Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation.

Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as "variants of uncertain significance" (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

Applications of artificial intelligence in clinical laboratory genomics.

The transition from analog to digital technologies in clinical laboratory genomics is ushering in an era of "big data" in ways that will exceed human capacity to rapidly and reproducibly analyze those data using conventional approaches. Accurately evaluating complex molecular data to facilitate timely diagnosis and management of genomic disorders will require supportive artificial intelligence methods. These are already being introduced into clinical laboratory genomics to identify variants in DNA sequencing data, predict the effects of DNA variants on protein structure and function to inform clinical interpretation of pathogenicity, link phenotype ontologies to genetic variants identified through exome or genome sequencing to help clinicians reach diagnostic answers faster, correlate genomic data with tumor staging and treatment approaches, utilize natural language processing to identify critical published medical literature during analysis of genomic data, and use interactive chatbots to identify individuals who qualify for genetic testing or to provide pre-test and post-test education. With careful and ethical development and validation of artificial intelligence for clinical laboratory genomics, these advances are expected to significantly enhance the abilities of geneticists to translate complex data into clearly synthesized information for clinicians to use in managing the care of their patients at scale.

Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation.

Defects in hydroxymethylbilane synthase (HMBS) can cause Acute Intermittent Porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ~⅓ of clinical HMBS variants are missense variants, and most clinically-reported HMBS missense variants are designated as "variants of uncertain significance" (VUS). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino-acid substitutions. The resulting variant effect maps generally agreed with biochemical expectation. However, the maps showed variants at the dimerization interface to be unexpectedly well tolerated, and suggested residue roles in active site dynamics that were supported by molecular dynamics simulations. Most importantly, these HMBS variant effect maps can help discriminate pathogenic from benign variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

Extracting phylogenetic dimensions of coevolution reveals hidden functional signals.

Despite the structural and functional information contained in the statistical coupling between pairs of residues in a protein, coevolution associated with function is often obscured by artifactual signals such as genetic drift, which shapes a protein's phylogenetic history and gives rise to concurrent variation between protein sequences that is not driven by selection for function. Here, we introduce a background model for phylogenetic contributions of statistical coupling that separates the coevolution signal due to inter-clade and intra-clade sequence comparisons and demonstrate that coevolution can be measured on multiple phylogenetic timescales within a single protein. Our method, nested coevolution (NC), can be applied as an extension to any coevolution metric. We use NC to demonstrate that poorly conserved residues can nonetheless have important roles in protein function. Moreover, NC improved the structural-contact predictions of several coevolution-based methods, particularly in subsampled alignments with fewer sequences. NC also lowered the noise in detecting functional sectors of collectively coevolving residues. Sectors of coevolving residues identified after application of NC were more spatially compact and phylogenetically distinct from the rest of the protein, and strongly enriched for mutations that disrupt protein activity. Thus, our conceptualization of the phylogenetic separation of coevolution provides the potential to further elucidate relationships among protein evolution, function, and genetic diseases.

Spectrum of splicing variants in disease genes and the ability of RNA analysis to reduce uncertainty in clinical interpretation.

The complexities of gene expression pose challenges for the clinical interpretation of splicing variants. To better understand splicing variants and their contribution to hereditary disease, we evaluated their prevalence, clinical classifications, and associations with diseases, inheritance, and functional characteristics in a 689,321-person clinical cohort and two large public datasets. In the clinical cohort, splicing variants represented 13% of all variants classified as pathogenic (P), likely pathogenic (LP), or variants of uncertain significance (VUSs). Most splicing variants were outside essential splice sites and were classified as VUSs. Among all individuals tested, 5.4% had a splicing VUS. If RNA analysis were to contribute supporting evidence to variant interpretation, we estimated that splicing VUSs would be reclassified in 1.7% of individuals in our cohort. This would result in a clinically significant result (i.e., P/LP) in 0.1% of individuals overall because most reclassifications would change VUSs to likely benign. In ClinVar, splicing VUSs were 4.8% of reported variants and could benefit from RNA analysis. In the Genome Aggregation Database (gnomAD), splicing variants comprised 9.4% of variants in protein-coding genes; most were rare, precluding unambiguous classification as benign. Splicing variants were depleted in genes associated with dominant inheritance and haploinsufficiency, although some genes had rare variants at essential splice sites or had common splicing variants that were most likely compatible with normal gene function. Overall, we describe the contribution of splicing variants to hereditary disease, the potential utility of RNA analysis for reclassifying splicing VUSs, and how natural variation may confound clinical interpretation of splicing variants.

Conservation of conformational dynamics across prokaryotic actins.

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.

RodZ modulates geometric localization of the bacterial actin MreB to regulate cell shape.

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.

Deep Phenotypic Mapping of Bacterial Cytoskeletal Mutants Reveals Physiological Robustness to Cell Size.

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.

Coupling between Protein Stability and Catalytic Activity Determines Pathogenicity of G6PD Variants.

G6PD deficiency, an enzymopathy affecting 7% of the world population, is caused by over 160 identified amino acid variants in glucose-6-phosphate dehydrogenase (G6PD). The clinical presentation of G6PD deficiency is diverse, likely due to the broad distribution of variants across the protein and the potential for multidimensional biochemical effects. In this study, we use bioinformatic and biochemical analyses to interpret the relationship between G6PD variants and their clinical phenotype. Using structural information and statistical analyses of known G6PD variants, we predict the molecular phenotype of five uncharacterized variants from a reference population database. Through multidimensional analysis of biochemical data, we demonstrate that the clinical phenotypes of G6PD variants are largely determined by a trade-off between protein stability and catalytic activity. This work expands the current understanding of the biochemical underpinnings of G6PD variant pathogenicity and suggests a promising avenue for correcting G6PD deficiency by targeting essential structural features of G6PD.

Rapid, precise quantification of bacterial cellular dimensions across a genomic-scale knockout library.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Strain Library Imaging Protocol for high-throughput, automated single-cell microscopy of large bacterial collections arrayed on multiwell plates.

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.

A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria.

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.

Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division.

To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division.

Systematic perturbation of cytoskeletal function reveals a linear scaling relationship between cell geometry and fitness.

Diversification of cell size is hypothesized to have occurred through a process of evolutionary optimization, but direct demonstrations of causal relationships between cell geometry and fitness are lacking. Here, we identify a mutation from a laboratory-evolved bacterium that dramatically increases cell size through cytoskeletal perturbation and confers a large fitness advantage. We engineer a library of cytoskeletal mutants of different sizes and show that fitness scales linearly with respect to cell size over a wide physiological range. Quantification of the growth rates of single cells during the exit from stationary phase reveals that transitions between "feast-or-famine" growth regimes are a key determinant of cell-size-dependent fitness effects. We also uncover environments that suppress the fitness advantage of larger cells, indicating that cell-size-dependent fitness effects are subject to both biophysical and metabolic constraints. Together, our results highlight laboratory-based evolution as a powerful framework for studying the quantitative relationships between morphology and fitness.

Effects of polymerization and nucleotide identity on the conformational dynamics of the bacterial actin homolog MreB.

The assembly of protein filaments drives many cellular processes, from nucleoid segregation, growth, and division in single cells to muscle contraction in animals. In eukaryotes, shape and motility are regulated through cycles of polymerization and depolymerization of actin cytoskeletal networks. In bacteria, the actin homolog MreB forms filaments that coordinate the cell-wall synthesis machinery to regulate rod-shaped growth and contribute to cellular stiffness through unknown mechanisms. Like actin, MreB is an ATPase and requires ATP to polymerize, and polymerization promotes nucleotide hydrolysis. However, it is unclear whether other similarities exist between MreB and actin because the two proteins share low sequence identity and have distinct cellular roles. Here, we use all-atom molecular dynamics simulations to reveal surprising parallels between MreB and actin structural dynamics. We observe that MreB exhibits actin-like polymerization-dependent structural changes, wherein polymerization induces flattening of MreB subunits, which restructures the nucleotide-binding pocket to favor hydrolysis. MreB filaments exhibited nucleotide-dependent intersubunit bending, with hydrolyzed polymers favoring a straighter conformation. We use steered simulations to demonstrate a coupling between intersubunit bending and the degree of flattening of each subunit, suggesting cooperative bending along a filament. Taken together, our results provide molecular-scale insight into the diversity of structural states of MreB and the relationships among polymerization, hydrolysis, and filament properties, which may be applicable to other members of the broad actin family.

Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization.

Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization.