Insights into the Structure and Protein Composition of Moorella thermoacetica Spores Formed at Different Temperatures

The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores.

PALYNOLOGICAL STUDIES OF ARTIFACTS FROM CATACOMBS No. 97 AND No. 98 FROM THE DARGAVS CEMETERY: ANALYSIS AND INTERPRETATION

Даргавский могильник является одним из уникальных памятников на юге России. где сохранились немногочисленные. но информативные находки из органики. В 2019 г. в ходе раскопок Терским отрядом ИА РАН катакомб № 97 (IX в.) и 98 (втор. пол. VIII - перв. пол. IX в.) для палинологического анализа отобрано три образца: № 1 - содержимое кожаного мешочка (катакомба № 97). № 2 - бусина из помета (катакомба № 97) и № 3 - грунт из стеклянного стакана (катакомба № 98). По данным палинологического анализа установлено содержимое кожаного мешочка (мука или зерно) и стеклянного стакана (напиток. содержащий культурные злаки. или хлебное изделие. накрывающее стакан). принадлежность бусины. изготовленной из помета мелкого рогатого скота, определен сезон погребения в катакомбе № 97 (два первых летних месяца). Наличие хелицер клещей в мешочке и телиоспор головни в стакане может свидетельствовать о зараженности запасов вредителями и патогенными грибами. Результаты определения шерсти из катакомбы № 97 позволяют предположить, что на погребенной была верхняя одежда из овечьей шкуры. Изученные находки тесно связаны с дохристианскими представлениями оставившего могильник раннесредневекового населения о непосредственной связи мира мертвых с плодородием и урожаем. The Dargavs cemetery is one of the unique sites in the South of Russia that has preserved few but informative organic residue. In 2019 during the excavations of catacombs No. 97 (9 century) and No. 98 (second half of the 8 - first half of the 9 century the Terskiy team of the Institute of Archaeology, RAS, selected three samples for palynological analysis. Sample No. 1 is contents of a small leather bag (Catacomb No. 97); Sample No. 2 is a bead from animal droppings (catacomb No. 97) and Sample No. 3 - soil from a glass tumbler (catacomb No. 98). The palynological analysis established the contents of the leather bag (flour or grains) and the glass tumbler (a drink containing cultivated gramineous plants or some bread stuff placed over the glass tumbler), the origin of the bead made from droppings of goats or sheep. The season of the burial in catacomb No. 97 was determined as the first two summer months. Presence of the chelicerae of ticks in the bag and teliospores of blight in the tumbler suggests that the stored food was contaminated with pests and pathogenic fungi. The determination of wool in catacomb No. 97 suggests that the buried person wore an outer coat made from sheep skin. The examined finds are closely related with pre-Christian beliefs of the early medieval population that has left this cemetery concerning direct links between the world of the dead and fertility and harvest.

Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network

COPII mediates Endoplasmic Reticulum to Golgi trafficking of thousands of cargoes. Five essential proteins assemble into a two-layer architecture, with the inner layer thought to regulate coat assembly and cargo recruitment, and the outer coat forming cages assumed to scaffold membrane curvature. Here we visualise the complete, membrane-assembled COPII coat by cryo-electron tomography and subtomogram averaging, revealing the full network of interactions within and between coat layers. We demonstrate the physiological importance of these interactions using genetic and biochemical approaches. Mutagenesis reveals that the inner coat alone can provide membrane remodelling function, with organisational input from the outer coat. These functional roles for the inner and outer coats significantly move away from the current paradigm, which posits membrane curvature derives primarily from the outer coat. We suggest these interactions collectively contribute to coat organisation and membrane curvature, providing a structural framework to understand regulatory mechanisms of COPII trafficking and secretion.

Combinatorial multivalent interactions drive cooperative assembly of the COPII coat

Protein secretion is initiated at the endoplasmic reticulum by the COPII coat, which self-assembles to form vesicles. Here, we examine the mechanisms by which a cargo-bound inner coat layer recruits and is organized by an outer scaffolding layer to drive local assembly of a stable structure rigid enough to enforce membrane curvature. An intrinsically disordered region in the outer coat protein, Sec31, drives binding with an inner coat layer via multiple distinct interfaces, including a newly defined charge-based interaction. These interfaces combinatorially reinforce each other, suggesting coat oligomerization is driven by the cumulative effects of multivalent interactions. The Sec31 disordered region could be replaced by evolutionarily distant sequences, suggesting plasticity in the binding interfaces. Such a multimodal assembly platform provides an explanation for how cells build a powerful yet transient scaffold to direct vesicle traffic.

Steady-state Regulation of Secretory Cargo Export by Inositol Trisphosphate Receptors and Penta EF Hand Proteins

ABSTRACTER Ca2+ regulates ER-to-Golgi transport machinery. Sustained Ca2+ signaling by inositol trisphosphate receptors (IP3Rs) leads to depression of cargo export through activation of penta EF hand protein (PEF) ALG-2 which reduces outer COPII coat at ER exit sites (ERES). However, we do not know whether tonic Ca2+ signals during steady-state conditions affect ER export rates and if so by what mechanisms. Here we report that partial depletion of IP3 receptors from NRK epithelial cells causes a marked increase of basal ER export of the transmembrane glycoprotein cargo VSV-G. The increased ER-to-Golgi transport required ALG-2 and was actuated by decreased peflin and increased ALG-2 at ER exit sites (ERES) – a condition previously demonstrated to stimulate COPII-dependent ER export. Upon IP3R depletion the amount of outer coat at ERES increased, the opposite to what occurs during ALG-2-dependent inhibition of secretion during agonist-driven Ca2+ signaling. The increased ER export correlated with reduced spontaneous cytosolic Ca2+ oscillations caused by the reduced number of Ca2+ release channels. IP3R depletion also unexpectedly resulted in partial depletion of ER luminal Ca2+ stores. The low Ca2+ conditions appeared to decrease both ALG-2 and peflin expression levels somewhat, but these were the only detectable expression changes in COPII trafficking machinery and the Ca2+ decrease had no detectable impact on ER stress. We conclude that at steady state, IP3Rs produce tonic Ca2+ signals that suppress the basal rate of ER export by maintaining lower outer coat targeting to ERES.

A multivalent fuzzy interface drives reversible COPII coat assembly

Protein secretion is initiated at the endoplasmic reticulum by the COPII coat, which self-assembles to form vesicles. Here, we examine the mechanisms by which the outer scaffolding layer of the coat drives local assembly of a structure rigid enough to enforce membrane curvature, yet able to readily disassemble at the Golgi. An intrinsically disordered region in the outer coat protein, Sec31, drives binding with an inner coat layer via multiple distinct interfaces. Interactions are individually dispensable but combinatorially reinforce each other, suggesting coat oligomerization is driven by the cumulative effects of multivalent interactions. Such a multimodal assembly platform could be readily reversed at the Golgi via perturbation of each individual interface. These design principles provide an explanation for how cells build a powerful yet transient scaffold to direct vesicle traffic.

A protein phosphorylation module patterns theBacillus subtilisspore outer coat

Assembly of theBacillus subtilisspore coat involves over 80 protein components, which self-organize into a basal layer, a lamellar inner coat, a striated electrondense outer coat and a more external crust. CotB is an abundant component of the outer coat. Its C-terminal moiety contains a region, termed SKRB, formed by a series of serine-rich repeats, which we show is phosphorylated by the coat-associated Ser/Thr kinase CotH at multiple Ser residues. Another coat protein, CotG, which contains a central repeat region, SKRG, interacts with the C-terminal moiety of CotB and promotes its phosphorylation by CotHin vivoand in a heterologous system. CotG itself is phosphorylated by CotH but phosphorylation is enhanced in the absence of CotB. Spores of acotHD288Astrain, producing an inactive form of the kinase, like those formed by acotGdeletion mutant, lack the characteristic pattern of electrondense outer coat striations, while retaining the crust. Specifically, in the absence of CotG or CotH activity, most of the outer coat proteins are assembled but form a layer of amorphous material that peels-off the spore if crust formation is genetically ablated. In contrast, deletion of the SKRBregion, has no major impact on the structure of the outer coat. Thus, phosphorylation of CotG by CotH is the principal factor establishing the structural pattern of the spore outer coat. The presence of thecotB/cotH/cotGcluster in several species closely related toB. subtilisand ofcotG-like proteins in nearly all spore-formers that also code for a CotH homologue hints at the importance of this protein phosphorylation module in the morphogenesis of the spore outer layers.

Flatworm-specific transcriptional regulators promote the specification of tegumental progenitors in Schistosoma mansoni

Schistosomes infect more than 200 million people. These parasitic flatworms rely on a syncytial outer coat called the tegument to survive within the vasculature of their host. Although the tegument is pivotal for their survival, little is known about maintenance of this tissue during the decades schistosomes survive in the bloodstream. Here, we demonstrate that the tegument relies on stem cells (neoblasts) to specify fusogenic progenitors that replace tegumental cells lost to turnover. Molecular characterization of neoblasts and tegumental progenitors led to the discovery of two flatworm-specific zinc finger proteins that are essential for tegumental cell specification. These proteins are homologous to a protein essential for neoblast-driven epidermal maintenance in free-living flatworms. Therefore, we speculate that related parasites (i.e., tapeworms and flukes) employ similar strategies to control tegumental maintenance. Since parasitic flatworms infect every vertebrate species, understanding neoblast-driven tegumental maintenance could identify broad-spectrum therapeutics to fight diseases caused by these parasites.