The relevance of sustainable laboratory practices.

Scientists are of key importance to the society to advocate awareness of the climate crisis and its underlying scientific evidence and provide solutions for a sustainable future. As much as scientific research has led to great achievements and benefits, traditional laboratory practices come with unintended environmental consequences. Scientists, while providing solutions to climate problems and educating the young innovators of the future, are also part of the problem: excessive energy consumption, (hazardous) waste generation, and resource depletion. Through their own research operations, science, research and laboratories have a significant carbon footprint and contribute to the climate crisis. Climate change requires a rapid response across all sectors of society, modeled by inspiring leaders. A broader scientific community that takes concrete actions would serve as an important step in convincing the general public of similar actions. Over the past years, grassroots movements across the sciences have recognized the overlooked impact of the scientific enterprise, and so-called Green Lab initiatives emerged seeking to address the environmental footprint of research. Driven by the voluntary efforts of researchers and staff, they educate peers, develop sustainability guidelines, write scientific publications and maintain accreditation frameworks. With this perspective we want to advocate for and spark leadership to promote a systemic change in laboratory practices and approach to research. Comprehensive evidence for the environmental impact of laboratories and their root-causes is presented, expanded with data from a current case study of the University of Groningen showcasing annual savings of 398 763 € as well as 477.1 tons of CO2e. This is followed by guidelines for sustainable lab practices and hands-on advice on how to achieve a systemic change at research institutions and industry. How can we expect industry, politics, and society to change, if we as scientists are not changing either? Scientists should lead by example and practice the change they want to see.

Contribution of different macromolecules to the diffusion of a 40 nm particle in Escherichia coli.

Due to the high concentration of proteins, nucleic acids, and other macromolecules, the bacterial cytoplasm is typically described as a crowded environment. However, the extent to which each of these macromolecules individually affects the mobility of macromolecular complexes, and how this depends on growth conditions, is presently unclear. In this study, we sought to quantify the crowding experienced by an exogenous 40 nm fluorescent particle in the cytoplasm of E. coli under different growth conditions. By performing single-particle tracking measurements in cells selectively depleted of DNA and/or mRNA, we determined the contribution to crowding of mRNA, DNA, and remaining cellular components, i.e., mostly proteins and ribosomes. To estimate this contribution to crowding, we quantified the difference of the particle's diffusion coefficient in conditions with and without those macromolecules. We found that the contributions of the three classes of components were of comparable magnitude, being largest in the case of proteins and ribosomes. We further found that the contributions of mRNA and DNA to crowding were significantly larger than expected based on their volumetric fractions alone. Finally, we found that the crowding contributions change only slightly with the growth conditions. These results reveal how various cellular components partake in crowding of the cytoplasm and the consequences this has for the mobility of large macromolecular complexes.

Quantifying intracellular glucose levels when yeast is grown in glucose media.

In Saccharomyces cerevisiae, intracellular glucose levels impact glucose transport and regulate carbon metabolism via various glucose sensors. To investigate mechanisms of glucose sensing, it is essential to know the intracellular glucose concentrations. Measuring intracellular glucose concentrations, however, is challenging when cells are grown on glucose, as glucose in the water phase around cells or stuck to the cell surface can be carried over during cell sampling and in the following attributed to intracellular glucose, resulting in an overestimation of intracellular glucose concentrations. Using lactose as a carryover marker in the growth medium, we found that glucose carryover originates from both the water phase and from sticking to the cell surface. Using a hexokinase null strain to estimate the glucose carryover from the cell surface, we found that glucose stuck on the cell surface only contributes a minor fraction of the carryover. To correct the glucose carryover, we revisited L-glucose as a carryover marker. Here, we found that L-glucose slowly enters cells. Thus, we added L-glucose to yeast cultures growing on uniformly 13C-labeled D-glucose only shortly before sampling. Using GC-MS to distinguish between the two differently labeled sugars and subtracting the carryover effect, we determined the intracellular glucose concentrations among two yeast strains with distinct kinetics of glucose transport to be at 0.89 mM in the wild-type strain and around 0.24 mM in a mutant with compromised glucose uptake. Together, our study provides insight into the origin of the glucose carryover effect and suggests that L-glucose added to the culture shortly before sampling is a possible method that yet has limitations with regard to measurement accuracy.

Improving photosynthetic efficiency toward food security: Strategies, advances, and perspectives.

Photosynthesis in crops and natural vegetation allows light energy to be converted into chemical energy and thus forms the foundation for almost all terrestrial trophic networks on Earth. The efficiency of photosynthetic energy conversion plays a crucial role in determining the portion of incident solar radiation that can be used to generate plant biomass throughout a growth season. Consequently, alongside the factors such as resource availability, crop management, crop selection, maintenance costs, and intrinsic yield potential, photosynthetic energy use efficiency significantly influences crop yield. Photosynthetic efficiency is relevant to sustainability and food security because it affects water use efficiency, nutrient use efficiency, and land use efficiency. This review focuses specifically on the potential for improvements in photosynthetic efficiency to drive a sustainable increase in crop yields. We discuss bypassing photorespiration, enhancing light use efficiency, harnessing natural variation in photosynthetic parameters for breeding purposes, and adopting new-to-nature approaches that show promise for achieving unprecedented gains in photosynthetic efficiency.

Temporal segregation of biosynthetic processes is responsible for metabolic oscillations during the budding yeast cell cycle.

Many cell biological and biochemical mechanisms controlling the fundamental process of eukaryotic cell division have been identified; however, the temporal dynamics of biosynthetic processes during the cell division cycle are still elusive. Here, we show that key biosynthetic processes are temporally segregated along the cell cycle. Using budding yeast as a model and single-cell methods to dynamically measure metabolic activity, we observe two peaks in protein synthesis, in the G1 and S/G2/M phase, whereas lipid and polysaccharide synthesis peaks only once, during the S/G2/M phase. Integrating the inferred biosynthetic rates into a thermodynamic-stoichiometric metabolic model, we find that this temporal segregation in biosynthetic processes causes flux changes in primary metabolism, with an acceleration of glucose-uptake flux in G1 and phase-shifted oscillations of oxygen and carbon dioxide exchanges. Through experimental validation of the model predictions, we demonstrate that primary metabolism oscillates with cell-cycle periodicity to satisfy the changing demands of biosynthetic processes exhibiting unexpected dynamics during the cell cycle.

Perspective: a stirring role for metabolism in cells.

Based on recent findings indicating that metabolism might be governed by a limit on the rate at which cells can dissipate Gibbs energy, in this Perspective, we propose a new mechanism of how metabolic activity could globally regulate biomolecular processes in a cell. Specifically, we postulate that Gibbs energy released in metabolic reactions is used to perform work, allowing enzymes to self-propel or to break free from supramolecular structures. This catalysis-induced enzyme movement will result in increased intracellular motion, which in turn can compromise biomolecular functions. Once the increased intracellular motion has a detrimental effect on regulatory mechanisms, this will establish a feedback mechanism on metabolic activity, and result in the observed thermodynamic limit. While this proposed explanation for the identified upper rate limit on cellular Gibbs energy dissipation rate awaits experimental validation, it offers an intriguing perspective of how metabolic activity can globally affect biomolecular functions and will hopefully spark new research.

Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis.

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters.

A New Sugar for an Old Phage: a c-di-GMP-Dependent Polysaccharide Pathway Sensitizes Escherichia coli for Bacteriophage Infection.

Bacteriophages are ubiquitous parasites of bacteria and major drivers of bacterial ecology and evolution. Despite an ever-growing interest in their biotechnological and therapeutic applications, detailed knowledge of the molecular mechanisms underlying phage-host interactions remains scarce. Here, we show that bacteriophage N4 exploits a novel surface glycan (NGR) as a receptor to infect its host Escherichia coli. We demonstrate that this process is regulated by the second messenger c-di-GMP and that N4 infection is specifically stimulated by the diguanylate cyclase DgcJ, while the phosphodiesterase PdeL effectively protects E. coli from N4-mediated killing. PdeL-mediated protection requires its catalytic activity to reduce c-di-GMP and includes a secondary role as a transcriptional repressor. We demonstrate that PdeL binds to and represses the promoter of the wec operon, which encodes components of the enterobacterial common antigen (ECA) exopolysaccharide pathway. However, only the acetylglucosamine epimerase WecB but none of the other ECA components is required for N4 infection. Based on this, we postulate that NGR is an N-acetylmannosamine-based carbohydrate polymer that is produced and exported to the cell surface of E. coli in a c-di-GMP-dependent manner, where it serves as a receptor for N4. This novel carbohydrate pathway is conserved in E. coli and other bacterial pathogens, serves as the primary receptor for various bacteriophages, and is induced at elevated temperature and by specific amino acid-based nutrients. These studies provide an entry point into understanding how bacteria use specific regulatory mechanisms to balance costs and benefits of highly conserved surface structures. IMPORTANCE Because bacterial surface glycans are in direct contact with the environment they can provide essential protective functions during infections or against competing bacteria. But such structures are also "Achilles' heels" since they can serve as primary receptors for bacteriophages. Bacteria thus need to carefully control the exposure of conserved surface glycans to balance costs and benefits. Here, we identify a novel exopolysaccharide that is widely conserved in E. coli and is used by N4 and related bacteriophages as primary receptor. We demonstrate that the synthesis of NGR (N4 glycan receptor) is tightly controlled by the second messenger c-di-GMP in a highly specific manner and by a single diguanylate cyclase. These studies provide an example of how bacteria can alleviate the strong selective pressure imposed on them by bacteriophages entering through conserved surface structures by carefully regulating their synthesis and secretion.

A Robust Method for Generating, Quantifying, and Testing Large Numbers of Escherichia coli Persisters.

Bacteria can exhibit phenotypes that render them tolerant against antibiotics. However, often only a few cells of a bacterial population show the so-called persister phenotype, which makes it difficult to study this health-threatening phenotype. We recently found that certain abrupt nutrient shifts generate Escherichia coli populations that consist almost entirely of antibiotic-tolerant cells. These nearly homogeneous persister cell populations enable assessment with population-averaging experimental methods, such as high-throughput methods. In this chapter, we provide a detailed protocol for generating a large fraction of tolerant cells using the nutrient-switch approach. Furthermore, we describe how to determine the fraction of cells that enter the tolerant state upon a sudden nutrient shift and we provide a new way to assess antibiotic tolerance using flow cytometry. We envision that these methods will facilitate research into the important and exciting phenotype of bacterial persister cells.

Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices.

Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.

Physical bioenergetics: Energy fluxes, budgets, and constraints in cells.

Cells are the basic units of all living matter which harness the flow of energy to drive the processes of life. While the biochemical networks involved in energy transduction are well-characterized, the energetic costs and constraints for specific cellular processes remain largely unknown. In particular, what are the energy budgets of cells? What are the constraints and limits energy flows impose on cellular processes? Do cells operate near these limits, and if so how do energetic constraints impact cellular functions? Physics has provided many tools to study nonequilibrium systems and to define physical limits, but applying these tools to cell biology remains a challenge. Physical bioenergetics, which resides at the interface of nonequilibrium physics, energy metabolism, and cell biology, seeks to understand how much energy cells are using, how they partition this energy between different cellular processes, and the associated energetic constraints. Here we review recent advances and discuss open questions and challenges in physical bioenergetics.

A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux.

RNA-based sensors for intracellular metabolites are a promising solution to the emerging issue of metabolic heterogeneity. However, their development, i.e., the conversion of an aptamer into an in vivo-functional intracellular metabolite sensor, still harbors challenges. Here, we accomplished this for the glycolytic flux-signaling metabolite, fructose-1,6-bisphosphate (FBP). Starting from in vitro selection of an aptamer, we constructed device libraries with a hammerhead ribozyme as actuator. Using high-throughput screening in yeast with fluorescence-activated cell sorting (FACS), next-generation sequencing, and genetic-environmental perturbations to modulate the intracellular FBP levels, we identified a sensor that generates ratiometric fluorescent readout. An abrogated response in sensor mutants and occurrence of two sensor conformations-revealed by RNA structural probing-indicated in vivo riboswitching activity. Microscopy showed that the sensor can differentiate cells with different glycolytic fluxes within yeast populations, opening research avenues into metabolic heterogeneity. We demonstrate the possibility to generate RNA-based sensors for intracellular metabolites for which no natural metabolite-binding RNA element exits.

Implications of initial physiological conditions for bacterial adaptation to changing environments.

This piece discusses how the different observations of two independent studies (Kotte et al, 2014; Basan et al, 2020), regarding population-level heterogeneity and lag times during diauxic shift, can be largely explained by different experimental protocols.

How bacteria recognise and respond to surface contact.

Bacterial biofilms can cause medical problems and issues in technical systems. While a large body of knowledge exists on the phenotypes of planktonic and of sessile cells in mature biofilms, our understanding of what happens when bacteria change from the planktonic to the sessile state is still very incomplete. Fundamental questions are unanswered: for instance, how do bacteria sense that they are in contact with a surface, and what are the very initial cellular responses to surface contact. Here, we review the current knowledge on the signals that bacteria could perceive once they attach to a surface, the signal transduction systems that could be involved in sensing the surface contact and the cellular responses that are triggered as a consequence to surface contact ultimately leading to biofilm formation. Finally, as the main obstacle in investigating the initial responses to surface contact has been the difficulty to experimentally study the dynamic response of single cells upon surface attachment, we also review recent experimental approaches that could be employed to study bacterial surface sensing, which ultimately could lead to an improved understanding of how biofilm formation could be prevented.

Manipulating rod-shaped bacteria with optical tweezers.

Optical tweezers have great potential in microbiology for holding and manipulating single cells under a microscope. However, the methodology to use optical tweezers for live cell studies is still at its infancy. In this work, we determined suitable parameters for stable trapping of single Escherichia coli bacteria, and identified the upper limits of IR-exposure that can be applied without affecting viability. We found that the maximum tolerable IR-exposure is 2.5-fold higher when employing oscillating instead of stationary optical trapping (20 J and 8 J, respectively). We found that good stability of cells in an oscillating trap is achieved when the effective trap length is 20% larger than the cell length, the oscillation frequency higher than 100 Hz and the trap oriented perpendicular to the medium flow direction. Further, we show, using an IR power just sufficient for stable holding, that bacteria remain viable during at least 30 min of holding in an oscillating trap. In this work, we established a method for long-term stable handling of single E. coli cells using optical tweezers. This work will pave the way for future use of optical tweezers in microbiology.

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor.

Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), we transplanted the B. subtilis FBP-binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.

Differential scaling between G1 protein production and cell size dynamics promotes commitment to the cell division cycle in budding yeast.

In the unicellular eukaryote Saccharomyces cerevisiae, Cln3-cyclin-dependent kinase activity enables Start, the irreversible commitment to the cell division cycle. However, the concentration of Cln3 has been paradoxically considered to remain constant during G1, due to the presumed scaling of its production rate with cell size dynamics. Measuring metabolic and biosynthetic activity during cell cycle progression in single cells, we found that cells exhibit pulses in their protein production rate. Rather than scaling with cell size dynamics, these pulses follow the intrinsic metabolic dynamics, peaking around Start. Using a viral-based bicistronic construct and targeted proteomics to measure Cln3 at the single-cell and population levels, we show that the differential scaling between protein production and cell size leads to a temporal increase in Cln3 concentration, and passage through Start. This differential scaling causes Start in both daughter and mother cells across growth conditions. Thus, uncoupling between two fundamental physiological parameters drives cell cycle commitment.

Inference of the High-Level Interaction Topology between the Metabolic and Cell-Cycle Oscillators from Single-Cell Dynamics.

Recent evidence suggests that the eukaryotic metabolism is an autonomous oscillator. Together with oscillating elements of the cyclin/CDK machinery, this oscillator might form a coupled oscillator system, from which cell-cycle control emerges. The topology of interactions between the metabolic oscillator and the elements of the cyclin/CDK machinery, however, remains unknown. Using single-cell metabolic and cell-cycle dynamics in yeast, and solving an inverse problem with a system of Kuramoto oscillators, we inferred how the metabolic oscillator interacts with the cyclin/CDK machinery. The identified and experimentally validated interaction topology shows that the early and late cell cycle are independently driven by metabolism. While in this topology, the S phase is coordinated by START. We obtained no support for a strong interaction between early and late cell cycle. The identified high-level interaction topology will guide future efforts to discover the molecular links between metabolism and the cell cycle.

Publisher Correction: Definitions and guidelines for research on antibiotic persistence.

In Figure 2b, the minimal duration for killing (MDK) 99% of tolerant cells was erroneously labelled as MDK99.99 instead of MDK99. This has now been corrected in all versions of the Review. The publisher apologizes to the authors and to readers for this error.