Handuo Shi

All Publications

• Abundance measurements reveal the balance between lysis and lysogeny in the human gut microbiome. bioRxiv : the preprint server for biology Lopez, J., McKeithen-Mead, S., Shi, H., Nguyen, T. H., Huang, K. C., Good, B. H. 2024

  Abstract

  The human gut contains diverse communities of bacteriophage, whose interactions with the broader microbiome and potential roles in human health are only beginning to be uncovered. Here, we combine multiple types of data to quantitatively estimate gut phage population dynamics and lifestyle characteristics in human subjects. Unifying results from previous studies, we show that an average human gut contains a low ratio of phage particles to bacterial cells (~1:100), but a much larger ratio of phage genomes to bacterial genomes (~4:1), implying that most gut phage are effectively temperate (e.g., integrated prophage, phage-plasmids, etc.). By integrating imaging and sequencing data with a generalized model of temperate phage dynamics, we estimate that phage induction and lysis occurs at a low average rate (~0.001-0.01 per bacterium per day), imposing only a modest fitness burden on their bacterial hosts. Consistent with these estimates, we find that the phage composition of a diverse synthetic community in gnotobiotic mice can be quantitatively predicted from bacterial abundances alone, while still exhibiting phage diversity comparable to native human microbiomes. These results provide a foundation for interpreting existing and future studies on links between the gut virome and human health.

  View details for DOI 10.1101/2024.09.27.614587

  View details for PubMedID 39386523

• Complex state transitions of the bacterial cell division protein FtsZ. Molecular biology of the cell Knapp, B. D., Shi, H., Huang, K. C. 2024: mbcE23110446

  Abstract

  The key bacterial cell division protein FtsZ can adopt multiple conformations and prevailing models suggest that transitions from the closed to open state are necessary for filament formation and stability. Using all-atom molecular dynamics simulations, we analyzed state transitions of Staphylococcus aureus FtsZ as a monomer, dimer, and hexamer. We found that monomers can adopt intermediate states but preferentially adopt a closed state that is robust to forced re-opening. Dimer subunits transitioned between open and closed states, and dimers with both subunits in the closed state remained highly stable, suggesting that open-state conformations are not necessary for filament formation. Mg2+ strongly stabilized the conformation of GTP-bound subunits and the dimer filament interface. Our hexamer simulations indicate that the plus end subunit preferentially closes and that other subunits can transition between states without affecting inter-subunit stability. We found that rather than being correlated with subunit opening, inter-subunit stability was strongly correlated with catalytic site interactions. By leveraging deep-learning models, we identified key intra-subunit interactions governing state transitions. Our findings suggest a greater range of possible monomer and filament states than previously considered, and offer new insights into the nuanced interplay between subunit states and the critical role of nucleotide hydrolysis and Mg2+ in FtsZ filament dynamics.

  View details for DOI 10.1091/mbc.E23-11-0446

  View details for PubMedID 39083352

• Profiling the human intestinal environment under physiological conditions. Nature Shalon, D., Culver, R. N., Grembi, J. A., Folz, J., Treit, P. V., Shi, H., Rosenberger, F. A., Dethlefsen, L., Meng, X., Yaffe, E., Aranda-Diaz, A., Geyer, P. E., Mueller-Reif, J. B., Spencer, S., Patterson, A. D., Triadafilopoulos, G., Holmes, S. P., Mann, M., Fiehn, O., Relman, D. A., Huang, K. C. 2023

  Abstract

  The spatiotemporal structure of the human microbiome1,2, proteome3 and metabolome4,5 reflects and determines regional intestinal physiology and may have implications for disease6. Yet, little is known about the distribution of microorganisms, their environment and their biochemical activity in the gut because of reliance on stool samples and limited access to only some regions of the gut using endoscopy in fasting or sedated individuals7. To address these deficiencies, we developed an ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion. Collection of 240 intestinal samples from 15 healthy individuals using the device and subsequent multi-omics analyses identified significant differences between bacteria, phages, host proteins and metabolites in the intestines versus stool. Certain microbial taxa were differentially enriched and prophage induction was more prevalent in the intestines than in stool. The host proteome and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundance predicted species that altered the bile acid pool through deconjugation. Furthermore, microbially conjugated bile acid concentrations exhibited amino acid-dependent trends that were not apparent in stool. Overall, non-invasive, longitudinal profiling of microorganisms, proteins and bile acids along the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in human physiology and disease.

  View details for DOI 10.1038/s41586-023-05989-7

  View details for PubMedID 37165188

• Physiological and regulatory convergence between osmotic and nutrient stress responses in microbes. Current opinion in cell biology Brauer, A. M., Shi, H., Levin, P. A., Huang, K. C. 2023; 81: 102170

  Abstract

  Bacterial cells are regularly confronted with simultaneous changes in environmental nutrient supply and osmolarity. Despite the importance of osmolarity and osmoregulation in bacterial physiology, the relationship between the cellular response to osmotic perturbations and other stresses has remained largely unexplored. Bacteria cultured in hyperosmotic conditions and bacteria experiencing nutrient stress exhibit similar physiological changes, including metabolic shutdown, increased protein instability, dehydration, and condensation of chromosomal DNA. In this review, we highlight overlapping molecular players between osmotic and nutrient stresses. These connections between two seemingly disparate stress response pathways reinforce the importance of central carbon metabolism as a control point for diverse aspects of homeostatic regulation. We identify important open questions for future research, emphasizing the pressing need to develop and exploit new methods for probing how osmolarity affects phylogenetically diverse species.

  View details for DOI 10.1016/j.ceb.2023.102170

  View details for PubMedID 37119759

• Previously uncharacterized rectangular bacterial structures in the dolphin mouth. Nature communications Dudek, N. K., Galaz-Montoya, J. G., Shi, H., Mayer, M., Danita, C., Celis, A. I., Viehboeck, T., Wu, G., Behr, B., Bulgheresi, S., Huang, K. C., Chiu, W., Relman, D. A. 2023; 14 (1): 2098

  Abstract

  Much remains to be explored regarding the diversity of uncultured, host-associated microbes. Here, we describe rectangular bacterial structures (RBSs) in the mouths of bottlenose dolphins. DNA staining revealed multiple paired bands within RBSs, suggesting the presence of cells dividing along the longitudinal axis. Cryogenic transmission electron microscopy and tomography showed parallel membrane-bound segments that are likely cells, encapsulated by an S-layer-like periodic surface covering. RBSs displayed unusual pilus-like appendages with bundles of threads splayed at the tips. We present multiple lines of evidence, including genomic DNA sequencing of micromanipulated RBSs, 16S rRNA gene sequencing, and fluorescence in situ hybridization, suggesting that RBSs are bacterial and distinct from the genera Simonsiella and Conchiformibius (family Neisseriaceae), with which they share similar morphology and division patterning. Our findings highlight the diversity of novel microbial forms and lifestyles that await characterization using tools complementary to genomics such as microscopy.

  View details for DOI 10.1038/s41467-023-37638-y

  View details for PubMedID 37055390

• Optimization of the 16S rRNA sequencing analysis pipeline for studying invitro communities of gut commensals. iScience Celis, A. I., Aranda-Diaz, A., Culver, R., Xue, K., Relman, D., Shi, H., Huang, K. C. 2022; 25 (4): 103907

  Abstract

  While microbial communities inhabit a wide variety of complex natural environments, invitro culturing enables highly controlled conditions and high-throughput interrogation for generating mechanistic insights. Invitro assemblies of gut commensals have recently been introduced as models for the intestinal microbiota, which plays fundamental roles in host health. However, a protocol for 16S rRNA sequencing and analysis of invitro samples that optimizes financial cost, time/effort, and accuracy/reproducibility has yet to be established. Here, we systematically identify protocol elements that have significant impact, introduce bias, and/or can be simplified. Our results indicate that community diversity and composition are generally unaffected by substantial protocol streamlining. Additionally, we demonstrate that a strictly aerobic halophile is an effective spike-in for estimating absolute abundances in communities of anaerobic gut commensals. This time- and money-saving protocol should accelerate discovery by increasing 16S rRNA data reliability and comparability and through the incorporation of absolute abundance estimates.

  View details for DOI 10.1016/j.isci.2022.103907

  View details for PubMedID 35340431

• Multiple conserved states characterize the twist landscape of the bacterial actin homolog MreB. Computational and structural biotechnology journal Knapp, B. D., Ward, M. D., Bowman, G. R., Shi, H., Huang, K. C. 2022; 20: 5838-5846

  Abstract

  Filament formation by cytoskeletal proteins is critical to their involvement in myriad cellular processes. The bacterial actin homolog MreB, which is essential for cell-shape determination in many rod-shaped bacteria, has served as a model system for studying the mechanics of cytoskeletal filaments. Previous molecular dynamics (MD) simulations revealed that the twist of MreB double protofilaments is dependent on the bound nucleotide, as well as binding to the membrane or the accessory protein RodZ, and MreB mutations that modulate twist also affect MreB spatial organization and cell shape. Here, we show that MreB double protofilaments can adopt multiple twist states during microsecond-scale MD simulations. A deep learning algorithm trained only on high- and low-twist states robustly identified all twist conformations across most perturbations of ATP-bound MreB, suggesting the existence of a conserved set of states whose occupancy is affected by each perturbation to MreB. Simulations replacing ATP with ADP indicated that twist states were generally stable after hydrolysis. These findings suggest a rich twist landscape that could provide the capacity to tune MreB activity and therefore its effects on cell shape.

  View details for DOI 10.1016/j.csbj.2022.10.008

  View details for PubMedID 36382191

• Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli. mBio Silvis, M. R., Rajendram, M., Shi, H., Osadnik, H., Gray, A. N., Cesar, S., Peters, J. M., Hearne, C. C., Kumar, P., Todor, H., Huang, K. C., Gross, C. A. 2021: e0256121

  Abstract

  CRISPR interference (CRISPRi) has facilitated the study of essential genes in diverse organisms using both high-throughput and targeted approaches. Despite the promise of this technique, no comprehensive arrayed CRISPRi library targeting essential genes exists for the model bacterium Escherichia coli, or for any Gram-negative species. Here, we built and characterized such a library. Each of the 500 strains in our E. coli library contains an inducible, chromosomally integrated single guide RNA (sgRNA) targeting an essential (or selected nonessential) gene and can be mated with a pseudo-Hfr donor strain carrying a dcas9 cassette to create a CRISPRi knockdown strain. Using this system, we built an arrayed library of CRISPRi strains and performed population and single-cell growth and morphology measurements as well as targeted follow-up experiments. These studies found that inhibiting translation causes an extended lag phase, identified new modulators of cell morphology, and revealed that the morphogene mreB is subject to transcriptional feedback regulation, which is critical for the maintenance of morphology. Our findings highlight canonical and noncanonical roles for essential genes in numerous aspects of cellular homeostasis. IMPORTANCE Essential genes make up only 5 to 10% of the genetic complement in most organisms but occupy much of their protein synthesis and account for almost all antibiotic targets. Despite the importance of essential genes, their intractability has, until recently, hampered efforts to study them. CRISPRi has facilitated the study of essential genes by allowing inducible and titratable depletion. However, all large-scale CRISPRi studies in Gram-negative bacteria thus far have used plasmids to express CRISPRi components and have been constructed in pools, limiting their utility for targeted assays and complicating the determination of antibiotic effects. Here, we use a modular method to construct an arrayed library of chromosomally integrated CRISPRi strains targeting the essential genes of the model bacterium Escherichia coli. This library enables targeted studies of essential gene depletions and high-throughput determination of antibiotic targets and facilitates studies targeting the outer membrane, an essential component that serves as the major barrier to antibiotics.

  View details for DOI 10.1128/mBio.02561-21

  View details for PubMedID 34634934

• Starvation induces shrinkage of the bacterial cytoplasm. Proceedings of the National Academy of Sciences of the United States of America Shi, H., Westfall, C. S., Kao, J., Odermatt, P. D., Anderson, S. E., Cesar, S., Sievert, M., Moore, J., Gonzalez, C. G., Zhang, L., Elias, J. E., Chang, F., Huang, K. C., Levin, P. A. 2021; 118 (24)

  Abstract

  Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli's inner membrane from the cell wall. Shrinkage was accompanied by an 17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.

  View details for DOI 10.1073/pnas.2104686118

  View details for PubMedID 34117124

• Precise regulation of the relative rates of surface area and volume synthesis in bacterial cells growing in dynamic environments. Nature communications Shi, H., Hu, Y., Odermatt, P. D., Gonzalez, C. G., Zhang, L., Elias, J. E., Chang, F., Huang, K. C. 2021; 12 (1): 1975

  Abstract

  The steady-state size of bacterial cells correlates with nutrient-determined growth rate. Here, we explore how rod-shaped bacterial cells regulate their morphology during rapid environmental changes. We quantify cellular dimensions throughout passage cycles of stationary-phase cells diluted into fresh medium and grown back to saturation. We find that cells exhibit characteristic dynamics in surface area to volume ratio (SA/V), which are conserved across genetic and chemical perturbations as well as across species and growth temperatures. A mathematical model with a single fitting parameter (the time delay between surface and volume synthesis) is quantitatively consistent with our SA/V experimental observations. The model supports that this time delay is due to differential expression of volume and surface-related genes, and that the first division after dilution occurs at a tightly controlled SA/V. Our minimal model thus provides insight into the connections between bacterial growth rate and cell shape in dynamic environments.

  View details for DOI 10.1038/s41467-021-22092-5

  View details for PubMedID 33785742

• Hyperosmotic Shock Transiently Accelerates Constriction Rate in Escherichia coli. Frontiers in microbiology Sun, J., Shi, H., Huang, K. C. 2021; 12: 718600

  Abstract

  Bacterial cells in their natural environments encounter rapid and large changes in external osmolality. For instance, enteric bacteria such as Escherichia coli experience a rapid decrease when they exit from host intestines. Changes in osmolality alter the mechanical load on the cell envelope, and previous studies have shown that large osmotic shocks can slow down bacterial growth and impact cytoplasmic diffusion. However, it remains unclear how cells maintain envelope integrity and regulate envelope synthesis in response to osmotic shocks. In this study, we developed an agarose pad-based protocol to assay envelope stiffness by measuring population-averaged cell length before and after a hyperosmotic shock. Pad-based measurements exhibited an apparently larger length change compared with single-cell dynamics in a microfluidic device, which we found was quantitatively explained by a transient increase in division rate after the shock. Inhibiting cell division led to consistent measurements between agarose pad-based and microfluidic measurements. Directly after hyperosmotic shock, FtsZ concentration and Z-ring intensity increased, and the rate of septum constriction increased. These findings establish an agarose pad-based protocol for quantifying cell envelope stiffness, and demonstrate that mechanical perturbations can have profound effects on bacterial physiology.

  View details for DOI 10.3389/fmicb.2021.718600

  View details for PubMedID 34489908

• Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth. mBio Atolia, E., Cesar, S., Arjes, H. A., Rajendram, M., Shi, H., Knapp, B. D., Khare, S., Aranda-Diaz, A., Lenski, R. E., Huang, K. C. 2020; 11 (5)

  Abstract

  Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth.IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

  View details for DOI 10.1128/mBio.01378-20

  View details for PubMedID 33082255

• The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America Grimm, J., Shi, H., Wang, W., Mitchell, A. M., Wingreen, N. S., Huang, K. C., Silhavy, T. J. 2020

  Abstract

  The outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and beta-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA*, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA*-mediated lysis suggested that PL transport can occur via a high-flux diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA*-mediated lysis. Deletion of yhdP from mlaA* reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of ∆yhdP cells was further compromised and ruptured before the IM during mlaA*-mediated death. These findings demonstrate the existence of a high-flux diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP.

  View details for DOI 10.1073/pnas.2015556117

  View details for PubMedID 33046656

• Pictures of Tongues Sticking Out. Trends in endocrinology and metabolism: TEM Shi, H., Huang, K. C. 2020

  Abstract

  Despite their small sizes, bacterial cells within a host-associated microbial community often form highly structured complexes determined by environmental factors and interspecies interactions. Wilbert et al. combined species-specific fluorescent labels and high-resolution microscopy to visualize human tongue dorsum microbiomes and to highlight their structure and dynamics.

  View details for DOI 10.1016/j.tem.2020.05.003

  View details for PubMedID 32475653

• Chiral twisting in a bacterial cytoskeletal polymer affects filament size and orientation. Nature communications Shi, H., Quint, D. A., Grason, G. M., Gopinathan, A., Huang, K. C. 2020; 11 (1): 1408

  Abstract

  In many rod-shaped bacteria, the actin homolog MreB directs cell-wall insertion and maintains cell shape, but it remains unclear how structural changes to MreB affect its organization in vivo. Here, we perform molecular dynamics simulations for Caulobacter crescentus MreB to extract mechanical parameters for inputs into a coarse-grained biophysical polymer model that successfully predicts MreB filament properties in vivo. Our analyses indicate that MreB double protofilaments can exhibit left-handed twisting that is dependent on the bound nucleotide and membrane binding; the degree of twisting correlates with the length and orientation of MreB filaments observed in vitro and in vivo. Our molecular dynamics simulations also suggest that membrane binding of MreB double protofilaments induces a stable membrane curvature of similar magnitude to that observed in vivo. Thus, our multiscale modeling correlates cytoskeletal filament size with conformational changes inferred from molecular dynamics simulations, providing a paradigm for connecting protein filament structure and mechanics to cellular organization and function.

  View details for DOI 10.1038/s41467-020-14752-9

  View details for PubMedID 32179732

• Klebsiella michiganensis transmission enhances resistance to Enterobacteriaceae gut invasion by nutrition competition. Nature microbiology Oliveira, R. A., Ng, K. M., Correia, M. B., Cabral, V., Shi, H., Sonnenburg, J. L., Huang, K. C., Xavier, K. B. 2020

  Abstract

  Intestinal microbiotas contain beneficial microorganisms that protect against pathogen colonization; treatment with antibiotics disrupts the microbiota and compromises colonization resistance. Here, we determine the impact of exchanging microorganisms between hosts on resilience to the colonization of invaders after antibiotic-induced dysbiosis. We assess the functional consequences of dysbiosis using a mouse model of colonization resistance against Escherichia coli. Antibiotics caused stochastic loss of members of the microbiota, but the microbiotas of co-housed mice remained more similar to each other compared with the microbiotas among singly housed animals. Strikingly, co-housed mice maintained colonization resistance after treatment with antibiotics, whereas most singly housed mice were susceptible to E. coli. The ability to retain or share the commensal Klebsiella michiganensis, a member of the Enterobacteriaceae family, was sufficient for colonization resistance after treatment with antibiotics. K. michiganensis generally outcompeted E. coli in vitro, but in vivo administration of galactitol-a nutrient that supports the growth of only E. coli-to bi-colonized gnotobiotic mice abolished the colonization-resistance capacity of K. michiganensis against E. coli, supporting the idea that nutrient competition is the primary interaction mechanism. K. michiganensis also hampered colonization of the pathogen Salmonella, prolonging host survival. Our results address functional consequences of the stochastic effects of microbiota perturbations, whereby microbial transmission through host interactions can facilitate reacquisition of beneficial commensals, minimizing the negative impact of antibiotics.

  View details for DOI 10.1038/s41564-019-0658-4

  View details for PubMedID 31959968

• AimB Is a Small Protein Regulator of Cell Size and MreB Assembly. Biophysical journal Werner, J. N., Shi, H. n., Hsin, J. n., Huang, K. C., Gitai, Z. n., Klein, E. A. 2020

  Abstract

  The MreB actin-like cytoskeleton assembles into dynamic polymers that coordinate cell shape in many bacteria. In contrast to most other cytoskeleton systems, few MreB-interacting proteins have been well characterized. Here, we identify a small protein from Caulobacter crescentus, an assembly inhibitor of MreB (AimB). AimB overexpression mimics inhibition of MreB polymerization, leading to increased cell width and MreB delocalization. Furthermore, aimB appears to be essential, and its depletion results in decreased cell width and increased resistance to A22, a small-molecule inhibitor of MreB assembly. Molecular dynamics simulations suggest that AimB binds MreB at its monomer-monomer protofilament interaction cleft and that this interaction is favored for C. crescentus MreB over Escherichia coli MreB because of a closer match in the degree of opening with AimB size, suggesting coevolution of AimB with MreB conformational dynamics in C. crescentus. We support this model through functional analysis of point mutants in both AimB and MreB, photo-cross-linking studies with site-specific unnatural amino acids, and species-specific activity of AimB. Together, our findings are consistent with AimB promoting MreB dynamics by inhibiting monomer-monomer assembly interactions, representing a new mechanism for regulating actin-like polymers and the first identification of a non-toxin MreB assembly inhibitor. Because AimB has only 104 amino acids and small proteins are often poorly characterized, our work suggests the possibility of more bacterial cytoskeletal regulators to be found in this class. Thus, like FtsZ and eukaryotic actin, MreB may have a rich repertoire of regulators to tune its precise assembly and dynamics.

  View details for DOI 10.1016/j.bpj.2020.04.029

  View details for PubMedID 32416080

• FtsZ-Independent Mechanism of Division Inhibition by the Small Molecule PC190723 in Escherichia coli. Advanced biosystems Khare, S., Hsin, J., Sorto, N. A., Nepomuceno, G. M., Shaw, J. T., Shi, H., Huang, K. C. 2019; 3 (11): e1900021

  Abstract

  While cell division is a critical process in cellular proliferation, very few antibiotics have been identified that target the bacterial cell-division machinery. Recent studies have shown that the small molecule PC190723 inhibits cell division in several Gram-positive bacteria, with a hypothesized mechanism of action involving direct targeting of the tubulin homolog FtsZ, which is essential for division in virtually all bacterial species. Here, it is shown that PC190723 also inhibits cell division in the Gram-negative bacterium Escherichia coli if the outer membrane permeability barrier is compromised genetically or chemically. The results show that the equivalent FtsZ mutations conferring PC190723 resistance in Staphylococcus aureus do not protect E. coli against PC190723, and that suppressors of PC190723 sensitivity in E. coli, which do not generically decrease outer membrane permeability, do not map to FtsZ or other division proteins. These suppressors display a wide range of morphological and growth phenotypes, and one exhibits a death phenotype in the stationary phase similar to that of a mutant with disrupted lipid homeostasis. Finally, a complementing FtsZ-msfGFP fusion is used to show that PC190723 does not affect the Z-ring structure. Taken together, the findings suggest that PC190723 inhibits growth and division in E. coli without targeting FtsZ. This study highlights the importance of utilizing a combination of genetic, chemical, and single-cell approaches to dissect the mechanisms of action of new antibiotics, which are not necessarily conserved across bacterial species.

  View details for DOI 10.1002/adbi.201900021

  View details for PubMedID 32648693

• FtsZ-Independent Mechanism of Division Inhibition by the Small Molecule PC190723 in Escherichia coli ADVANCED BIOSYSTEMS Khare, S., Hsin, J., Sorto, N. A., Nepomuceno, G. M., Shaw, J. T., Shi, H., Huang, K. 2019; 3 (11)

  View details for DOI 10.1002/adbi.201900021

  View details for Web of Science ID 000498192300009

• Chromosome Organization: Making Room in a Crowd. Current biology : CB Shi, H., Huang, K. C. 2019; 29 (13): R630–R632

  Abstract

  Despite their small size and lack of membrane-based DNA encapsulation, prokaryotic cells still organize and scale their nucleoid in specific subcellular regions. Two studies show that the DNA-free regions in prokaryotes are full of large biomolecules, which exclude DNA via entropic forces.

  View details for DOI 10.1016/j.cub.2019.06.002

  View details for PubMedID 31287980

• Conservation of conformational dynamics across prokaryotic actins. PLoS computational biology Ng, N., Shi, H., Colavin, A., Huang, K. C. 2019; 15 (4): e1006683

  Abstract

  The actin family of cytoskeletal proteins is essential to the physiology of virtually all archaea, bacteria, and eukaryotes. While X-ray crystallography and electron microscopy have revealed structural homologies among actin-family proteins, these techniques cannot probe molecular-scale conformational dynamics. Here, we use all-atom molecular dynamic simulations to reveal conserved dynamical behaviors in four prokaryotic actin homologs: MreB, FtsA, ParM, and crenactin. We demonstrate that the majority of the conformational dynamics of prokaryotic actins can be explained by treating the four subdomains as rigid bodies. MreB, ParM, and FtsA monomers exhibited nucleotide-dependent dihedral and opening angles, while crenactin monomer dynamics were nucleotide-independent. We further show that the opening angle of ParM is sensitive to a specific interaction between subdomains. Steered molecular dynamics simulations of MreB, FtsA, and crenactin dimers revealed that changes in subunit dihedral angle lead to intersubunit bending or twist, suggesting a conserved mechanism for regulating filament structure. Taken together, our results provide molecular-scale insights into the nucleotide and polymerization dependencies of the structure of prokaryotic actins, suggesting mechanisms for how these structural features are linked to their diverse functions.

  View details for PubMedID 30951524

• RodZ modulates geometric localization of the bacterial actin MreB to regulate cell shape NATURE COMMUNICATIONS Colavin, A., Shi, H., Huang, K. 2018; 9: 1280

  Abstract

  In the rod-shaped bacterium Escherichia coli, the actin-like protein MreB localizes in a curvature-dependent manner and spatially coordinates cell-wall insertion to maintain cell shape, although the molecular mechanism by which cell width is regulated remains unknown. Here we demonstrate that the membrane protein RodZ regulates the biophysical properties of MreB and alters the spatial organization of E. coli cell-wall growth. The relative expression levels of MreB and RodZ change in a manner commensurate with variations in growth rate and cell width, and RodZ systematically alters the curvature-based localization of MreB and cell width in a concentration-dependent manner. We identify MreB mutants that alter the bending properties of MreB filaments in molecular dynamics simulations similar to RodZ binding, and show that these mutants rescue rod-like shape in the absence of RodZ alone or in combination with wild-type MreB. Thus, E. coli can control its shape and dimensions by differentially regulating RodZ and MreB to alter the patterning of cell-wall insertion, highlighting the rich regulatory landscape of cytoskeletal molecular biophysics.

  View details for PubMedID 29599448

• How to Build a Bacterial Cell: MreB as the Foreman of E. coli Construction CELL Shi, H., Bratton, B. P., Gitai, Z., Huang, K. 2018; 172 (6): 1294–1305

  Abstract

  Cell shape matters across the kingdoms of life, and cells have the remarkable capacity to define and maintain specific shapes and sizes. But how are the shapes of micron-sized cells determined from the coordinated activities of nanometer-sized proteins? Here, we review general principles that have surfaced through the study of rod-shaped bacterial growth. Imaging approaches have revealed that polymers of the actin homolog MreB play a central role. MreB both senses and changes cell shape, thereby generating a self-organizing feedback system for shape maintenance. At the molecular level, structural and computational studies indicate that MreB filaments exhibit tunable mechanical properties that explain their preference for certain geometries and orientations along the cylindrical cell body. We illustrate the regulatory landscape of rod-shape formation and the connectivity between cell shape, cell growth, and other aspects of cell physiology. These discoveries provide a framework for future investigations into the architecture and construction of microbes.

  View details for PubMedID 29522748

  View details for PubMedCentralID PMC5846203

• Deep Phenotypic Mapping of Bacterial Cytoskeletal Mutants Reveals Physiological Robustness to Cell Size CURRENT BIOLOGY Shi, H., Colavin, A., Bigos, M., Tropini, C., Monds, R. D., Huang, K. 2017; 27 (22): 3419-+

  Abstract

  Size is a universally defining characteristic of all living cells and tissues and is intrinsically linked with cell genotype, growth, and physiology. Many mutations have been identified to alter cell size, but pleiotropic effects have largely hampered our ability to probe how cell size specifically affects fundamental cellular properties, such as DNA content and intracellular localization. To systematically interrogate the impact of cell morphology on bacterial physiology, we used fluorescence-activated cell sorting to enrich a library of hundreds of Escherichia coli mutants in the essential cytoskeletal protein MreB for subtle changes in cell shape, cumulatively spanning ∼5-fold variation in average cell volume. Critically, pleiotropic effects in the mutated library are most likely minimized because only one gene was mutated and because growth rate was unaffected, thereby allowing us to query the general effects of morphology on cellular physiology over a large range of cell sizes with high resolution. We discovered linear scaling of the abundance of DNA and the key division protein FtsZ with cell volume, a strong dependency of sensitivity to specific antibiotics on cell width, and a simple correlation between MreB localization pattern and cell width. Our systematic, quantitative approach reveals complex and dynamic links between bacterial morphology and physiology and should be generally applicable for probing size-related genotype-phenotype relationships.

  View details for PubMedID 29103935

• Rapid, precise quantification of bacterial cellular dimensions across a genomic-scale knockout library. BMC biology Ursell, T., Lee, T. K., Shiomi, D., Shi, H., Tropini, C., Monds, R. D., Colavin, A., Billings, G., Bhaya-Grossman, I., Broxton, M., Huang, B. E., Niki, H., Huang, K. C. 2017; 15 (1): 17-?

  Abstract

  The determination and regulation of cell morphology are critical components of cell-cycle control, fitness, and development in both single-cell and multicellular organisms. Understanding how environmental factors, chemical perturbations, and genetic differences affect cell morphology requires precise, unbiased, and validated measurements of cell-shape features.Here we introduce two software packages, Morphometrics and BlurLab, that together enable automated, computationally efficient, unbiased identification of cells and morphological features. We applied these tools to bacterial cells because the small size of these cells and the subtlety of certain morphological changes have thus far obscured correlations between bacterial morphology and genotype. We used an online resource of images of the Keio knockout library of nonessential genes in the Gram-negative bacterium Escherichia coli to demonstrate that cell width, width variability, and length significantly correlate with each other and with drug treatments, nutrient changes, and environmental conditions. Further, we combined morphological classification of genetic variants with genetic meta-analysis to reveal novel connections among gene function, fitness, and cell morphology, thus suggesting potential functions for unknown genes and differences in modes of action of antibiotics.Morphometrics and BlurLab set the stage for future quantitative studies of bacterial cell shape and intracellular localization. The previously unappreciated connections between morphological parameters measured with these software packages and the cellular environment point toward novel mechanistic connections among physiological perturbations, cell fitness, and growth.

  View details for DOI 10.1186/s12915-017-0348-8

  View details for PubMedID 28222723

  View details for PubMedCentralID PMC5320674

• Strain Library Imaging Protocol for high-throughput, automated single-cell microscopy of large bacterial collections arrayed on multiwell plates. Nature protocols Shi, H., Colavin, A., Lee, T. K., Huang, K. C. 2017; 12 (2): 429-438

  Abstract

  Single-cell microscopy is a powerful tool for studying gene functions using strain libraries, but it suffers from throughput limitations. Here we describe the Strain Library Imaging Protocol (SLIP), which is a high-throughput, automated microscopy workflow for large strain collections that requires minimal user involvement. SLIP involves transferring arrayed bacterial cultures from multiwell plates onto large agar pads using inexpensive replicator pins and automatically imaging the resulting single cells. The acquired images are subsequently reviewed and analyzed by custom MATLAB scripts that segment single-cell contours and extract quantitative metrics. SLIP yields rich data sets on cell morphology and gene expression that illustrate the function of certain genes and the connections among strains in a library. For a library arrayed on 96-well plates, image acquisition can be completed within 4 min per plate.

  View details for DOI 10.1038/nprot.2016.181

  View details for PubMedID 28125106

• Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells NATURE COMMUNICATIONS Lee, T. K., Meng, K., Shi, H., Huang, K. C. 2016; 7

  Abstract

  The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation. We observed heterogeneous localization dynamics of penicillin-binding protein (PBP) 1A, the synthase predominantly associated with cell wall elongation, with individual PBP1A molecules distributed between mobile and immobile populations. Perturbations to PBP1A activity, either directly through antibiotics or indirectly through PBP1A's interaction with its lipoprotein activator or other synthases, shifted the fraction of mobile molecules. Our results suggest that multiple levels of regulation control the activity of enzymes to coordinate peptidoglycan synthesis.

  View details for DOI 10.1038/ncomms13170

  View details for Web of Science ID 000385916600001

  View details for PubMedID 27774981

  View details for PubMedCentralID PMC5078992

• 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

• Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sutterlin, H. A., Shi, H., May, K. L., Miguel, A., Khare, S., Huang, K. C., Silhavy, T. J. 2016; 113 (11): E1565-E1574

  View details for DOI 10.1073/pnas.1601375113

  View details for Web of Science ID 000372014200020