How to Build a Bacterial Cell: MreB as the Foreman of E. coli Construction.

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.

Regulated Stochasticity in a Bacterial Signaling Network Permits Tolerance to a Rapid Environmental Change.

Microbial populations can maximize fitness in dynamic environments through bet hedging, a process wherein a subpopulation assumes a phenotype not optimally adapted to the present environment but well adapted to an environment likely to be encountered. Here, we show that oxygen induces fluctuating expression of the trimethylamine oxide (TMAO) respiratory system of Escherichia coli, diversifying the cell population and enabling a bet-hedging strategy that permits growth following oxygen loss. This regulation by oxygen affects the variance in gene expression but leaves the mean unchanged. We show that the oxygen-sensitive transcription factor IscR is the key regulator of variability. Oxygen causes IscR to repress expression of a TMAO-responsive signaling system, allowing stochastic effects to have a strong effect on the output of the system and resulting in heterogeneous expression of the TMAO reduction machinery. This work reveals a mechanism through which cells regulate molecular noise to enhance fitness.

Bifunctional Immunity Proteins Protect Bacteria against FtsZ-Targeting ADP-Ribosylating Toxins.

ADP-ribosylation of proteins can profoundly impact their function and serves as an effective mechanism by which bacterial toxins impair eukaryotic cell processes. Here, we report the discovery that bacteria also employ ADP-ribosylating toxins against each other during interspecies competition. We demonstrate that one such toxin from Serratia proteamaculans interrupts the division of competing cells by modifying the essential bacterial tubulin-like protein, FtsZ, adjacent to its protomer interface, blocking its capacity to polymerize. The structure of the toxin in complex with its immunity determinant revealed two distinct modes of inhibition: active site occlusion and enzymatic removal of ADP-ribose modifications. We show that each is sufficient to support toxin immunity; however, the latter additionally provides unprecedented broad protection against non-cognate ADP-ribosylating effectors. Our findings reveal how an interbacterial arms race has produced a unique solution for safeguarding the integrity of bacterial cell division machinery against inactivating post-translational modifications.

Redox-Based Regulation of Bacterial Development and Behavior.

Severe changes in the environmental redox potential, and resulting alterations in the oxidation states of intracellular metabolites and enzymes, have historically been considered negative stressors, requiring responses that are strictly defensive. However, recent work in diverse organisms has revealed that more subtle changes in the intracellular redox state can act as signals, eliciting responses with benefits beyond defense and detoxification. Changes in redox state have been shown to influence or trigger chromosome segregation, sporulation, aerotaxis, and social behaviors, including luminescence as well as biofilm establishment and dispersal. Connections between redox state and complex behavior allow bacteria to link developmental choices with metabolic state and coordinate appropriate responses. Promising future directions for this area of study include metabolomic analysis of species- and condition-dependent changes in metabolite oxidation states and elucidation of the mechanisms whereby the redox state influences circadian regulation.

Collective Space-Sensing Coordinates Pattern Scaling in Engineered Bacteria.

Scale invariance refers to the maintenance of a constant ratio of developing organ size to body size. Although common, its underlying mechanisms remain poorly understood. Here, we examined scaling in engineered Escherichia coli that can form self-organized core-ring patterns in colonies. We found that the ring width exhibits perfect scale invariance to the colony size. Our analysis revealed a collective space-sensing mechanism, which entails sequential actions of an integral feedback loop and an incoherent feedforward loop. The integral feedback is implemented by the accumulation of a diffusive chemical produced by a colony. This accumulation, combined with nutrient consumption, sets the timing for ring initiation. The incoherent feedforward is implemented by the opposing effects of the domain size on the rate and duration of ring maturation. This mechanism emphasizes a role of timing control in achieving robust pattern scaling and provides a new perspective in examining the phenomenon in natural systems.

Sugar Synthesis from CO2 in Escherichia coli.

Can a heterotrophic organism be evolved to synthesize biomass from CO2 directly? So far, non-native carbon fixation in which biomass precursors are synthesized solely from CO2 has remained an elusive grand challenge. Here, we demonstrate how a combination of rational metabolic rewiring, recombinant expression, and laboratory evolution has led to the biosynthesis of sugars and other major biomass constituents by a fully functional Calvin-Benson-Bassham (CBB) cycle in E. coli. In the evolved bacteria, carbon fixation is performed via a non-native CBB cycle, while reducing power and energy are obtained by oxidizing a supplied organic compound (e.g., pyruvate). Genome sequencing reveals that mutations in flux branchpoints, connecting the non-native CBB cycle to biosynthetic pathways, are essential for this phenotype. The successful evolution of a non-native carbon fixation pathway, though not yet resulting in net carbon gain, strikingly demonstrates the capacity for rapid trophic-mode evolution of metabolism applicable to biotechnology. PAPERCLIP.

Relative Rates of Surface and Volume Synthesis Set Bacterial Cell Size.

Many studies have focused on the mechanisms underlying length and width determination in rod-shaped bacteria. Here, we focus instead on cell surface area to volume ratio (SA/V) and demonstrate that SA/V homeostasis underlies size determination. We propose a model whereby the instantaneous rates of surface and volume synthesis both scale with volume. This model predicts that these relative rates dictate SA/V and that cells approach a new steady-state SA/V exponentially, with a decay constant equal to the volume growth rate. To test this, we exposed diverse bacterial species to sublethal concentrations of a cell wall biosynthesis inhibitor and observed dose-dependent decreases in SA/V. Furthermore, this decrease was exponential and had the expected decay constant. The model also quantitatively describes SA/V alterations induced by other chemical, nutritional, and genetic perturbations. We additionally present evidence for a surface material accumulation threshold underlying division, sensitizing cell length to changes in SA/V requirements.

Visualizing chaperonin function in situ by cryo-electron tomography.

Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding1-3. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES4-6. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55-70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL-GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL-GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells.

Stromal Cells Covering Omental Fat-Associated Lymphoid Clusters Trigger Formation of Neutrophil Aggregates to Capture Peritoneal Contaminants.

The omentum is a visceral adipose tissue rich in fat-associated lymphoid clusters (FALCs) that collects peritoneal contaminants and provides a first layer of immunological defense within the abdomen. Here, we investigated the mechanisms that mediate the capture of peritoneal contaminants during peritonitis. Single-cell RNA sequencing and spatial analysis of omental stromal cells revealed that the surface of FALCs were covered by CXCL1+ mesothelial cells, which we termed FALC cover cells. Blockade of CXCL1 inhibited the recruitment and aggregation of neutrophils at FALCs during zymosan-induced peritonitis. Inhibition of protein arginine deiminase 4, an enzyme important for the release of neutrophil extracellular traps, abolished neutrophil aggregation and the capture of peritoneal contaminants by omental FALCs. Analysis of omental samples from patients with acute appendicitis confirmed neutrophil recruitment and bacterial capture at FALCs. Thus, specialized omental mesothelial cells coordinate the recruitment and aggregation of neutrophils to capture peritoneal contaminants.

An integrated approach reveals regulatory controls on bacterial translation elongation.

Ribosomes elongate at a nonuniform rate during translation. Theoretical models and experiments disagree on the in vivo determinants of elongation rate and the mechanism by which elongation rate affects protein levels. To resolve this conflict, we measured transcriptome-wide ribosome occupancy under multiple conditions and used it to formulate a whole-cell model of translation in E. coli. Our model predicts that elongation rates at most codons during nutrient-rich growth are not limited by the intracellular concentrations of aminoacyl-tRNAs. However, elongation pausing during starvation for single amino acids is highly sensitive to the kinetics of tRNA aminoacylation. We further show that translation abortion upon pausing accounts for the observed ribosome occupancy along mRNAs during starvation. Abortion reduces global protein synthesis, but it enhances the translation of a subset of mRNAs. These results suggest a regulatory role for aminoacylation and abortion during stress, and our study provides an experimentally constrained framework for modeling translation.

A constant size extension drives bacterial cell size homeostasis.

Cell size control is an intrinsic feature of the cell cycle. In bacteria, cell growth and division are thought to be coupled through a cell size threshold. Here, we provide direct experimental evidence disproving the critical size paradigm. Instead, we show through single-cell microscopy and modeling that the evolutionarily distant bacteria Escherichia coli and Caulobacter crescentus achieve cell size homeostasis by growing, on average, the same amount between divisions, irrespective of cell length at birth. This simple mechanism provides a remarkably robust cell size control without the need of being precise, abating size deviations exponentially within a few generations. This size homeostasis mechanism is broadly applicable for symmetric and asymmetric divisions, as well as for different growth rates. Furthermore, our data suggest that constant size extension is implemented at or close to division. Altogether, our findings provide fundamentally distinct governing principles for cell size and cell-cycle control in bacteria.

Antiviral type III CRISPR signalling via conjugation of ATP and SAM.

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements1,2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against mobile genetic elements either directly, via the integral histidine-aspartate (HD) nuclease domain3-5 or indirectly, via synthesis of cyclic oligoadenylate second messengers to activate diverse ancillary effectors6-9. A subset of type III CRISPR systems encode an uncharacterized CorA-family membrane protein and an associated NrN family phosphodiesterase that are predicted to function in antiviral defence. Here we demonstrate that the CorA-associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against mobile genetic elements when expressed in Escherichia coli. However, B. fragilis Cmr does not synthesize cyclic oligoadenylate species on activation, instead generating S-adenosyl methionine (SAM)-AMP (SAM is also known as AdoMet) by conjugating ATP to SAM via a phosphodiester bond. Once synthesized, SAM-AMP binds to the CorA effector, presumably leading to cell dormancy or death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR-associated phosphodiesterases or a SAM-AMP lyase, potentially providing an 'off switch' analogous to cyclic oligoadenylate-specific ring nucleases10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.

(p)ppGpp controls bacterial persistence by stochastic induction of toxin-antitoxin activity.

Persistence refers to the phenomenon in which isogenic populations of antibiotic-sensitive bacteria produce rare cells that transiently become multidrug tolerant. Whether slow growth in a rare subset of cells underlies the persistence phenotype has not be examined in wild-type bacteria. Here, we show that an exponentially growing population of wild-type Escherichia coli cells produces rare cells that stochastically switch into slow growth, that the slow-growing cells are multidrug tolerant, and that they are able to resuscitate. The persistence phenotype depends hierarchically on the signaling nucleotide (p)ppGpp, Lon protease, inorganic polyphosphate, and toxin-antitoxins. We show that the level of (p)ppGpp varies stochastically in a population of exponentially growing cells and that the high (p)ppGpp level in rare cells induces slow growth and persistence. (p)ppGpp triggers slow growth by activating toxin-antitoxin loci through a regulatory cascade depending on inorganic polyphosphate and Lon protease.

Peptidoglycan maturation controls outer membrane protein assembly.

Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial for the maintenance of cellular integrity and enable survival in challenging environments1-5. The function of the outer membrane is dependent on outer membrane proteins (OMPs), which are inserted into the membrane by the ß-barrel assembly machine6,7 (BAM). Growing Escherichia coli cells segregate old OMPs towards the poles by a process known as binary partitioning, the basis of which is unknown8. Here we demonstrate that peptidoglycan underpins the spatiotemporal organization of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little effect on its activity, leading to preferential insertion of OMPs at division sites. The synchronization of OMP biogenesis with cell wall growth results in the binary partitioning of OMPs as cells divide. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation, a potential vulnerability that could be exploited in future antibiotic design.

Stress Can Induce Transcription of Toxin-Antitoxin Systems without Activating Toxin.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in bacterial genomes, but their functions are controversial. Although they are frequently postulated to regulate cell growth following stress, few null phenotypes for TA systems have been reported. Here, we show that TA transcript levels can increase substantially in response to stress, but toxin is not liberated. We find that the growth of an Escherichia coli strain lacking ten TA systems encoding endoribonuclease toxins is not affected following exposure to six stresses that each trigger TA transcription. Additionally, using RNA sequencing, we find no evidence of mRNA cleavage following stress. Stress-induced transcription arises from antitoxin degradation and relief of transcriptional autoregulation. Importantly, although free antitoxin is readily degraded in vivo, antitoxin bound to toxin is protected from proteolysis, preventing release of active toxin. Thus, transcription is not a reliable marker of TA activity, and TA systems do not strongly promote survival following individual stresses.

BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates.

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

Observation of universal ageing dynamics in antibiotic persistence.

Stress responses allow cells to adapt to changes in external conditions by activating specific pathways1. Here we investigate the dynamics of single cells that were subjected to acute stress that is too strong for a regulated response but not lethal. We show that when the growth of bacteria is arrested by acute transient exposure to strong inhibitors, the statistics of their regrowth dynamics can be predicted by a model for the cellular network that ignores most of the details of the underlying molecular interactions. We observed that the same stress, applied either abruptly or gradually, can lead to totally different recovery dynamics. By measuring the regrowth dynamics after stress exposure on thousands of cells, we show that the model can predict the outcome of antibiotic persistence measurements. Our results may account for the ubiquitous antibiotic persistence phenotype2, as well as for the difficulty in attempts to link it to specific genes3. More generally, our approach suggests that two different cellular states can be observed under stress: a regulated state, which prepares cells for fast recovery, and a disrupted cellular state due to acute stress, with slow and heterogeneous recovery dynamics. The disrupted state may be described by general properties of large random networks rather than by specific pathway activation. Better understanding of the disrupted state could shed new light on the survival and evolution of cells under stress.

ATP-Dependent Dynamic Protein Aggregation Regulates Bacterial Dormancy Depth Critical for Antibiotic Tolerance.

Cell dormancy is a widespread mechanism used by bacteria to evade environmental threats, including antibiotics. Here we monitored bacterial antibiotic tolerance and regrowth at the single-cell level and found that each individual survival cell shows different "dormancy depth," which in return regulates the lag time for cell resuscitation after removal of antibiotic. We further established that protein aggresome-a collection of endogenous protein aggregates-is an important indicator of bacterial dormancy depth, whose formation is promoted by decreased cellular ATP level. For cells to leave the dormant state and resuscitate, clearance of protein aggresome and recovery of proteostasis are required. We revealed that the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the lag time for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.

Metabolomics-Driven Exploration of the Chemical Drug Space to Predict Combination Antimicrobial Therapies.

Alternative to the conventional search for single-target, single-compound treatments, combination therapies can open entirely new opportunities to fight antibiotic resistance. However, combinatorial complexity prohibits experimental testing of drug combinations on a large scale, and methods to rationally design combination therapies are lagging behind. Here, we developed a combined experimental-computational approach to predict drug-drug interactions using high-throughput metabolomics. The approach was tested on 1,279 pharmacologically diverse drugs applied to the gram-negative bacterium Escherichia coli. Combining our metabolic profiling of drug response with previously generated metabolic and chemogenomic profiles of 3,807 single-gene deletion strains revealed an unexpectedly large space of inhibited gene functions and enabled rational design of drug combinations. This approach is applicable to other therapeutic areas and can unveil unprecedented insights into drug tolerance, side effects, and repurposing. The compendium of drug-associated metabolome profiles is available at https://zampierigroup.shinyapps.io/EcoPrestMet, providing a valuable resource for the microbiological and pharmacological communities.

Escherichia coli self-organizes developmental rosettes.

Rosettes are self-organizing, circular multicellular communities that initiate developmental processes, like organogenesis and embryogenesis, in complex organisms. Their formation results from the active repositioning of adhered sister cells and is thought to distinguish multicellular organisms from unicellular ones. Though common in eukaryotes, this multicellular behavior has not been reported in bacteria. In this study, we found that Escherichia coli forms rosettes by active sister-cell repositioning. After division, sister cells "fold" to actively align at the 2- and 4-cell stages of clonal division, thereby producing rosettes with characteristic quatrefoil configuration. Analysis revealed that folding follows an angular random walk, composed of ~1 µm strokes and directional randomization. We further showed that this motion was produced by the flagellum, the extracellular tail whose rotation generates swimming motility. Rosette formation was found to require de novo flagella synthesis suggesting it must balance the opposing forces of Ag43 adhesion and flagellar propulsion. We went on to show that proper rosette formation was required for subsequent morphogenesis of multicellular chains, rpoS gene expression, and formation of hydrostatic clonal-chain biofilms. Moreover, we found self-folding rosette-like communities in the standard motility assay, indicating that this behavior may be a general response to hydrostatic environments in E. coli. These findings establish self-organization of clonal rosettes by a prokaryote and have implications for evolutionary biology, synthetic biology, and medical microbiology.