 2 weeks using this approach. We expect this protocol will lower the barrier to generating comprehensive, ordered mutant libraries for many species in the human microbiota, allowing for new investigations into genotype-phenotype relationships within this important microbial ecosystem.

  View details for DOI 10.1038/s41596-021-00531-3

  View details for PubMedID 34021295

• Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs. Molecular cell Shiver, A. L., Osadnik, H., Peters, J. M., Mooney, R. A., Wu, P. I., Henry, K. K., Braberg, H., Krogan, N. J., Hu, J. C., Landick, R., Huang, K. C., Gross, C. A. 2021; 81 (10): 2201

  Abstract

  The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and demonstrate that some RNA polymerase mutants both decrease average cell length and prevent killing by cell-wall targeting antibiotics. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships invivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.

  View details for DOI 10.1016/j.molcel.2021.04.027

  View details for PubMedID 34019789

• Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments. Cell reports Liu, H. n., Shiver, A. L., Price, M. N., Carlson, H. K., Trotter, V. V., Chen, Y. n., Escalante, V. n., Ray, J. n., Hern, K. E., Petzold, C. J., Turnbaugh, P. J., Huang, K. C., Arkin, A. P., Deutschbauer, A. M. 2021; 34 (9): 108789

  Abstract

  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.

  View details for DOI 10.1016/j.celrep.2021.108789

  View details for PubMedID 33657378

• Genetic interaction mapping informs integrative structure determination of protein complexes SCIENCE Braberg, H., Echeverria, I., Bohn, S., Cimermancic, P., Shiver, A., Alexander, R., Xu, J., Shales, M., Dronamraju, R., Jiang, S., Dwivedi, G., Bogdanoff, D., Chaung, K. K., Huttenhain, R., Wang, S., Mavor, D., Pellarin, R., Schneidman, D., Bader, J. S., Fraser, J. S., Morris, J., Haber, J. E., Strahl, B. D., Gross, C. A., Dai, J., Boeke, J. D., Sali, A., Krogan, N. J. 2020; 370 (6522): 1294-+

  Abstract

  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.

  View details for DOI 10.1126/science.aaz4910

  View details for Web of Science ID 000597271300037

  View details for PubMedID 33303586

• Mechanically resolved imaging of bacteria using expansion microscopy. PLoS biology Lim, Y., Shiver, A. L., Khariton, M., Lane, K. M., Ng, K. M., Bray, S. R., Qin, J., Huang, K. C., Wang, B. 2019; 17 (10): e3000268

  Abstract

  Imaging dense and diverse microbial communities has broad applications in basic microbiology and medicine, but remains a grand challenge due to the fact that many species adopt similar morphologies. While prior studies have relied on techniques involving spectral labeling, we have developed an expansion microscopy method (muExM) in which bacterial cells are physically expanded prior to imaging. We find that expansion patterns depend on the structural and mechanical properties of the cell wall, which vary across species and conditions. We use this phenomenon as a quantitative and sensitive phenotypic imaging contrast orthogonal to spectral separation to resolve bacterial cells of different species or in distinct physiological states. Focusing on host-microbe interactions that are difficult to quantify through fluorescence alone, we demonstrate the ability of muExM to distinguish species through an in vitro defined community of human gut commensals and in vivo imaging of a model gut microbiota, and to sensitively detect cell-envelope damage caused by antibiotics or previously unrecognized cell-to-cell phenotypic heterogeneity among pathogenic bacteria as they infect macrophages.

  View details for DOI 10.1371/journal.pbio.3000268

  View details for PubMedID 31622337

• Cutting the Gordian Knot of the Microbiota MOLECULAR CELL Vasquez, K. S., Shiver, A. L., Huang, K. 2018; 70 (5): 765–67

  Abstract

  The gut microbiota plays a central role in human health. Studies by Tramontano et al. (2018) and Maier et al. (2018) improve our understanding of the metabolism and pharmaceutical impact of human gut bacteria through high-throughput screening of growth in the presence of different nutrients and drugs, respectively.

  View details for PubMedID 29883604

• A genome-wide screen in Escherichia coli reveals that ubiquinone is a key antioxidant for metabolism of long-chain fatty acids JOURNAL OF BIOLOGICAL CHEMISTRY Agrawal, S., Jaswal, K., Shiver, A. L., Balecha, H., Patra, T., Chaba, R. 2017; 292 (49): 20086–99

  Abstract

  Long-chain fatty acids (LCFAs) are used as a rich source of metabolic energy by several bacteria including important pathogens. Because LCFAs also induce oxidative stress, which may be detrimental to bacterial growth, it is imperative to understand the strategies employed by bacteria to counteract such stresses. Here, we performed a genetic screen in Escherichia coli on the LCFA, oleate, and compared our results with published genome-wide screens of multiple non-fermentable carbon sources. This large-scale analysis revealed that among components of the aerobic electron transport chain (ETC), only genes involved in the biosynthesis of ubiquinone, an electron carrier in the ETC, are highly required for growth in LCFAs when compared with other carbon sources. Using genetic and biochemical approaches, we show that this increased requirement of ubiquinone is to mitigate elevated levels of reactive oxygen species generated by LCFA degradation. Intriguingly, we find that unlike other ETC components whose requirement for growth is inversely correlated with the energy yield of non-fermentable carbon sources, the requirement of ubiquinone correlates with oxidative stress. Our results therefore suggest that a mechanism in addition to the known electron carrier function of ubiquinone is required to explain its antioxidant role in LCFA metabolism. Importantly, among the various oxidative stress combat players in E. coli, ubiquinone acts as the cell's first line of defense against LCFA-induced oxidative stress. Taken together, our results emphasize that ubiquinone is a key antioxidant during LCFA metabolism and therefore provides a rationale for investigating its role in LCFA-utilizing pathogenic bacteria.

  View details for PubMedID 29042439

  View details for PubMedCentralID PMC5723998

• A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria CELL Peters, J. M., Colavin, A., Shi, H., Czarny, T. L., Larson, M. H., Wong, S., Hawkins, J. S., Lu, C. H., Koo, B., Marta, E., Shiver, A. L., Whitehead, E. H., Weissman, J. S., Brown, E. D., Qi, L. S., Huang, K. C., Gross, C. A. 2016; 165 (6): 1493-1506

  Abstract

  Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.

  View details for DOI 10.1016/j.cell.2016.05.003

  View details for Web of Science ID 000377045400021

  View details for PubMedID 27238023

  View details for PubMedCentralID PMC4894308