A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit

Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55–10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range.publishe

SARS-CoV-2 nsp3 and nsp4 are minimal constituents of a pore spanning replication organelle

Funding Information: We thank the Infectious Diseases Imaging Platform (IDIP) at the Center for Integrative Infectious Disease Research Heidelberg, the cryo-EM network at the Heidelberg University (HD-cryoNET) and Heidelberg University Electron Microscopy Core Facility for support and assistance. The authors gratefully acknowledge the data storage service SDS@hd supported by the Ministry of Science, Research, and the Arts Baden-Württemberg (MWK), the German Research Foundation (DFG) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG. This work was supported by a research grant from the Chica and Heinz Schaller Foundation (Schaller Research Group Leader Program). Work of P.C. and R.B. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project no. 240245660–SFB1129 and project number 437060729 (PC and MWM). In addition, work of R.B. is supported by DFG project no. 272983813–TRR 179) and by the project “Virological and immunological determinants of COVID-19 pathogenesis – lessons to get prepared for future pandemics (KA1-Co-02 “COVIPA”)”, a grant from the Helmholtz Association’s Initiative and Networking Fund. V.P. is supported by a European Molecular Biology Organization (EMBO) long-term fellowship (ALTF454-2020). LZ and JM are supported by CoVLP project of the Flagship Initiative Engineering Molecular Systems. ZH received support for this work from FCT - Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, 510 UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and from a joint research agreement with the Okinawa Institute of Science and Technology. Funding Information: We thank the Infectious Diseases Imaging Platform (IDIP) at the Center for Integrative Infectious Disease Research Heidelberg, the cryo-EM network at the Heidelberg University (HD-cryoNET) and Heidelberg University Electron Microscopy Core Facility for support and assistance. The authors gratefully acknowledge the data storage service SDS@hd supported by the Ministry of Science, Research, and the Arts Baden-Württemberg (MWK), the German Research Foundation (DFG) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG. This work was supported by a research grant from the Chica and Heinz Schaller Foundation (Schaller Research Group Leader Program). Work of P.C. and R.B. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project no. 240245660–SFB1129 and project number 437060729 (PC and MWM). In addition, work of R.B. is supported by DFG project no. 272983813–TRR 179) and by the project “Virological and immunological determinants of COVID-19 pathogenesis – lessons to get prepared for future pandemics (KA1-Co-02 “COVIPA”)”, a grant from the Helmholtz Association’s Initiative and Networking Fund. V.P. is supported by a European Molecular Biology Organization (EMBO) long-term fellowship (ALTF454-2020). LZ and JM are supported by CoVLP project of the Flagship Initiative Engineering Molecular Systems. ZH received support for this work from FCT - Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, 510 UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and from a joint research agreement with the Okinawa Institute of Science and Technology. Publisher Copyright: © 2023, The Author(s).Coronavirus replication is associated with the remodeling of cellular membranes, resulting in the formation of double-membrane vesicles (DMVs). A DMV-spanning pore was identified as a putative portal for viral RNA. However, the exact components and the structure of the SARS-CoV-2 DMV pore remain to be determined. Here, we investigate the structure of the DMV pore by in situ cryo-electron tomography combined with subtomogram averaging. We identify non-structural protein (nsp) 3 and 4 as minimal components required for the formation of a DMV-spanning pore, which is dependent on nsp3-4 proteolytic cleavage. In addition, we show that Mac2-Mac3-DPUP-Ubl2 domains are critical for nsp3 oligomerization and crown integrity which influences membrane curvature required for biogenesis of DMVs. Altogether, SARS-CoV-2 nsp3-4 have a dual role by driving the biogenesis of replication organelles and assembly of DMV-spanning pores which we propose here to term replicopores.publishersversionpublishe

Conformational changes in the essential E. coli septal cell wall synthesis complex suggest an activation mechanism

Funding Information: The authors thank all members of the Hensel, Xiao, and Lau laboratories for helpful discussions and feedback on the manuscript. Z.H. received support for this work from FCT—Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020, Z.H.) and LS4FUTURE Associated Laboratory (LA/P/0087/2020, Z.H.), from a joint research agreement with the Okinawa Institute of Science and Technology, and from the Google Cloud Research Credits program with the award GCP20210916. S.F.C. received support from FCT Fundação para a Ciência e a Tecnologia, I.P., through a PhD fellowship (PD/BD/135480/2018, SMC). Work in the Xiao laboratory was supported by NIH F32GM143895 (B.M.B.), NIH T32GM007445 (J.W.M), and R35GM136436 (J.X.). Work in the Lau laboratory was funded by the Johns Hopkins Catalyst Award (to A.Y.L.); NIH T32GM135131 (to R.A.Y.). Anton 2 computer time (MCB130045P) was provided by the Pittsburgh Supercomputing Center (PSC) through NIH grant R01GM116961 (to A.Y.L.); the Anton 2 machine at PSC was generously made available by D.E. Shaw Research. We also used resources provided by Advanced Research Computing at Hopkins (ARCH) at Johns Hopkins University. Funding Information: The authors thank all members of the Hensel, Xiao, and Lau laboratories for helpful discussions and feedback on the manuscript. Z.H. received support for this work from FCT—Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020, Z.H.) and LS4FUTURE Associated Laboratory (LA/P/0087/2020, Z.H.), from a joint research agreement with the Okinawa Institute of Science and Technology, and from the Google Cloud Research Credits program with the award GCP20210916. S.F.C. received support from FCT Fundação para a Ciência e a Tecnologia, I.P., through a PhD fellowship (PD/BD/135480/2018, SMC). Work in the Xiao laboratory was supported by NIH F32GM143895 (B.M.B.), NIH T32GM007445 (J.W.M), and R35GM136436 (J.X.). Work in the Lau laboratory was funded by the Johns Hopkins Catalyst Award (to A.Y.L.); NIH T32GM135131 (to R.A.Y.). Anton 2 computer time (MCB130045P) was provided by the Pittsburgh Supercomputing Center (PSC) through NIH grant R01GM116961 (to A.Y.L.); the Anton 2 machine at PSC was generously made available by D.E. Shaw Research. We also used resources provided by Advanced Research Computing at Hopkins (ARCH) at Johns Hopkins University. Publisher Copyright: © 2023, The Author(s).The bacterial divisome is a macromolecular machine composed of more than 30 proteins that controls cell wall constriction during division. Here, we present a model of the structure and dynamics of the core complex of the E. coli divisome, supported by a combination of structure prediction, molecular dynamics simulation, single-molecule imaging, and mutagenesis. We focus on the septal cell wall synthase complex formed by FtsW and FtsI, and its regulators FtsQ, FtsL, FtsB, and FtsN. The results indicate extensive interactions in four regions in the periplasmic domains of the complex. FtsQ, FtsL, and FtsB support FtsI in an extended conformation, with the FtsI transpeptidase domain lifted away from the membrane through interactions among the C-terminal domains. FtsN binds between FtsI and FtsL in a region rich in residues with superfission (activating) and dominant negative (inhibitory) mutations. Mutagenesis experiments and simulations suggest that the essential domain of FtsN links FtsI and FtsL together, potentially modulating interactions between the anchor-loop of FtsI and the putative catalytic cavity of FtsW, thus suggesting a mechanism of how FtsN activates the cell wall synthesis activities of FtsW and FtsI.publishersversionpublishe

Entropy-driven formation of a chiral liquid-crystalline phase of helical filaments

Author Posting. © The Authors, 2006. This article is posted here by permission of American Physical Society for personal use, not for redistribution. The definitive version was published in Physical Review Letters 96 (2006): 018305, doi:10.1103/PhysRevLett.96.018305.We study the liquid-crystalline phase behavior of a concentrated suspension of helical flagella isolated from Salmonella typhimurium. Flagella are prepared with different polymorphic states, some of which have a pronounced helical character while others assume a rodlike shape. We show that the static phase behavior and dynamics of chiral helices are very different when compared to simpler achiral hard rods. With increasing concentration, helical flagella undergo an entropy-driven first order phase transition to a liquid-crystalline state having a novel chiral symmetry.M. S. and R. O. are supported by NIH Grant No. EB002583

On the value of preprints: an early career researcher perspective

Peer-reviewed journal publication is the main means for academic researchers in the life sciences to create a permanent, public record of their work. These publications are also the de facto currency for career progress, with a strong link between journal brand recognition and perceived value. The current peer-review process can lead to long delays between submission and publication, with cycles of rejection, revision and resubmission causing redundant peer review. This situation creates unique challenges for early career researchers (ECRs), who rely heavily on timely publication of their work to gain recognition for their efforts. ECRs face changes in the academic landscape including the increased interdisciplinarity of life sciences research, expansion of the researcher population and consequent shifts in employer and funding demands. The publication of preprints, publicly available scientific manuscripts posted on dedicated preprint servers prior to journal managed peer-review, can play a key role in addressing these ECR challenges. Preprinting benefits include rapid dissemination of academic work, open access, establishing priority or concurrence, receiving feedback and facilitating collaborations. While there is a growing appreciation for and adoption of preprints, a minority of all articles in life sciences and medicine are preprinted. The current low rate of preprint submissions in life sciences and ECR concerns regarding preprinting needs to be addressed. We provide a perspective from an interdisciplinary group of early career researchers on the value of preprints and advocate the wide adoption of preprints to advance knowledge and facilitate career development

Cell constriction requires processive septal peptidoglycan synthase movement independent of FtsZ treadmilling in Staphylococcus aureus

Funding Information: We thank members of the Pinho lab, P. Pereira (ITQB-NOVA), S. Filipe (FCT-NOVA) and J. Xiao (Johns Hopkins University) for helpful discussions; L. Lavis (Janelia Research Campus, Ashburn) for the generous gift of JF549-HTL, JF549-cpSTL and JFX650-STL; E. Harry (University of Technology, Sydney) for providing the anti-FtsZ antibody; T. Roemer (Merck) for providing DMPI; M. S. VanNieuwenhze (Indiana University) for providing HADA; and the Electron Microscopy Facility and Genomics Unit of Instituto Gulbenkian de Ciência. This study was funded by the European Research Council (ERC) through grant ERC-2017-CoG-771709 (to M.G.P.), by Fundação para a Ciência e a Tecnologia (FCT) through MOSTMICRO-ITQB R and D Unit (UIDB/04612/2020, UIDP/04612/2020 to ITQB-NOVA) and LS4FUTURE Associated Laboratory (LA/P/0087/2020 to ITQB-NOVA); by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 839596 (to S.S.), by the European Molecular Biology Organization (EMBO) through award ALTF 673-2018 (to S.S.), by The Company of Biologists Ltd. (Journal of Cell Science) under the travelling fellowship agreement JCSTF1911323 (to S.S.), and by FCT through contract 2022.03033.CEECIND (to S.S.). R.H.’s contributions were supported by the Gulbenkian Foundation, the ERC (grant agreement no. 101001332) and the EMBO installation grant (EMBO-2020-IG-4734). Extended Data Figure was created with Biorender.com . Publisher Copyright: © The Author(s) 2024.Bacterial cell division requires recruitment of peptidoglycan (PG) synthases to the division site by the tubulin homologue, FtsZ. Septal PG synthases promote septum growth. FtsZ treadmilling is proposed to drive the processive movement of septal PG synthases and septal constriction in some bacteria; however, the precise mechanisms spatio-temporally regulating PG synthase movement and activity and FtsZ treadmilling are poorly understood. Here using single-molecule imaging of division proteins in the Gram-positive pathogen Staphylococcus aureus, we showed that the septal PG synthase complex FtsW/PBP1 and its putative activator protein, DivIB, move with similar velocity around the division site. Impairing FtsZ treadmilling did not affect FtsW or DivIB velocities or septum constriction rates. Contrarily, PG synthesis inhibition decelerated or stopped directional movement of FtsW and DivIB, and septum constriction. Our findings suggest that a single population of processively moving FtsW/PBP1 associated with DivIB drives cell constriction independently of FtsZ treadmilling in S. aureus.publishersversioninpres

In Vivo Structure of the E. coli FtsZ-ring Revealed by Photoactivated Localization Microscopy (PALM)

The FtsZ protein, a tubulin-like GTPase, plays a pivotal role in prokaryotic cell division. In vivo it localizes to the midcell and assembles into a ring-like structure-the Z-ring. The Z-ring serves as an essential scaffold to recruit all other division proteins and generates contractile force for cytokinesis, but its supramolecular structure remains unknown. Electron microscopy (EM) has been unsuccessful in detecting the Z-ring due to the dense cytoplasm of bacterial cells, and conventional fluorescence light microscopy (FLM) has only provided images with limited spatial resolution (200–300 nm) due to the diffraction of light. Hence, given the small sizes of bacteria cells, identifying the in vivo structure of the Z-ring presents a substantial challenge. Here, we used photoactivated localization microscopy (PALM), a single molecule-based super-resolution imaging technique, to characterize the in vivo structure of the Z-ring in E. coli. We achieved a spatial resolution of ∼35 nm and discovered that in addition to the expected ring-like conformation, the Z-ring of E. coli adopts a novel compressed helical conformation with variable helical length and pitch. We measured the thickness of the Z-ring to be ∼110 nm and the packing density of FtsZ molecules inside the Z-ring to be greater than what is expected for a single-layered flat ribbon configuration. Our results strongly suggest that the Z-ring is composed of a loose bundle of FtsZ protofilaments that randomly overlap with each other in both longitudinal and radial directions of the cell. Our results provide significant insight into the spatial organization of the Z-ring and open the door for further investigations of structure-function relationships and cell cycle-dependent regulation of the Z-ring

Energy injustice and Nordic electric mobility: inequality, elitism, and externalities in the electrification of vehicle-to-grid (V2G) transport

Much research on electric mobility transitions has been descriptive or positive, rather than normative or critical, assessing the deeper ethical, justice, or moral issues that arise. To address this gap, this study qualitatively assesses the ongoing transition to Nordic electric vehicles (EVs) and vehicle-to-grid (V2G) systems. It does so through the various lenses of distributive justice, procedural justice, cosmopolitan justice, and recognition justice. It asks: what are the types of injustices associated with electric mobility and V2G? In what ways do emerging patterns of electric mobility worsen socio-environmental risks or vulnerabilities? Based on original primary data collected from 257 experts across Denmark, Finland, Iceland, Norway, and Sweden, the study finds that electric mobility can erode elements of distributive justice for being accessible only to the rich, and for raising risks related to privacy, hacking, and cyberterrorism. Electric mobility may contravene aspects of procedural justice by reinforcing exclusion and elitism in national planning. It can erode cosmopolitan justice by producing negative environmental externalities, and exacerbating rural (and global) vulnerability. It may threaten recognition justice through unemployment, disruption to traditional businesses, and the entrenchment of patriarchy. Thankfully, the study also proposes a suite of policy mechanisms to address many of these concerns