Mutational robustness and the role of buffer genes in evolvability.

Organisms rely on mutations to fuel adaptive evolution. However, many mutations impose a negative effect on fitness. Cells may have therefore evolved mechanisms that affect the phenotypic effects of mutations, thus conferring mutational robustness. Specifically, so-called buffer genes are hypothesized to interact directly or indirectly with genetic variation and reduce its effect on fitness. Environmental or genetic perturbations can change the interaction between buffer genes and genetic variation, thereby unmasking the genetic variation's phenotypic effects and thus providing a source of variation for natural selection to act on. This review provides an overview of our understanding of mutational robustness and buffer genes, with the chaperone gene HSP90 as a key example. It discusses whether buffer genes merely affect standing variation or also interact with de novo mutations, how mutational robustness could influence evolution, and whether mutational robustness might be an evolved trait or rather a mere side-effect of complex genetic interactions.

Protocol to train a support vector machine for the automatic curation of bacterial cell detections in microscopy images.

Manual curation of bacterial cell detections in microscopy images remains a time-consuming and laborious task. This work offers a comprehensive, step-by-step tutorial on training a support vector machine to autonomously distinguish between good and bad cell detections. Jupyter notebooks are included to perform feature extraction, labeling, and training of the machine learning model. This method can readily be incorporated into profiling pipelines aimed at extracting a multitude of features across large collections of individual cells, strains, and species. For complete details on the use and execution of this protocol, please refer to Govers et al.1.

Apparent simplicity and emergent robustness in the control of the Escherichia coli cell cycle.

To examine how bacteria achieve robust cell proliferation across diverse conditions, we developed a method that quantifies 77 cell morphological, cell cycle, and growth phenotypes of a fluorescently labeled Escherichia coli strain and >800 gene deletion derivatives under multiple nutrient conditions. This approach revealed extensive phenotypic plasticity and deviating mutant phenotypes were often nutrient dependent. From this broad phenotypic landscape emerged simple and robust unifying rules (laws) that connect DNA replication initiation, nucleoid segregation, FtsZ ring formation, and cell constriction to specific aspects of cell size (volume, length, or added length) at the population level. Furthermore, completion of cell division followed the initiation of cell constriction after a constant time delay across strains and nutrient conditions, identifying cell constriction as a key control point for cell size determination. Our work provides a population-level description of the governing principles by which E. coli integrates cell cycle processes and growth rate with cell size to achieve its robust proliferative capability. A record of this paper's transparent peer review process is included in the supplemental information.

Protein aggregates act as a deterministic disruptor during bacterial cell size homeostasis.

Mechanisms underlying deviant cell size fluctuations among clonal bacterial siblings are generally considered to be cryptic and stochastic in nature. However, by scrutinizing heat-stressed populations of the model bacterium Escherichia coli, we uncovered the existence of a deterministic asymmetry in cell division that is caused by the presence of intracellular protein aggregates (PAs). While these structures typically locate at the cell pole and segregate asymmetrically among daughter cells, we now show that the presence of a polar PA consistently causes a more distal off-center positioning of the FtsZ division septum. The resulting increased length of PA-inheriting siblings persists over multiple generations and could be observed in both E. coli and Bacillus subtilis populations. Closer investigation suggests that a PA can physically perturb the nucleoid structure, which subsequently leads to asymmetric septation.

Multiple tethers of organelle contact sites are involved in α-synuclein toxicity in yeast.

The protein α-synuclein (α-syn) is one of the major factors linked to Parkinson's disease, yet how its misfolding and deposition contribute to the pathology remains largely elusive. Recently, contact sites among organelles were implicated in the development of this disease. Here, we used the budding yeast Saccharomyces cerevisiae, in which organelle contact sites have been characterized extensively, as a model to investigate their role in α-syn cytotoxicity. We observed that lack of specific tethers that anchor the endoplasmic reticulum to the plasma membrane resulted in cells with increased resistance to α-syn expression. Additionally, we found that strains lacking two dual-function proteins involved in contact sites, Mdm10 and Vps39, were resistant to the expression of α-syn. In the case of Mdm10, we found that this is related to its function in mitochondrial protein biogenesis and not to its role as a contact site tether. In contrast, both functions of Vps39, in vesicular transport and as a tether of the vacuole-mitochondria contact site, were required to support α-syn toxicity. Overall, our findings support that interorganelle communication through membrane contact sites is highly relevant for α-syn-mediated toxicity.

Ethanol induces replication fork stalling and membrane stress in immortalized laryngeal cells.

Although ethanol is a class I carcinogen and is linked to more than 700,000 cancer incidences, a clear understanding of the molecular mechanisms underlying ethanol-related carcinogenesis is still lacking. Further understanding of ethanol-related cell damage can contribute to reducing or treating alcohol-related cancers. Here, we investigated the effects of both short- and long-term exposure of human laryngeal epithelial cells to different ethanol concentrations. RNA sequencing shows that ethanol altered gene expression patterns in a time- and concentration-dependent way, affecting genes involved in ribosome biogenesis, cytoskeleton remodeling, Wnt signaling, and transmembrane ion transport. Additionally, ethanol induced a slower cell proliferation, a delayed cell cycle progression, and replication fork stalling. In addition, ethanol exposure resulted in morphological changes, which could be associated with membrane stress. Taken together, our data yields a comprehensive view of molecular changes associated with ethanol stress in epithelial cells of the upper aerodigestive tract.

Assessing persister awakening dynamics following antibiotic treatment in E. coli.

Given the low fraction of antibiotic-tolerant persisters and the transient nature of the persister phenotype, identifying molecular mechanisms underlying persister state exit, also called "awakening," is challenging. Here, we describe how persister awakening kinetics can be quantified at the single-cell level, enabling the identification of genes that are important for persister survival following antibiotic treatment. We report step-by-step sample preparation, dynamic recording, and data analysis. Although the setup is flexible, time-lapse microscopy requires a minimal number of persisters being present. For complete details on the use and execution of this protocol, please refer to Wilmaerts et al. (2022).

Chromosome choreography during the non-binary cell cycle of a predatory bacterium.

In bacteria, the dynamics of chromosome replication and segregation are tightly coordinated with cell-cycle progression and largely rely on specific spatiotemporal arrangement of the chromosome. Whereas these key processes are mostly investigated in species that divide by binary fission, they remain mysterious in bacteria producing larger number of descendants. Here, we establish the predatory bacterium Bdellovibrio bacteriovorus as a model to investigate the non-binary processing of a circular chromosome. We found that its single chromosome is highly compacted in a polarized nucleoid that excludes freely diffusing proteins during the non-proliferative stage of the cell cycle. A binary-like cycle of DNA replication and asymmetric segregation is followed by multiple asynchronous rounds of replication and progressive ParABS-dependent partitioning, uncoupled from cell division. Finally, we provide the first evidence for an on-off behavior of the ParB protein, which localizes at the centromere in a cell-cycle-regulated manner. Altogether, our findings support a model of complex chromosome choreography leading to the generation of variable, odd, or even numbers of offspring and highlight the adaptation of conserved mechanisms to achieve non-binary reproduction.

Interconnecting solvent quality, transcription, and chromosome folding in Escherichia coli.

All cells fold their genomes, including bacterial cells, where the chromosome is compacted into a domain-organized meshwork called the nucleoid. How compaction and domain organization arise is not fully understood. Here, we describe a method to estimate the average mesh size of the nucleoid in Escherichia coli. Using nucleoid mesh size and DNA concentration estimates, we find that the cytoplasm behaves as a poor solvent for the chromosome when the cell is considered as a simple semidilute polymer solution. Monte Carlo simulations suggest that a poor solvent leads to chromosome compaction and DNA density heterogeneity (i.e., domain formation) at physiological DNA concentration. Fluorescence microscopy reveals that the heterogeneous DNA density negatively correlates with ribosome density within the nucleoid, consistent with cryoelectron tomography data. Drug experiments, together with past observations, suggest the hypothesis that RNAs contribute to the poor solvent effects, connecting chromosome compaction and domain formation to transcription and intracellular organization.

Caulobacter crescentus: model system extraordinaire.

In scientific research, we often rely on well-established model systems to tackle important questions. In this context, extensive characterization of specific bacterial species such as Escherichia coli and Bacillus subtilis has provided a vast amount of knowledge that extends well beyond the biology of these two organisms. However, the bacterial world is large and extremely diverse, necessitating the development of additional models that complement the classical rod-shaped and symmetrically dividing systems. Caulobacter crescentus is a species that has met this need effectively, as its dimorphic lifestyle showcases distinctive features, including cellular asymmetry and differentiation during the cell cycle. Studying C. crescentus has reformed our understanding of bacterial intracellular organization, cellular development, and cell-cycle regulation. These findings have, in turn, stimulated studies in other bacteria, shedding light on how protein function and cell morphology can evolve and diversify. Studies in C. crescentus have also deepened our knowledge of other topics (e.g. cell mechanosensing, motility, and bacterial aging), while opening the door to biotechnological innovations. In this Primer, we provide some general background to this peculiar bacterium and highlight specific features that have contributed to its rise as a versatile bacterial model. This Primer is not meant to be exhaustive on any topic and is instead intended to provide a taste of the power of C. crescentus as a model system to explore a diverse range of topics.

Borrelia burgdorferi peptidoglycan is a persistent antigen in patients with Lyme arthritis.

Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PGBb) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PGBb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PGBb, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PGBb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PGBb In addition, systemic administration of PGBb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PGBb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PGBb fragments during growth suggests a potential role for PGBb in the immunopathogenesis of other Lyme disease manifestations.

Nucleoid Size Scaling and Intracellular Organization of Translation across Bacteria.

The scaling of organelles with cell size is thought to be exclusive to eukaryotes. Here, we demonstrate that similar scaling relationships hold for the bacterial nucleoid. Despite the absence of a nuclear membrane, nucleoid size strongly correlates with cell size, independent of changes in DNA amount and across various nutrient conditions. This correlation is observed in diverse bacteria, revealing a near-constant ratio between nucleoid and cell size for a given species. As in eukaryotes, the nucleocytoplasmic ratio in bacteria varies greatly among species. This spectrum of nucleocytoplasmic ratios is independent of genome size, and instead it appears linked to the average population cell size. Bacteria with different nucleocytoplasmic ratios have a cytoplasm with different biophysical properties, impacting ribosome mobility and localization. Together, our findings identify new organizational principles and biophysical features of bacterial cells, implicating the nucleocytoplasmic ratio and cell size as determinants of the intracellular organization of translation.

Stress-induced protein aggregates shape population heterogeneity in bacteria.

The concept of phenotypic heterogeneity preparing a subpopulation of isogenic cells to better cope with anticipated stresses has been well established. However, less is known about how stress itself can drive subsequent cellular individualization in clonal populations. In this perspective, we focus on the impact of stress-induced cellular protein aggregates, and how their segregation and disaggregation can act as a deterministic incentive for heterogeneity in the population emerging from a stressed ancestor.

Transition between fermentation and respiration determines history-dependent behavior in fluctuating carbon sources.

Cells constantly adapt to environmental fluctuations. These physiological changes require time and therefore cause a lag phase during which the cells do not function optimally. Interestingly, past exposure to an environmental condition can shorten the time needed to adapt when the condition re-occurs, even in daughter cells that never directly encountered the initial condition. Here, we use the molecular toolbox of Saccharomyces cerevisiae to systematically unravel the molecular mechanism underlying such history-dependent behavior in transitions between glucose and maltose. In contrast to previous hypotheses, the behavior does not depend on persistence of proteins involved in metabolism of a specific sugar. Instead, presence of glucose induces a gradual decline in the cells' ability to activate respiration, which is needed to metabolize alternative carbon sources. These results reveal how trans-generational transitions in central carbon metabolism generate history-dependent behavior in yeast, and provide a mechanistic framework for similar phenomena in other cell types.

Bimodal Expression of the Salmonella Typhimurium spv Operon.

The well-studied spv operon of Salmonellatyphimurium is important for causing full virulence in mice and both the regulation and function of the Spv proteins have been characterized extensively over the past several decades. Using quantitative single-cell fluorescence microscopy, we demonstrate the spv regulon to display a bimodal expression pattern that originates in the bimodal expression of the SpvR activator. The spv expression pattern is influenced by growth conditions and the specific Styphimurium strain used, but does not require Salmonella-specific virulence regulators. By monitoring real-time promoter kinetics, we reveal that SpvA has the ability to impart negative feedback on spvABCD expression without affecting spvR expression. Together, our data suggest that the SpvA protein counteracts the positive feedback loop imposed by SpvR, and could thus be responsible for dampening spvABCD expression and coordinating virulence protein production in time. The results presented here yield new insights in the intriguing regulation of the spv operon and adds this operon to the growing list of virulence factors exhibiting marked expression heterogeneity in Styphimurium.

Protein aggregates encode epigenetic memory of stressful encounters in individual Escherichia coli cells.

Protein misfolding and aggregation are typically perceived as inevitable and detrimental processes tied to a stress- or age-associated decline in cellular proteostasis. A careful reassessment of this paradigm in the E. coli model bacterium revealed that the emergence of intracellular protein aggregates (PAs) was not related to cellular aging but closely linked to sublethal proteotoxic stresses such as exposure to heat, peroxide, and the antibiotic streptomycin. After removal of the proteotoxic stress and resumption of cellular proliferation, the polarly deposited PA was subjected to limited disaggregation and therefore became asymmetrically inherited for a large number of generations. Many generations after the original PA-inducing stress, the cells inheriting this ancestral PA displayed a significantly increased heat resistance compared to their isogenic, PA-free siblings. This PA-mediated inheritance of heat resistance could be reproduced with a conditionally expressed, intracellular PA consisting of an inert, aggregation-prone mutant protein, validating the role of PAs in increasing resistance and indicating that the resistance-conferring mechanism does not depend on the origin of the PA. Moreover, PAs were found to confer robustness to other proteotoxic stresses, as imposed by reactive oxygen species or streptomycin exposure, suggesting a broad protective effect. Our findings therefore reveal the potential of intracellular PAs to serve as long-term epigenetically inheritable and functional memory elements, physically referring to a previous cellular insult that occurred many generations ago and meanwhile improving robustness to a subsequent proteotoxic stress. The latter is presumably accomplished through the PA-mediated asymmetric inheritance of protein quality control components leading to their specific enrichment in PA-bearing cells.

Genomewide phenotypic analysis of growth, cell morphogenesis, and cell cycle events in Escherichia coli.

Cell size, cell growth, and cell cycle events are necessarily intertwined to achieve robust bacterial replication. Yet, a comprehensive and integrated view of these fundamental processes is lacking. Here, we describe an image-based quantitative screen of the single-gene knockout collection of Escherichia coli and identify many new genes involved in cell morphogenesis, population growth, nucleoid (bulk chromosome) dynamics, and cell division. Functional analyses, together with high-dimensional classification, unveil new associations of morphological and cell cycle phenotypes with specific functions and pathways. Additionally, correlation analysis across ~4,000 genetic perturbations shows that growth rate is surprisingly not predictive of cell size. Growth rate was also uncorrelated with the relative timings of nucleoid separation and cell constriction. Rather, our analysis identifies scaling relationships between cell size and nucleoid size and between nucleoid size and the relative timings of nucleoid separation and cell division. These connections suggest that the nucleoid links cell morphogenesis to the cell cycle.

Rapid phenotypic individualization of bacterial sister cells.

A growing bacterium typically divides into two genetically identical and morphologically similar sister cells and eventually gives rise to a clonal population. Nevertheless, significant phenotypic differentiation among isogenic cells frequently occurs, with the resulting heterogeneity in cellular behavior often ensuring population level growth and survival in complex and unpredictable environments. Although several mechanisms underlying the generation of phenotypic heterogeneity have been elucidated, the speed with which identical sister cells tend to phenotypically diverge from each other has so far remained unaddressed. Using Escherichia coli as a model organism, we therefore examined the timing and dynamics of phenotypic individualization among sister cells by scrutinizing and modeling microscopically tracked clonally growing populations before and after a semi-lethal heat challenge. This analysis revealed that both survival probability and post-stress physiology of sister cells shift from highly similar to uncorrelated within the first decile of their cell cycles. This nearly-immediate post-fission randomization of sister cell fates highlights the potential of stochastic fluctuations during clonal growth to rapidly generate phenotypically independent individuals.

Impact of high hydrostatic pressure on bacterial proteostasis.

High hydrostatic pressure (HHP) is an important factor that limits microbial growth in deep-sea ecosystems to specifically adapted piezophiles. Furthermore, HHP treatment is used as a novel food preservation technique because of its ability to inactivate pathogenic and spoilage bacteria while minimizing the loss of food quality. Disruption of protein homeostasis (i.e. proteostasis) as a result of HHP-induced conformational changes in ribosomes and proteins has been considered as one of the limiting factors for both microbial growth and survival under HHP conditions. This work therefore reviews the effects of sublethal (≤100MPa) and lethal (>100MPa) pressures on protein synthesis, structure, and functionality in bacteria. Furthermore, current understanding on the mechanisms adopted by piezophiles to maintain proteostasis in HHP environments and responses developed by atmospheric-adapted bacteria to protect or restore proteostasis after HHP exposure are discussed.

Intracellular movement of protein aggregates reveals heterogeneous inactivation and resuscitation dynamics in stressed populations of Escherichia coli.

Inactivation of bacterial pathogens is of critical importance in fields ranging from antimicrobial therapy to food preservation. The efficacy of an antimicrobial treatment is often experimentally determined through viable plate counts that inherently provide a poor focus on the mechanisms and distribution of (sub)lethal injury and subsequent inactivation or resuscitation behavior of the stressed cells, which are increasingly important features for the proper understanding and design of inactivation strategies. In this report, we employ a live cell biology approach focusing on the energy-dependent motion of intracellular protein aggregates to investigate the heterogeneity within heat stressed Escherichia coli populations. As such, we were able to identify differential dynamics of cellular resuscitation and inactivation that are impossible to distinguish using more traditional approaches. Moreover, our data indicate the existence of late-resuscitating cells that remain physiologically active and are able to persist in the presence of antibiotics before resuscitation.