No abstract

The replicative DNA polymerase PolIIIα from Escherichia coli is a uniquely fast and processive enzyme. For its activity it relies on the DNA sliding clamp β, the proofreading exonuclease ε and the C-terminal domain of the clamp loader subunit τ. Due to the dynamic nature of the four-protein complex it has long been refractory to structural characterization. Here we present the 8 Å resolution cryo-electron microscopy structures of DNA-bound and DNA-free states of the PolIII-clamp-exonuclease-τc complex. The structures show how the polymerase is tethered to the DNA through multiple contacts with the clamp and exonuclease. A novel contact between the polymerase and clamp is made in the DNA bound state, facilitated by a large movement of the polymerase tail domain and τc. These structures provide crucial insights into the organization of the catalytic core of the replisome and form an important step towards determining the structure of the complete holoenzyme.DOI: http://dx.doi.org/10.7554/eLife.11134.001

Faithful DNA replication is essential to all forms of life and depends on the action of 3'-5' exonucleases that remove misincorporated nucleotides from the newly synthesized strand. However, how the DNA is transferred from the polymerase to the exonuclease active site is not known. Here we present the cryo-EM structure of the editing mode of the catalytic core of the Escherichia coli replisome, revealing a dramatic distortion of the DNA whereby the polymerase thumb domain acts as a wedge that separates the two DNA strands. Importantly, NMR analysis of the DNA substrate shows that the presence of a mismatch increases the fraying of the DNA, thus enabling it to reach the exonuclease active site. Therefore the mismatch corrects itself, whereas the exonuclease subunit plays a passive role. Hence, our work provides unique insights into high-fidelity replication and establishes a new paradigm for the correction of misincorporated nucleotides.

The tetanus neurotoxin (TeNT) is a highly potent toxin produced by that inhibits neurotransmission of inhibitory interneurons, causing spastic paralysis in the tetanus disease. TeNT differs from the other clostridial neurotoxins by its unique ability to target the central nervous system by retrograde axonal transport. The crystal structure of the tetanus toxin reveals a "closed" domain arrangement stabilised by two disulphide bridges, and the molecular details of the toxin's interaction with its polysaccharide receptor. An integrative analysis combining X-ray crystallography, solution scattering and single particle electron cryo-microscopy reveals pH-mediated domain rearrangements that may give TeNT the ability to adapt to the multiple environments encountered during intoxication, and facilitate binding to distinct receptors.

The tetanus neurotoxin (TeNT) is a highly potent toxin produced by Clostridium tetani that inhibits neurotransmission of inhibitory interneurons, causing spastic paralysis in the tetanus disease. TeNT differs from the other clostridial neurotoxins by its unique ability to target the central nervous system by retrograde axonal transport. The crystal structure of the tetanus toxin reveals a "closed" domain arrangement stabilised by two disulphide bridges, and the molecular details of the toxin's interaction with its polysaccharide receptor. An integrative analysis combining X-ray crystallography, solution scattering and single particle electron cryo-microscopy reveals pH-mediated domain rearrangements that may give TeNT the ability to adapt to the multiple environments encountered during intoxication, and facilitate binding to distinct receptors.

In coming decades, drought is expected to expand globally owing to increased evaporation and reduced rainfall. Understanding, predicting, and controlling crop plants’ rhizosphere has the potential to manipulate its responses to environmental stress. Our plant growth-promoting rhizobacteria (PGPR) are isolated from a natural laboratory, ‘The Evolution Canyon’, Israel, (EC), from the wild progenitors of cereals, where they have been co-habituating with their hosts for long periods of time. The study revealed that commercial TM50 silica particles (SN) triggered the PGPR production of exopolysaccharides (EPS) containing D-glucuronate (D-GA). The increased EPS content increased the PGPR water-holding capacity (WHC) and osmotic pressure of the biofilm matrix, which led to enhanced plant biomass in drought-stressed growth environments. Light- and cryo-electron- microscopic studies showed that, in the presence of silica (SN) particles, bacterial morphology is changed, indicating that SNs are associated with significant reprogramming in bacteria. The findings encourage the development of large-scale methods for isolate formulation with natural silicas that ensure higher WHC and hyperosmolarity under field conditions. Osmotic pressure involvement of holobiont cohabitation is also discussed.

Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4’s broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria.

14In coming decades drought is expected to expand globally owing to increased 15 evaporation and reduced rainfall. In order to reduce the vulnerability of agricultural 16 systems we need to understand the crop plant growth environment. Understanding, 17 predicting and controlling the rhizosphere has potential to harness plant microbe 18 interactions, improve plant responses to environmental stress and mitigate effects of 19 climate change. Our plant growth-promoting rhizobacteria (PGPR) are isolated from 20 the natural laboratory 'Evolution Canyon' Israel (EC). The endophytic rhizobacteria 21 from the wild progenitors of cereals have been co-habituated with their hosts for long 22 periods of time. The study revealed that silica particles (SN) triggered the PGPR 23 production of exopolysaccharides (EPS) containing D-glucuronate (D-GA). This leads 24 to increased plant biomass accumulation in drought-stressed growth environments. 25The PGPR increased EPS content increases the water holding capacity (WHC) and 26 osmotic pressure of the biofilm matrix. Light-and electron-microscopic studies show 27 that in the presence of SN particles, bacterial morphology is changed, indicating that 28 SNs are associated with significant reprogramming in bacteria. 29The results here show that the production of EPS containing D-GA is induced by SN 30 treatment. The findings encourage formulation of cells considering microencapsulation 31 with materials that ensure higher WHC and hyperosmolarity under field conditions. 32Our results illustrate the importance of considering natural soil nanoparticles in the 33 application of PGPR. Osmotic pressure involvement of holobiont cohabitation is 34 discussed. 35 36 65 keeper for the bacterial drought tolerance enhancement (Timmusk, Kim et al. 2015).66 EC is a well described natural laboratory where microbes have co-habituated with 67 the hosts over a long period of time (Nevo 2012). Reciprocity between the organisms 68 is generally accepted (Gilbert, Bosch et al. 2015, Gilbert 2016, Rice, Kallonen et al. 69 2016, Gilbert 2019) and organisms that have co-evolved within the environment are 70 more robust to environmental stress situations (Robinson, Klein et al. 2018, Vega 71 2019). Microbes found in extreme habitats developed a different strategy to adapt to 72 such conditions through evolution. Holobiotic complex relationships in the given 73 framework, not robotic associations of genes, are becoming more and more accepted 74 in developmental and evolutionary biology (Gilbert, Bosch et al. 2015, Gilbert 2016, 75 Rice, Kallonen et al. 2016, Gilbert 2019). Hence, understanding the framework of 76 adaptation to extreme environments is gaining interest in a period of environment 77 change (Robinson, Klein et al. 2018, Vega 2019) . 78 Our recent plant nanointerface interaction research focusing on native soil particles 79 shows that silica nanoparticles (SN) promote PGPR attachment and plant 80 colonization, and this effect is translated into crop yield enhancement under stress 81 condit...