A fully sequenced collection of homozygous EMS mutants for forward and reverse genetic screens in Arabidopsis thaliana.

Genetic screens are powerful tools for biological research and are one of the reasons for the success of the thale cress Arabidopsis thaliana as a research model. Here, we describe the whole-genome sequencing of 871 Arabidopsis lines from the Homozygous EMS Mutant (HEM) collection as a novel resource for forward and reverse genetics. With an average 576 high-confidence mutations per HEM line, over three independent mutations altering protein sequences are found on average per gene in the collection. Pilot reverse genetics experiments on reproductive, developmental, immune and physiological traits confirmed the efficacy of the tool for identifying both null, knockdown and gain-of-function alleles. The possibility of conducting subtle repeated phenotyping and the immediate availability of the mutations will empower forward genetic approaches. The sequence resource is searchable with the ATHEM web interface (https://lipm-browsers.toulouse.inra.fr/pub/ATHEM/), and the biological material is distributed by the Versailles Arabidopsis Stock Center.

The structural landscape and diversity of Pyricularia oryzae MAX effectors revisited.

Magnaporthe AVRs and ToxB-like (MAX) effectors constitute a family of secreted virulence proteins in the fungus Pyricularia oryzae (syn. Magnaporthe oryzae), which causes blast disease on numerous cereals and grasses. In spite of high sequence divergence, MAX effectors share a common fold characterized by a ß-sandwich core stabilized by a conserved disulfide bond. In this study, we investigated the structural landscape and diversity within the MAX effector repertoire of P. oryzae. Combining experimental protein structure determination and in silico structure modeling we validated the presence of the conserved MAX effector core domain in 77 out of 94 groups of orthologs (OG) identified in a previous population genomic study. Four novel MAX effector structures determined by NMR were in remarkably good agreement with AlphaFold2 (AF2) predictions. Based on the comparison of the AF2-generated 3D models we propose a classification of the MAX effectors superfamily in 20 structural groups that vary in the canonical MAX fold, disulfide bond patterns, and additional secondary structures in N- and C-terminal extensions. About one-third of the MAX family members remain singletons, without strong structural relationship to other MAX effectors. Analysis of the surface properties of the AF2 MAX models also highlights the high variability within the MAX family at the structural level, potentially reflecting the wide diversity of their virulence functions and host targets.

New recognition specificity in a plant immune receptor by molecular engineering of its integrated domain.

Plant nucleotide-binding and leucine-rich repeat domain proteins (NLRs) are immune sensors that recognize pathogen effectors. Here, we show that molecular engineering of the integrated decoy domain (ID) of an NLR can extend its recognition spectrum to a new effector. We relied for this on detailed knowledge on the recognition of the Magnaporthe oryzae effectors AVR-PikD, AVR-Pia, and AVR1-CO39 by, respectively, the rice NLRs Pikp-1 and RGA5. Both receptors detect their effectors through physical binding to their HMA (Heavy Metal-Associated) IDs. By introducing into RGA5_HMA the AVR-PikD binding residues of Pikp-1_HMA, we create a high-affinity binding surface for this effector. RGA5 variants carrying this engineered binding surface perceive the new ligand, AVR-PikD, and still recognize AVR-Pia and AVR1-CO39 in the model plant N. benthamiana. However, they do not confer extended disease resistance specificity against M. oryzae in transgenic rice plants. Altogether, our study provides a proof of concept for the design of new effector recognition specificities in NLRs through molecular engineering of IDs.

Resolving the activation mechanism of the D99N antiterminator LicT protein.

LicT is an antiterminator protein of the BglG family whose members are key players in the control of carbohydrate catabolism in bacteria. These antiterminators are generally composed of three modules, an N-terminal RNA-binding domain (CAT) followed by two homologous regulation modules (PRD1 and PRD2) that control the RNA binding activity of the effector domain via phosphorylation on conserved histidines. Although several structures of isolated domains of BglG-like proteins have been described, no structure containing CAT and at least one PRD simultaneously has yet been reported in an active state, precluding detailed understanding of signal transduction between modules. To fulfill this gap, we recently reported the complete NMR sequence assignment of a constitutively active mutant (D99N) CAT-PRD1*, which contains the effector domain and the first regulation domain of LicT. As a follow-up, we have determined and report here the 3D solution structure of this active, dimeric LicT construct (40 kDa). The structure reveals how the mutation constrains the PRD1 regulation domain into an active conformation which is transduced to CAT via a network of negatively charged residues belonging to PRD1 dimeric interface and to the linker region. In addition, our data support a model where BglG-type antitermination regulatory modules can only adopt a single conformation compatible with the active structure of the effector domain, regardless of whether activation is mediated by mutation on the first or second PRD. The linker between the effector and regulation modules appears to function as an adaptable hinge tuning the position of the functional modules.

Modular Imaging Scaffold for Single-Particle Electron Microscopy.

Technological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and to raise novel challenges, specifically related to sample preparation and heterogeneous macromolecular assemblies such as DNA-protein, protein-protein, and membrane protein assemblies. Here, we built a V-shaped DNA origami as a scaffolding molecular system to template proteins at user-defined positions in space. This template positions macromolecular assemblies of various sizes, juxtaposes combinations of biomolecules into complex arrangements, isolates biomolecules in their active state, and stabilizes membrane proteins in solution. In addition, the design can be engineered to tune DNA mechanical properties by exerting a controlled piconewton (pN) force on the molecular system and thus adapted to characterize mechanosensitive proteins. The binding site can also be specifically customized to accommodate the protein of interest, either interacting spontaneously with DNA or through directed chemical conjugation, increasing the range of potential targets for single-particle EM investigation. We assessed the applicability for five different proteins. Finally, as a proof of principle, we used RNAP protein to validate the approach and to explore the compatibility of the template with cryo-EM sample preparation.

An oligomeric switch controls the Mrr-induced SOS response in E. coli.

Mrr from Escherichia coli K12 is a type IV restriction endonuclease whose role is to recognize and cleave foreign methylated DNA. Beyond this protective role, Mrr can inflict chromosomal DNA damage that elicits the SOS response in the host cell upon heterologous expression of specific methyltransferases such as M.HhaII, or after exposure to high pressure (HP). Activation of Mrr in response to these perturbations involves an oligomeric switch that dissociates inactive homo-tetramers into active dimers. Here we used scanning number and brightness (sN&B) analysis to determine in vivo the stoichiometry of a constitutively active Mrr mutant predicted to be dimeric and examine other GFP-Mrr mutants compromised in their response to either M.HhaII activity or HP shock. We also observed in vitro the direct pressure-induced tetramer dissociation by HP fluorescence correlation spectroscopy of purified GFP-Mrr. To shed light on the linkages between subunit interactions and activity of Mrr and its variants, we built a structural model of the full-length tetramer bound to DNA. Similar to functionally related endonucleases, the conserved DNA cleavage domain would be sequestered by the DNA recognition domain in the Mrr inactive tetramer, dissociating into an enzymatically active dimer upon interaction with multiple DNA sites.

Quantitative High-Resolution Imaging of Live Microbial Cells at High Hydrostatic Pressure.

The majority of the Earth's microbial biomass exists in the deep biosphere, in the deep ocean, and within the Earth's crust. Although other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold the pressure on the Earth's surface. The mechanisms of pressure adaptation are also important in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here, we describe implementation of a high-pressure sample chamber with a two-photon scanning microscope system, allowing for the first time, to our knowledge, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for fluorescence lifetime imaging microscopy with phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning number and brightness and raster scanning imaging correlation spectroscopy) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in Escherichia coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. These results provide a proof of principle that quantitative, high-resolution imaging of live microbial cells can be carried out at pressures equivalent to those in the deepest ocean trenches.

NMR chemical shift assignment of a constitutively active fragment of the antitermination protein LicT.

LicT belongs to an essential family of bacterial antitermination proteins which bind to nascent mRNAs in order to stimulate transcription of sugar-metabolizing operons. As most of other antitermination proteins involved in carbohydrate metabolism, LicT is composed of a N-terminal RNA-binding module (CAT) and two homologous regulatory modules (PRD1 and PRD2). The activity of the CAT effector module is controlled by antagonist phosphorylations by the phosphotransferase system on conserved histidines of the two C-terminal PRDs in response to available carbon sources. Previous studies on truncated and mutant constructs have provided partial structural insight into the mechanism of signal transduction between the N-terminal RNA-binding domain and the two regulation modules. However, no structure at atomic resolution has been ever solved that contain the RNA-binding domain and a regulation module. We report the NMR assignment of a constitutively active fragment of LicT, named D99A-CAT-PRD1 or CAT-PRD1*. This fragment is composed of the RNA-binding module and the first N-terminal regulation module which bears the mutation of Asp99 to an asparagine. It is dimeric as the native protein, with a 40 kD molecular weight. The D99N mutation is sufficient to endow this fragment with a high RNA-binding constitutive activity, in a phosphorylation-free context. The assignment reported here should set the base of future NMR investigation of signal transduction between the regulatory module and the effector module in the active state of the protein, and in the long term enable the structural study of the full length protein structure in interaction with its target RNA.

Structural Insights into of the Allosteric Activation of the LicT Antiterminator by PTS-Mediated Phosphorylation.

LicT belongs to an essential family of bacterial transcriptional antitermination proteins controlling the expression of sugar-metabolizing operons. When activated, they bind to nascent mRNAs, preventing premature arrest of transcription. The RNA binding capacity of the N-terminal domain CAT is controlled by phosphorylations of two homologous regulation modules by the phosphotransferase system (PTS). Previous studies on truncated and mutant proteins provided partial insight into the mechanism of signal transduction between the effector and regulatory modules. We report here the conformational and functional investigation on the allosteric activation of full-length LicT. Combining fluorescence anisotropy and NMR, we find a tight correlation between LicT RNA binding capacity and CAT closure upon PTS-mediated phosphorylation and phosphomimetic mutations. Our study highlights fine structural differences between activation processes. Furthermore, the NMR study of full-length proteins points to the back and forth propagation of structural restraints from the RNA binding to the distal regulatory module.

High pressure activation of the Mrr restriction endonuclease in Escherichia coli involves tetramer dissociation.

A sub-lethal hydrostatic pressure (HP) shock of ∼100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M.HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M.HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M.HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M.HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer.

Competitive folding of RNA structures at a termination-antitermination site.

Antitermination is a regulatory process based on the competitive folding of terminator-antiterminator structures that can form in the leader region of nascent transcripts. In the case of the Bacillus subtilis licS gene involved in ß-glucosides utilization, the binding of the antitermination protein LicT to a short RNA hairpin (RAT) prevents the formation of an overlapping terminator and thereby allows transcription to proceed. Here, we monitored in vitro the competition between termination and antitermination by combining bulk and single-molecule fluorescence-based assays using labeled RNA oligonucleotide constructs of increasing length that mimic the progressive transcription of the terminator invading the antiterminator hairpin. Although high affinity binding is abolished as soon as the antiterminator basal stem is disrupted by the invading terminator, LicT can still bind and promote closing of the partially unfolded RAT hairpin. However, binding no longer occurs once the antiterminator structure has been disrupted by the full-length terminator. Based on these findings, we propose a kinetic competition model for the sequential events taking place at the termination-antitermination site, where LicT needs to capture its RAT target before completion of the terminator to remain tightly bound during RNAP pausing, before finally dissociating irreversibly from the elongated licS transcript.

A part toolbox to tune genetic expression in Bacillus subtilis.

Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium Escherichia coli, such libraries are lacking for the Gram-positive model Bacillus subtilis, a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting ∼14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 µM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis.

The RYMV-encoded viral suppressor of RNA silencing P1 is a zinc-binding protein with redox-dependent flexibility.

Viral suppressors of RNA interference (VSRs) target host gene silencing pathways, thereby operating important roles in the viral cycle and in host cells, in which they counteract host innate immune responses. However, the molecular mechanisms of VSRs are poorly understood. We provide here biochemical and biophysical features of the dual suppressor/activator VSR P1 protein encoded by the rice yellow mottle virus. In silico analyses of P1 suggested common features with zinc finger proteins and native mass spectrometry unambiguously confirmed that recombinant P1 binds reversibly two zinc atoms, each with a different strength. Additionally, we demonstrate that the reaction of P1 with H2O2 leads to zinc release, disulfide bond formation, and protein oligomerization. A reversible protein modification by redox alterations has only been described for a limited number of zinc finger proteins and has never been reported for VSRs. Those reported here for P1 might be a general feature of Cys-rich VSRs and could be a key regulatory mechanism for the control of RNA silencing.

Interactions in gene expression networks studied by two-photon fluorescence fluctuation spectroscopy.

Fluorescence fluctuation techniques have proven to be extremely useful in the characterization of biomolecular interactions. Here an overview of recent applications of two-photon fluorescence fluctuation to the study of protein-nucleic acid complexes implicated in translational and transcriptional regulation is presented. In particular, the issue of the stoichiometry of the complexes is addressed using fluorescence (cross) correlation spectroscopy (F(C)CS) for the in vitro studies of one RNA-binding protein (the bacterial ribosomal protein L20) and two transcriptional repressors (CggR and CcpN, implicated in the control of the central carbon metabolism in Bacillus subtilis). Then, the application of two-photon scanning number and brightness (2psN&B) analysis of fluorescence microscopy measurements in single cells is presented. Multiple technical aspects related to the adaptation of this method to live bacteria are discussed. This approach was used to count the number of fluorescent protein molecules produced from different inducible promoters in B. subtilis reporter strains, in hundreds of individual cells under both permissive and repressive conditions. We present a case study in which the stochastic activity of glycolytic and gluconeogenic gene promoters could be quantified in vivo by 2psN&B and be related to the repression mechanisms proposed from in vitro studies.

Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET.

The control of transcription termination by RNA-binding proteins that modulate RNA-structures is an important regulatory mechanism in bacteria. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an anti-terminator RNA hairpin that overlaps an intrinsic terminator located in the 5'-mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this anti-termination/termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY anti-terminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we could monitor directly their opening or closing state, and thus investigate the effects on this equilibrium of the binding of anti-termination proteins or terminator-mimicking oligonucleotides. We show that the anti-terminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the anti-terminator hairpins determines both the affinity and specificity of the anti-termination protein binding. Finally, we show that stabilization of the anti-terminator hairpin, by an extended basal stem or anti-termination protein binding can efficiently counteract the competing effect of the terminator-mimic.

Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states.

Assessing gene expression noise in order to obtain mechanistic insights requires accurate quantification of gene expression on many individual cells over a large dynamic range. We used a unique method based on 2-photon fluorescence fluctuation microscopy to measure directly, at the single cell level and with single-molecule sensitivity, the absolute concentration of fluorescent proteins produced from the two Bacillus subtilis promoters that control the switch between glycolysis and gluconeogenesis. We quantified cell-to-cell variations in GFP concentrations in reporter strains grown on glucose or malate, including very weakly transcribed genes under strong catabolite repression. Results revealed strong transcriptional bursting, particularly for the glycolytic promoter. Noise pattern parameters of the two antagonistic promoters controlling the nutrient switch were differentially affected on glycolytic and gluconeogenic carbon sources, discriminating between the different mechanisms that control their activity. Our stochastic model for the transcription events reproduced the observed noise patterns and identified the critical parameters responsible for the differences in expression profiles of the promoters. The model also resolved apparent contradictions between in vitro operator affinity and in vivo repressor activity at these promoters. Finally, our results demonstrate that negative feedback is not noise-reducing in the case of strong transcriptional bursting.

Absolute quantification of gene expression in individual bacterial cells using two-photon fluctuation microscopy.

Quantification of promoter activity or protein expression in gene regulatory networks is generally achieved via measurement of fluorescent protein (FP) intensity, which is related to the true FP concentration by an unknown scaling factor, thereby limiting analysis and interpretation. Here, using approaches originally developed for eukaryotic cells, we show that two-photon (2p) fluorescence fluctuation microscopy, specifically scanning number and brightness (sN&B) analysis, can be applied to determine the absolute concentrations of diffusing FPs in live bacterial cells. First, we demonstrate the validity of the approach, despite the small size of the bacteria, using the central pixels and spatial averaging. We established the lower detection limit at or below 75 nM (~3 molecules of FP/vol(ex)) and the upper detection limit at approximately 10 µM, which can be extended using intensity measurements. We found that the uncertainty inherent in our measurements (<5%) was smaller than the high cell-cell variations observed for stochastic leakage from FP fusions of the lac promoter in the repressed state or the 10 to 25% variation observed on induction. This demonstrates that a reliable and absolute measure of transcriptional noise can be made using our approach, which should make it particularly appropriate for the investigation of stochasticity in gene expression networks.

Prevention of cross-talk in conserved regulatory systems: identification of specificity determinants in RNA-binding anti-termination proteins of the BglG family.

Each family of signal transduction systems requires specificity determinants that link individual signals to the correct regulatory output. In Bacillus subtilis, a family of four anti-terminator proteins controls the expression of genes for the utilisation of alternative sugars. These regulatory systems contain the anti-terminator proteins and a RNA structure, the RNA anti-terminator (RAT) that is bound by the anti-terminator proteins. We have studied three of these proteins (SacT, SacY, and LicT) to understand how they can transmit a specific signal in spite of their strong structural homology. A screen for random mutations that render SacT capable to bind a RNA structure recognized by LicT only revealed a substitution (P26S) at one of the few non-conserved residues that are in contact with the RNA. We have randomly modified this position in SacT together with another non-conserved RNA-contacting residue (Q31). Surprisingly, the mutant proteins could bind all RAT structures that are present in B. subtilis. In a complementary approach, reciprocal amino acid exchanges have been introduced in LicT and SacY at non-conserved positions of the RNA-binding site. This analysis revealed the key role of an arginine side-chain for both the high affinity and specificity of LicT for its cognate RAT. Introduction of this Arg at the equivalent position of SacY (A26) increased the RNA binding in vitro but also resulted in a relaxed specificity. Altogether our results suggest that this family of anti-termination proteins has evolved to reach a compromise between RNA binding efficacy and specific interaction with individual target sequences.

Physical basis of the inducer-dependent cooperativity of the Central glycolytic genes Repressor/DNA complex.

The Central glycolytic genes Repressor (CggR) from Bacillus subtilis belongs to the SorC family of transcription factors that control major carbohydrate metabolic pathways. Recent studies have shown that CggR binds as a tetramer to its tandem operator DNA sequences and that the inducer metabolite, fructose 1,6-bisphosphate (FBP), reduces the binding cooperativity of the CggR/DNA complex. Here, we have determined the effect of FBP on the size, shape and stoichiometry of CggR complexes with full-length and half-site operator sequence by small-angle X-ray scattering, size-exclusion chromatography, fluorescence cross-correlation spectroscopy and noncovalent mass spectrometry (MS). Our results show that CggR forms a compact tetrameric assembly upon binding to either the full-length operator or two half-site DNAs and that FBP triggers a tetramer-dimer transition that leaves a single dimer on the half-site or two physically independent dimers on the full-length target. Although the binding of other phospho-sugars was evidenced by MS, only FBP was found to completely disrupt dimer-dimer contacts. We conclude that inducer-dependent dimer-dimer bridging interactions constitute the physical basis for CggR cooperative binding to DNA and the underlying repression mechanism. This work provides experimental evidences for a cooperativity-based regulation model that should apply to other SorC family members.

Combination of noncovalent mass spectrometry and traveling wave ion mobility spectrometry reveals sugar-induced conformational changes of central glycolytic genes repressor/DNA complex.

The central glycolytic genes repressor (CggR) is a 37 kDa transcriptional repressor protein which plays a key role in Bacillus subtilis glycolysis by regulating the transcription of the gapA operon. Fructose-1,6-bisphosphate (FBP), identified as the effector sugar, has been shown to abolish the binding cooperativity of CggR to its DNA target and to modify the conformational dynamics of the CggR/DNA complex. In the present study, noncovalent mass spectrometry (MS) was used to obtain deeper insights into FBP-dependent CggR/DNA interactions. The effect of FBP binding on CggR alone and on CggR/DNA complexes was examined using automated chip-based nanoelectrospray MS and traveling wave ion mobility mass spectrometry (IM-MS). Our results revealed that tetrameric CggR dissociates into dimers upon FBP binding. Moreover, FBP binding to CggR/DNA complexes triggers disruption of intermolecular protein/protein interactions within the complex, significantly modifying its conformation as evidenced by a 5% increase of its collision cross section. For the first time, the use of IM-MS is reported to probe ligand-induced conformational modifications of a protein/DNA complex with an emphasis on the comparison with solution-based techniques.