The Arabidopsis thaliana AtHMA1 protein is a member of the P IB -ATPase family, which is implicated in heavy metal transport. However, sequence analysis reveals that AtHMA1 possesses a predicted stalk segment present in SERCA (sarcoplas- Plant Ca 2ϩ and heavy metal-ATPases belong to the superfamily of P-ATPases (1, 2). Their common characteristic is the presence of a phosphorylated intermediary in the catalytic cycle. In Arabidopsis, the heavy metal-ATPases belong to the P IB subfamily and normally have eight predicted transmembrane domains, whereas the Ca 2ϩ -ATPases are part of the P IIA and P IIB subfamilies and have ten predicted transmembrane domains (2). A common feature among the P IB -ATPases is the presence of a CPX motif, which is though to play a role in metal translocation, as well as putative metal-binding domains located at the amino or carboxyl terminus (3). On the other hand, P IIB ATPases have a calmodulin-binding domain that regulate their activity; however, the P IIA do not have this domain (4). The most important difference between the calcium and heavy metal-ATPases is their substrate specificity (1). However, in vitro metal transport studies performed with membrane fractions isolated from seedlings suggest that one or more members of the superfamily of P-ATPases are capable of transporting calcium and heavy metals. The studies showed competitive inhibition between active transport of heavy metals (such as copper and cadmium) and calcium.mic2 Interestingly, both transport activities were inhibited by the sesquiterpene lactone thapsigargin, a potent and specific inhibitor of SERCA 3 -type pumps (5-7). To date no one has described plant ion pumps that transport calcium and heavy metals in biochemical terms, nor have scientists described genes encoding for ion pumps inhibited by thapsigargin or plant mutants with thapsigargin-sensitive/tolerant phenotypes. Based on the results of biochemical assays suggesting the existence of thapsigargin-sensitive Ca 2ϩ /heavy metal-ATPase, we searched for potential candidate proteins in the Arabidopsis genome. This was made possible by the highly conserved "stalk segment" or S3 sequence adjacent to the third transmembrane segment of the SERCA pumps (8, 9) composed of amino acids DEF-GEQLSK (5-7). This sequence was almost complete and was annotated as a stalk segment (using the topology prediction software ARAMEMNON) in the Arabidopsis heavy metal pump AtHMA1 (At4g37270). This pump belongs to the subclass of zinc/cobalt/cadmium/lead-ATPases and is the most divergent metal pump of the Arabidopsis P IB -ATPases (1, 2, 10, 11) (see Fig. 1). It lacks an amino-terminal heavy metal-binding domain, such as GMXCXXC or GICC(T/S)SE, which is often found in other members of the group. It has an intramembranous SPC instead of the CP(C/H/S) motif located at the putative metal transporting site of P IB -ATPases (11, 12). The pump possesses other structural characteristics related to heavy metal binding and transport, such as a poly-H motif commonly found in zinc-binding pro...

The prokaryotic oxidation of reduced inorganic sulfur compounds (RISCs) is a topic of utmost importance from a biogeochemical and industrial perspective. Despite sulfur oxidizing bacterial activity is largely known, no quantitative approaches to biological RISCs oxidation have been made, gathering all the complex abiotic and enzymatic stoichiometry involved. Even though in the case of neutrophilic bacteria such as Paracoccus and Beggiatoa species the RISCs oxidation systems are well described, there is a lack of knowledge for acidophilic microorganisms. Here, we present the first experimentally validated stoichiometric model able to assess RISCs oxidation quantitatively in Acidithiobacillus thiooxidans (strain DSM 17318), the archetype of the sulfur oxidizing acidophilic chemolithoautotrophs. This model was built based on literature and genomic analysis, considering a widespread mix of formerly proposed RISCs oxidation models combined and evaluated experimentally. Thiosulfate partial oxidation by the Sox system (SoxABXYZ) was placed as central step of sulfur oxidation model, along with abiotic reactions. This model was coupled with a detailed stoichiometry of biomass production, providing accurate bacterial growth predictions. In silico deletion/inactivation highlights the role of sulfur dioxygenase as the main catalyzer and a moderate function of tetrathionate hydrolase in elemental sulfur catabolism, demonstrating that this model constitutes an advanced instrument for the optimization of At. thiooxidans biomass production with potential use in biohydrometallurgical and environmental applications.

Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with potential therapeutic applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of Nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent.

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Bacteria of the genus Prosthecobacter express homologs of eukaryotic ␣-and ␤-tubulin, called BtubA and BtubB (BtubA/B), that have been observed to assemble into filaments in the presence of GTP. BtubA/B polymers are proposed to be composed in vitro by two to six protofilaments in contrast to that in vivo, where they have been reported to form 5-protofilament tubes named bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may be derived from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is reversible and that BtubA/B folding does not require chaperones. To better understand BtubA/B filament behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro BtubA/B assembly using a combination of polymerization kinetics assays and microscopy. Like eukaryotic microtubules, BtubA/B filaments exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by BtubA/B polymerization drives a stochastic mechanism of filament disassembly that occurs via polymer breakage and/or fast continuous depolymerization. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of BtubA/B filament fragments. Unlike MTs, polymerization of BtubA/B requires KCl, which reduces the critical concentration for BtubA/B assembly and induces it to form stable mixed-orientation bundles in the absence of any additional BtubA/B-binding proteins. The complex dynamics that we observe in stabilized and unstabilized BtubA/B filaments may reflect common properties of an ancestral eukaryotic tubulin polymer.IMPORTANCE Microtubules are polymers within all eukaryotic cells that perform critical functions; they segregate chromosomes, organize intracellular transport, and support the flagella. These functions rely on the remarkable range of tunable dynamic behaviors of microtubules. Bacterial tubulin A and B (BtubA/B) are evolutionarily related proteins that form polymers. They are proposed to be evolved from the ancestral eukaryotic tubulin, a missing link in microtubule evolution. Using microscopy and biochemical approaches to characterize BtubA/B assembly in vitro, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules but differ in how they self-associate into bundles and how this bundling affects their stability. Our results demonstrate the diversity of mechanisms through which tubulin homologs promote filament dynamics and monomer turnover.