Benjamin P. Bratton

Genetic screens are powerful approaches to unveiling new biological insights and ordered redundant transposon libraries have emerged as a primary tool for performing screens with known genetic saturation. Newer sequencing approaches based on combinatorial pooling have lowered the cost and time required to generate these libraries. Caulobacter crescentus is a gram-negative bacterium that has served as a model for understanding bacterial physiology with a myriad of genetic tools. To add to this collection of tools, we created CauloKO—the first ordered, transposon library in C. crescentus . CauloKO includes insertion mutants in 86% of all non-essential genes and 77% of all open reading frames of strain CB15. CauloKO insertion mutants were validated using Sanger sequencing. We also present phenotypic analysis of the CauloKO library using a crystal violet screen for biofilm mutants, which both confirmed previous results and identified new mutants for future studies. This combined approach revealed that the CauloKO library shows promise for screening applications, particularly for phenotypes that require monoclonal populations of cells. IMPORTANCE: Ordered transposon mutant libraries have dramatically advanced the ability to study other systems, and the potential for combining such a library with the other genome-wide tools available in Caulobacter crescentus makes such a library particularly important for this system. Our work also establishes a novel method for analyzing sequencing data for ordered library construction purposes, and a first-of-its-kind dynamic, online platform for cataloging mutants within the library. Furthermore, our findings further confirmed results of previously published studies, but also identified potential novel regulators of biofilm formation in C. crescentus that will be important starting points for future investigations.

Understanding the mechanisms that dictate the localization of cytoskeletal filaments is crucial for elucidating cell shape regulation in prokaryotes. The actin homolog MreB plays a pivotal role in maintaining the shape of many rod-shaped bacteria such as Escherichia coli by directing cell-wall synthesis according to local curvature cues. However, the basis of MreB’s curvature-dependent localization has remained elusive. Here, we develop a biophysical model for the energetics of a filament binding to a surface that integrates the complex interplay between filament twist and bending and the two-dimensional surface geometry. Our model predicts that the spatial localization of a filament like MreB with substantial intrinsic twist is governed by both the mean and Gaussian curvatures of the cell envelope, which strongly covary in rod-shaped cells. Using molecular dynamics simulations to estimate the mechanical properties of MreB filaments, we show that their thermodynamic preference for regions with lower mean and Gaussian curvatures matches experimental observations for physiologically relevant filament lengths of ~50 nm. We find that the experimentally measured statistical curvature preference is maintained in the absence of filament motion and after a cycle of depolymerization, repolymerization, and membrane rebinding, indicating that equilibrium energetics can explain MreB localization. These findings provide critical insights into the physical principles underlying cytoskeletal filament localization and suggest design principles for synthetic shape-sensing nanomaterials.

The universally conserved α-oxoaldehydes glyoxal (GO) and methylglyoxal (MGO) are toxic metabolic byproducts whose accumulation can lead to cell death. In the absence of a known, natural inducer of the GO-specific response in prokaryotes, we exploited RNA-seq to define a GO response in the bacterial pathogen Pseudomonas aeruginosa. The highest upregulated operon consisted of the known glyoxalase (gloA2) and an antibiotic monooxygenase (ABM) domain of unknown function - renamed here A ldehyde r esponsive q uorum-sensing I nhibitor (ArqI). The arqI-gloA2 operon is highly specific to GO induction and ArqI protein responds by migrating to the flagellar pole. An ArqI atomic structure revealed several unique features to the ABM family, including a ‘pinwheel’ hexamer harboring a GO-derived post-translational modification on a conserved arginine residue (Arg49). Induction of ArqI abrogates production of the Pseudomonas Quinolone Signal (PQS) quorum sensing molecule and was found to directly interact with PqsA; the first enzyme in the PQS biosynthesis pathway. Finally, we use a sepsis model of infection to reveal a survival requirement for arqI-gloA2 in blood-rich organs (heart, spleen, liver and lung). Here we define a global GO response in a pathogen, identify and characterize the first GO-specific operon and implicate its role in PQS production and host survival.

The crowded bacterial cytoplasm is composed of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial genetically encoded multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (−3,240 to +2,700 e) in live Escherichia coli cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears subdiffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent subdiffusion arises from geometrical confinement within the nucleoid and by the cell boundary.

Understanding the mechanisms that dictate the localization of cytoskeletal filaments is crucial for elucidating cell shape regulation in prokaryotes. The actin homolog MreB plays a pivotal role in maintaining the shape of many rod-shaped bacteria such as Escherichia coli by directing cell-wall synthesis according to local curvature cues. However, the basis of MreB’s curvature-dependent localization has remained elusive. Here, we develop a biophysical model for the energetics of filament binding to a surface that integrates the complex interplay between filament twist and bending and the two-dimensional surface geometry. Our model predicts that the spatial localization of a filament like MreB with substantial intrinsic twist is governed by both the mean and Gaussian curvatures of the cell envelope, which strongly covary in rod-shaped cells. Using molecular dynamics simulations to estimate the mechanical properties of MreB filaments, we show that their thermodynamic preference for regions with lower mean and Gaussian curvatures matches experimental observations for physiologically relevant filament lengths of ∼50 nm. We find that the experimentally measured statistical curvature preference is maintained in the absence of filament motion and after a cycle of depolymerization, repolymerization, and membrane rebinding, indicating that equilibrium energetics can explain MreB localization. These findings provide critical insights into the physical principles underlying cytoskeletal filament localization, and suggest new design principles for synthetic shape sensing nanomaterials. Significance statement: The protein MreB, a homolog of eukaryotic actin, regulates the shape of bacteria like Escherichia coli by guiding new cell-wall insertion based on local curvature cues. However, the mechanism by which a nanometer-scale MreB filament “senses” the micron-scale curvature of the cell wall has remained a mystery. We introduce a biophysical model of the energetics of twisted and bent filaments bound to curved surfaces, which predicts that localization of filaments like MreB is sensitive to both mean and Gaussian curvature. The model captures experimentally measured curvature enrichment patterns and explains how MreB naturally localizes to saddle-shaped regions without energy-consuming processes. Beyond cell shape regulation, our work suggests design principles for synthetic systems that can sense and respond to surface shape.

Fluctuating environments that consist of regular cycles of co-occurring stress are a common challenge faced by cellular populations. For a population to thrive in constantly changing conditions, an ability to coordinate a rapid cellular response is essential. Here, we identify a mutation conferring an arginine-to-histidine (Arg to His) substitution in the transcription terminator Rho. The rho R109H mutation frequently arose in Escherichia coli populations experimentally evolved under repeated long-term starvation conditions, during which the accumulation of metabolic waste followed by transfer into fresh media results in drastic environmental pH fluctuations associated with feast and famine. Metagenomic sequencing revealed that populations containing the rho mutation also possess putative loss-of-function mutations in ydcI , which encodes a recently characterized transcription factor associated with pH homeostasis. Genetic reconstructions of these mutations show that the rho allele confers plasticity via an alkaline-induced reduction of Rho function that, when found in tandem with a Δ ydcI allele, leads to intracellular alkalization and genetic assimilation of Rho mutant function. We further identify Arg to His substitutions at analogous sites in rho alleles from species that regularly experience neutral to alkaline pH fluctuations in their environments. Our results suggest that Arg to His substitutions in Rho may serve to rapidly coordinate complex physiological responses through pH sensing and shed light on how cellular populations use environmental cues to coordinate rapid responses to complex, fluctuating environments.

Photoperiodic time measurement is the ability of plants and animals to measure differences in day versus night length (photoperiod) and use that information to anticipate critical seasonal transformations, such as annual temperature cycles. This timekeeping phenomenon triggers adaptive responses in higher organisms, such as gonadal stimulation, flowering, and hibernation. Unexpectedly, we observed this capability in cyanobacteria—unicellular prokaryotes with generation times as short as 5 to 6 hours. Cyanobacteria exposed to short, winter-like days developed enhanced resistance to cold mediated by desaturation of membrane lipids and differential programs of gene transcription, including stress response pathways. As in eukaryotes, this photoperiodic timekeeping required an intact circadian clockwork and developed over multiple cycles of photoperiod. Therefore, photoperiodic timekeeping evolved in much simpler organisms than previously appreciated and enabled genetic responses to stresses that recur seasonally.

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis.

The crowded bacterial cytoplasm is comprised of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial Genetically Encoded Multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (−2160 to +1800 e) in live Escherichia coli cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears sub-diffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent sub-diffusion arises from geometrical confinement within the nucleoid and by the cell boundary.

Fluctuating environments that consist of regular cycles of co-occurring stress are a common challenge faced by cellular populations. For a population to thrive in constantly changing conditions, an ability to coordinate a rapid cellular response is essential. Here, we identify a mutation conferring an arginine-to-histidine (Arg to His) substitution in the transcription terminator Rho. The rho R109H mutation frequently arose in E. coli populations experimentally evolved under repeated long-term starvation conditions, during which feast and famine result in drastic environmental pH fluctuations. Metagenomic sequencing revealed that populations containing the rho mutation also possess putative loss-of-function mutations in ydcI , which encodes a recently characterized transcription factor associated with pH homeostasis. Genetic reconstructions of these mutations show that the rho allele confers a plastic alkaline-induced reduction of Rho function that, when found in tandem with a Δ ydcI allele, leads to intracellular alkalinization and genetic assimilation of Rho mutant function. We further identify Arg to His substitutions at analogous sites in rho alleles from species originating from fluctuating alkaline environments. Our results suggest that Arg to His substitutions in global regulators of gene expression can serve to rapidly coordinate complex responses through pH sensing and shed light on how cellular populations across the tree of life use environmental cues to coordinate rapid responses to complex, fluctuating environments.