Nanoscale dynamics of peptidoglycan assembly during the cell cycle of Streptococcus pneumoniae.

Dynamics of cell elongation and septation are key determinants of bacterial morphogenesis. These processes are intimately linked to peptidoglycan synthesis performed by macromolecular complexes called the elongasome and the divisome. In rod-shaped bacteria, cell elongation and septation, which are dissociated in time and space, have been well described. By contrast, in ovoid-shaped bacteria, the dynamics and relationships between these processes remain poorly understood because they are concomitant and confined to a nanometer-scale annular region at midcell. Here, we set up a metabolic peptidoglycan labeling approach using click chemistry to image peptidoglycan synthesis by single-molecule localization microscopy in the ovoid bacterium Streptococcus pneumoniae. Our nanoscale-resolution data reveal spatiotemporal features of peptidoglycan assembly and fate along the cell cycle and provide geometrical parameters that we used to construct a morphogenesis model of the ovoid cell. These analyses show that septal and peripheral peptidoglycan syntheses first occur within a single annular region that later separates in two concentric regions and that elongation persists after septation is completed. In addition, our data reveal that freshly synthesized peptidoglycan is remodeled all along the cell cycle. Altogether, our work provides evidence that septal peptidoglycan is synthesized from the beginning of the cell cycle and is constantly remodeled through cleavage and insertion of material at its periphery. The ovoid-cell morphogenesis would thus rely on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct successive phases of peripheral and septal synthesis

A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis

During spore formation in Bacillus subtilis a transenvelope complex is assembled across the double membrane that separates the mother cell and forespore. This complex (called the "A-Q complex") is required to maintain forespore development and is composed of proteins with remote homology to components of type II, III, and IV secretion systems found in Gram-negative bacteria. Here, we show that one of these proteins, SpoIIIAG, which has remote homology to ring-forming proteins found in type III secretion systems, assembles into an oligomeric ring in the periplasmic-like space between the two membranes. Three-dimensional reconstruction of images generated by cryo-electron microscopy indicates that the SpoIIIAG ring has a cup-and-saucer architecture with a 6-nm central pore. Structural modeling of SpoIIIAG generated a 24-member ring with dimensions similar to those of the EM-derived saucer. Point mutations in the predicted oligomeric interface disrupted ring formation in vitro and impaired forespore gene expression and efficient spore formation in vivo. Taken together, our data provide strong support for the model in which the A-Q transenvelope complex contains a conduit that connects the mother cell and forespore. We propose that a set of stacked rings spans the intermembrane space, as has been found for type III secretion systems

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

18 pags., 6 figs., 1 tab. -- Open Access funded by Creative Commons Atribution Licence 4.0Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation.This work used the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from INSTRUCT (“Innovative EM/NMR approach for the characterization of the drug target ClpP APPID: 301“), FRISBI (ANR-10-INSB-05-02), and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank the ESRF for beamtime at ID30A and ID23-1. Funding: This work was supported by Spanish Ministerio de Economia y Competitividad (BFU2016-78232-P) and Instituto de Salud Carlos III co-funded by European Union (PI15/00663 and PI18/00349, ERDF/ ESF, “Investing in your future”). This work was financially supported by the European Research Council (ERC-Stg-2012-311318 to P.S.). J.F. is supported by an EMBO long-term post-doctoral fellowship (ALTF441-2017)

A dynamic, ring-forming MucB / RseB-like protein influences spore shape in Bacillus subtilis.

How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ΔssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely supresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development

Structural insights into ring-building motif domains involved in bacterial sporulation.

Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for "Ring-Building Motifs". During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the β-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions

Structure and deformation of the Kermadec forearc in response to subduction of the Pacific oceanic plate

The Tonga-Kermadec forearc is deforming in response to on-going subduction of the Pacific Plate beneath the Indo-Australian Plate. Previous research has focussed on the structural development of the forearc where large bathymetric features such as the Hikurangi Plateau and Louisville Ridge seamount chain are being subducted. Consequently, knowledge of the ‘background’ forearc in regions of normal plate convergence is limited. We report on an ∼250-km-long multichannel seismic reflection profile that was shot perpendicular to the Tonga-Kermadec trench at ∼28°S to determine the lateral and temporal variations in the structure, stratigraphy and deformation of the Kermadec forearc resulting solely from Pacific Plate subduction. Interpretation of the seismic profile, in conjunction with regional swath bathymetry data, shows that the Pacific Plate exhibits horst and graben structures that accommodate bending-induced extensional stresses, generated as the trenchward dip of the crust increases. Trench infill is also much thicker than expected at 1 km which, we propose, results from increased sediment flux into and along the trench. Pervasive normal faulting of the mid-trench slope most likely accommodates the majority of the observed forearc extension in response to basal subduction erosion, and a structural high is located between the mid- and upper-trench slopes. We interpret this high as representing a dense and most likely structurally robust region of crust lying beneath this region. Sediment of the upper-trench slope documents depositional hiatuses and on-going uplift of the arc. Strong along-arc currents appear to erode the Kermadec volcanic arc and distribute this sediment to the surrounding basins, while currents over the forearc redistribute deposits as sediment waves. Minor uplift of the transitional Kermadec forearc, observed just to the north of the profile, appears to relate to an underlying structural trend as well as subduction of the Louisville Ridge seamount chain 250 km to the north. Relative uplift of the Kermadec arc is observed from changes in the tilt of upper-trench slope deposits and extensional faulting of the basement immediately surrounding the Louisville Ridge

Identification of temporal consistency in rating curve data : Bidirectional Reach (BReach)

In this paper, a methodology is developed to identify consistency of rating curve data based on a quality analysis of model results. This methodology, called Bidirectional Reach (BReach), evaluates results of a rating curve model with randomly sampled parameter sets in each observation. The combination of a parameter set and an observation is classified as nonacceptable if the deviation between the accompanying model result and the measurement exceeds observational uncertainty. Based on this classification, conditions for satisfactory behavior of a model in a sequence of observations are defined. Subsequently, a parameter set is evaluated in a data point by assessing the span for which it behaves satisfactory in the direction of the previous (or following) chronologically sorted observations. This is repeated for all sampled parameter sets and results are aggregated by indicating the endpoint of the largest span, called the maximum left (right) reach. This temporal reach should not be confused with a spatial reach (indicating a part of a river). The same procedure is followed for each data point and for different definitions of satisfactory behavior. Results of this analysis enable the detection of changes in data consistency. The methodology is validated with observed data and various synthetic stage-discharge data sets and proves to be a robust technique to investigate temporal consistency of rating curve data. It provides satisfying results despite of low data availability, errors in the estimated observational uncertainty, and a rating curve model that is known to cover only a limited part of the observations

Compensatory Evolution of pbp Mutations Restores the Fitness Cost Imposed by β-Lactam Resistance in Streptococcus pneumoniae

The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae

Informing UK governance of resilience to climate risks: improving the local evidence-base

International assessments of evidence on climate change (e.g. Intergovernmental Panel on Climate Change, IPCC) or national climate change risk assessments (e.g. UK Climate Change Risk Assessment, CCRA) do not offer a sufficiently granular perspective on climate impacts to adequately inform governance of resilience to climate risks at the local level. Using an analysis of UK decision-makers managing and responding to heatwaves and flood risks, this paper argues how more robust local evidence is needed to inform decision-making regarding adaptation options for enhancing local resilience. We identify evidence gaps and issues relating to local climate change impacts, including sources and quality of evidence used, adequacy and accessibility of evidence available, ill-communicated evidence and conflicting or misused evidence. A lack of appreciation regarding how scientific evidence and personal judgement can mutually enhance the quality of decision-making underpins all of these gaps. Additionally, we find that the majority of evidence currently used is reductively based upon socio-economic and physical characteristics of climate risks. We argue that a step change is needed in local climate resilience that moves beyond current physical and socio-economic risk characterisation to a more inclusive co-constitution of social and politically defined climate risks at the local scale that are better aligned with the local impacts felt and needs of stakeholders