Physicochemical surface properties of Chlorella vulgaris: a multiscale assessment, from electrokinetic and proton uptake descriptors to intermolecular adhesion forces.

Given the growing scientific and industrial interests in green microalgae, a comprehensive understanding of the forces controlling the colloidal stability of these bioparticles and their interactions with surrounding aqueous microenvironment is required. Accordingly, we addressed here the electrostatic and hydrophobic surface properties of Chlorella vulgaris from the population down to the individual cell levels. We first investigated the organisation of the electrical double layer at microalgae surfaces on the basis of electrophoresis measurements. Interpretation of the results beyond zeta-potential framework underlined the need to account for both the hydrodynamic softness of the algae cells and the heterogeneity of their interface formed with the outer electrolyte solution. We further explored the nature of the structural charge carriers at microalgae interfaces through potentiometric proton titrations. Extraction of the electrostatic descriptors of interest from such data was obscured by cell physiology processes and dependence thereof on prevailing measurement conditions, which includes light, temperature and medium salinity. As an alternative, cell electrostatics was successfully evaluated at the cellular level upon mapping the molecular interactions at stake between (positively and negatively) charged atomic force microscopy tips and algal surface via chemical force microscopy. A thorough comparison between charge-dependent tip-to-algae surface adhesion and hydrophobicity level of microalgae surface evidenced that the contribution of electrostatics to the overall interaction pattern is largest, and that the electrostatic/hydrophobic balance can be largely modulated by pH. Overall, the combination of multiscale physicochemical approaches allowed a drawing of some of the key biosurface properties that govern microalgae cell-cell and cell-surface interactions.

Taxonomic and functional responses of soil and root bacterial communities associated with poplar exposed to a contamination gradient of phenanthrene.

Polycyclic aromatic hydrocarbon (PAH) contamination of industrial wasteland soils affects microbial diversity, but little is known about the dose-response effects of such contaminants on taxonomic and functional diversities of rhizospheric and plant endophytic bacteria. This study focused on the response of soil and root bacterial communities associated to poplar grown in a contamination gradient of phenanthrene (PHE). It was hypothesized that the increase in contamination would modify gradually the bacterial diversity and functions. The effects of the PHE contamination were limited to soil communities and did not affect the poplar root endophytome where Streptomyces and Cutibacterium were the most abundant genera. Along the PHE gradient, alpha-diversity indices decreased and the community structure of soil bacteria at the taxonomic level shifted. The abundance of genes involved in PAH-degradation pathways and the relative proportion of certain microbial taxa such as Polaromonas, Sphingopyxis, Peredibacter, Phenylobacterium, Ramlibacter, Sphingomonas, and Pseudomonas, often described as potential PAH biodegraders, increased with the PHE concentration in the soil community. Conversely, the contamination negatively impacted other taxa like Nocardioides, Streptomyces, Gaiella, Solirubrobacter, Bradyrhizobium, and Nitrospira. Functional inference and enzymatic activity measurements revealed that some bacterial functions related to carbon, nitrogen and phosphorus cycles were modified in soil throughout the PHE gradient. This study allowed a deeper understanding of the complex plant-bacteria interactions in the case of soil PAH contamination and the potential impact on soil functioning.

Probing the mechanism of the peroxiredoxin decamer interaction with its reductase sulfiredoxin from the single molecule to the solution scale.

Peroxiredoxins from the Prx1 subfamily (Prx) are highly regulated multifunctional proteins involved in oxidative stress response, redox signaling and cell protection. Prx is a homodimer that associates into a decamer. The monomer C-terminus plays intricate roles in Prx catalytic functions, decamer stability and interaction with its redox partner, the small reductase sulfiredoxin (Srx), that regulates the switching between Prx cellular functions. As only static structures of covalent Prx-Srx complexes have been reported, whether Srx binding dissociates the decameric assembly and how Prx subunit flexibility impacts complex formation are unknown. Here, we assessed the non-covalent interaction mechanism and dynamics in the solution of Saccharomyces cerevisiae Srx with the ten subunits of Prx Tsa1 at the decamer level via a combination of multiscale biophysical approaches including native mass spectrometry. We show that the ten subunits of the decamer can be saturated by ten Srx molecules and that the Tsa1 decamer in complex with Srx does not dissociate in solution. Furthermore, the binding events of atomic force microscopy (AFM) tip-grafted Srx molecules to Tsa1 individual subunits were relevant to the interactions between free molecules in solution. Combined with protein engineering and rapid kinetics, the observation of peculiar AFM force-distance signatures revealed that Tsa1 C-terminus flexibility controls Tsa1/Srx two-step binding and dynamics and determines the force-induced dissociation of Srx from each subunit of the decameric complex in a sequential or concerted mode. This combined approach from the solution to the single-molecule level offers promising prospects for understanding oligomeric protein interactions with their partners.

Dynamics of a Key Conformational Transition in the Mechanism of Peroxiredoxin Sulfinylation.

Peroxiredoxins from the Prx1 subfamily (Prx) are moonlighting peroxidases that operate in peroxide signaling and are regulated by sulfinylation. Prxs offer a major model of protein-thiol oxidative modification. They react with H2O2 to form a sulfenic acid intermediate that either engages into a disulfide bond, committing the enzyme into its peroxidase cycle, or again reacts with peroxide to produce a sulfinic acid that inactivates the enzyme. Sensitivity to sulfinylation depends on the kinetics of these two competing reactions and is critically influenced by a structural transition from a fully folded (FF) to locally unfolded (LU) conformation. Analysis of the reaction of the Tsa1 Saccharomyces cerevisiae Prx with H2O2 by Trp fluorescence-based rapid kinetics revealed a process linked to the FF/LU transition that is kinetically distinct from disulfide formation and suggested that sulfenate formation facilitates local unfolding. Use of mutants of distinctive sensitivities and of different peroxide substrates showed that sulfinylation sensitivity is not coupled to the resolving step kinetics but depends only on the sulfenic acid oxidation and FF-to-LU transition rate constants. In addition, stabilization of the active site FF conformation, the determinant of sulfinylation kinetics, is only moderately influenced by the Prx C-terminal tail dynamics that determine the FF → LU kinetics. From these two parameters, the relative sensitivities of Prxs toward hyperoxidation with different substrates can be predicted, as confirmed by in vitro and in vivo patterns of sulfinylation.

TREM-1 multimerization is essential for its activation on monocytes and neutrophils.

The triggering receptor expressed on myeloid cells-1 (TREM-1) is a receptor expressed on innate immune cells. By promoting the amplification of inflammatory signals that are initially triggered by Toll-like receptors (TLRs), TREM-1 has been characterized as a major player in the pathophysiology of acute and chronic inflammatory diseases, such as septic shock, myocardial infarction, atherosclerosis, and inflammatory bowel diseases. However, the molecular events leading to the activation of TREM-1 in innate immune cells remain unknown. Here, we show that TREM-1 is activated by multimerization and that the levels of intracellular Ca2+ release, reactive oxygen species, and cytokine production correlate with the degree of TREM-1 aggregation. TREM-1 activation on primary human monocytes by LPS required a two-step process consisting of upregulation followed by clustering of TREM-1 at the cell surface, in contrast to primary human neutrophils, where LPS induced a rapid cell membrane reorganization of TREM-1, which confirmed that TREM-1 is regulated differently in primary human neutrophils and monocytes. In addition, we show that the ectodomain of TREM-1 is able to homooligomerize in a concentration-dependent manner, which suggests that the clustering of TREM-1 on the membrane promotes its oligomerization. We further show that the adapter protein DAP12 stabilizes TREM-1 surface expression and multimerization. TREM-1 multimerization at the cell surface is also mediated by its endogenous ligand, a conclusion supported by the ability of the TREM-1 inhibitor LR12 to limit TREM-1 multimerization. These results provide evidence for ligand-induced, receptor-mediated dimerization of TREM-1. Collectively, our findings uncover the mechanisms necessary for TREM-1 activation in monocytes and neutrophils.

CRDSAT Generated by pCARGHO: A New Efficient Lectin-Based Affinity Tag Method for Safe, Simple, and Low-Cost Protein Purification.

Purification of recombinant proteins remains a bottleneck for downstream processing. The authors engineered a new galectin 3 truncated form (CRDSAT ), functionally and structurally characterized, with preserved solubility and lectinic activity. Taking advantage of these properties, the authors designed an expression vector (pCARGHO), suitable for CRDSAT -tagged protein expression in prokaryotes. CRDSAT binds to lactose-Sepharose with a high specificity and facilitates solubilization of fusion proteins. This tag is structurally stable and can be easily removed from fusion proteins using TEV protease. Furthermore, due to their basic isoelectric point (pI), CRDSAT , and TEV are efficiently eliminated using cationic exchange chromatography. When pI of the protein of interest (POI) and CRDSAT are close, other chromatographic methods are successfully tested. Using CRDSAT tag, the authors purified several proteins from prokaryote and eukaryote origin and demonstrated as examples, the preservation of both Escherichia coli Thioredoxin 1 and human CDC25Bcd activities. Overall, yields of proteins obtained after tag removal are about 5-50 mg per litre of bacterial culture. Our purification method displays various advantages described herein that may greatly interest academic laboratories, biotechnology, and pharmaceutical companies.

Opposing signals differentially regulate transcript stability in Aspergillus nidulans.

A good model for gene regulation, requiring the organism to monitor a complex and changing environment and respond in a precise and rapid way, is nitrogen metabolism in Aspergillus nidulans. This involves co-ordinated expression of hundreds of genes, many dependent on the transcription factor AreA, which monitors the nitrogen state of the cell. AreA activity is in part modulated by differential degradation of its transcript in response to intracellular glutamine. Here we report that glutamine triggers synchronized degradation of a large subset of transcripts involved in nitrogen metabolism. Among these are all four genes involved in the assimilation of nitrate. Significantly, we show that two of these transcripts, niaD and niiA, are stabilized by intracellular nitrate, directly reinforcing transcriptional regulation. Glutamine-signalled degradation and the nitrate-dependent stabilization of the niaD transcript are effected at the level of deadenylation and are dependent on its 3' UTR. When glutamine and nitrate are both present, nitrate stabilization is predominant, ensuring that nitrate and the toxic intermediate nitrite are removed from the cell. Regulated transcript stability is therefore an integral part of the adaptive response. This represents the first example of distinct physiological signals competing to differentially regulate transcripts at the level of deadenylation.