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Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci.

Providing inert materials with a biochemical function, for example using proteins, is a cornerstone technology underlying many applications. However, the controlled construction of protein thin films remains a major challenge. Here, an innovative solvent-free approach for protein deposition is reported, using lysozyme as a model. By diverting a time-of-flight secondary ion mass spectrometer (ToF-SIMS) from its standard analytical function, large argon clusters were used to achieve protein transfer. A target consisting of a pool of proteins was bombarded with 10 keV Ar5000+ ions, and the ejected proteins were collected on a silicon wafer. The ellipsoidal deposition pattern was evidenced by ToF-SIMS analysis, while SDS-PAGE electrophoresis confirmed the presence of intact lysozyme on the collector. Finally, enzymatic activity assays demonstrated the preservation of the three-dimensional structure of the transferred proteins. These results pave the way to well-controlled protein deposition using ion beams and to the investigation of more complex multilayer architectures.

Although synthetic pesticides are still used to control insect pests, greater efforts have been made to develop healthier and more environmentally friendly pesticides. Because of their insecticidal properties, botanical essential oils (EOs) are considered as promising alternatives to the use of synthetic insecticides. However, little is known about mechanisms underlying the insecticidal activity of most these natural compounds. In the present study, we evaluated the contact toxicity and the modes of action of the EO from Mentha arvensis against the granary weevil, Sitophilus granarius L. (Coleoptera: Curculionidae), a cosmopolitan insect pest that causes extensive damage to stored cereals. M. arvensis EO caused high contact toxicity in S. granarius adults, resulting in a rapid paralysis and rapid alteration of walking behavior. Our label-free quantitative proteomics approach revealed that M. arvensis EO induced dramatic physiological changes in exposed insects. The majority of the differentially expressed proteins (DEPs) were upregulated and are related to the development and functioning of the muscular and nervous systems, cellular respiration, protein synthesis, and detoxification. These results suggest that M. arvensis essential oil is capable of affecting a variety of biological processes, and shed light on the repair mechanisms put in place in surviving insects to counter the damage inflicted. This work opens new perspectives on the proposed mechanisms of insecticidal activity of a promising EO for controlling pests of stored cereals and may represent a first step in the development of novel bio-rational insecticides.

The coupling between mitochondrial respiration and photosynthesis plays an important role in the energetic physiology of green plants and some secondary-red photosynthetic eukaryotes(diatoms), allowing an efficient CO2 assimilation and optimal growth. - Using the flagellate Euglena gracilis, we first tested if photosynthesis–respiration coupling occurs in this species harbouring secondary green plastids (i.e. originated from an endosymbiosis between a green alga and a phagotrophic euglenozoan). Second, we tested how the trophic state (mixotrophy and photoautotrophy) of the cell alters the mechanisms involved in the photosynthesis–respiration coupling. - Energetic coupling between photosynthesis and respiration was determined by testing the effect of respiratory inhibitors on photosynthesis, and measuring the simultaneous variation of photosynthesis and respiration rates as a function of temperature (i.e. thermal response curves). The mechanism involved in the photosynthesis–respiration coupling was assessed by combining proteomics, biophysical and cytological analyses. - Our work shows that there is photosynthesis–respiration coupling and membrane contacts between mitochondria and chloroplasts in E. gracilis. However, whereas in mixotrophy adjustment of the chloroplast ATP/NADPH ratio drives the interaction, in photoautotrophy the coupling is conditioned by CO2 limitation and photorespiration. This indicates that maintenance of photosynthesis–respiration coupling, through plastic metabolic responses, is key to E. gracilis functioning under changing environmental conditions.

Plasma membrane (PM) intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water and small solutes. The functional importance of the PM organisation of PIPs in the interaction with other cellular structures is not completely understood. We performed a pull-down assay using maize (Zea mays) suspension cells expressing YFP-ZmPIP2;5 and validated the protein interactions by yeast split-ubiquitin and bimolecular fluorescence complementation assays. We expressed interacting proteins tagged with fluorescent proteins in Nicotiana benthamiana leaves and performed water transport assays in oocytes. Finally, a phylogenetic analysis was conducted. The PM-located ZmPIP2;5 physically interacts with the endoplasmic reticulum (ER) resident ZmVAP27-1. This interaction requires the ZmVAP27-1 cytoplasmic major sperm domain. ZmPIP2;5 and ZmVAP27-1 localise in close vicinity in ER–PM contact sites (EPCSs) and endocytic structures upon exposure to salt stress conditions. This interaction enhances PM water permeability in oocytes. Similarly, the Arabidopsis ZmVAP27-1 paralogue, AtVAP27-1, interacts with the AtPIP2;7 aquaporin. Together, these data indicate that the PIP2–VAP27 interaction in EPCSs is evolutionarily conserved, and suggest that VAP27 might stabilise the aquaporins and guide their endocytosis in response to salt stress.

HOXA1 belongs to the HOX family of transcription factors which are key regulators of animal development. Little is known about the molecular pathways controlling HOXA1. Recent data from our group revealed distinct partner proteins interacting with HOXA1. Among them, OGT is an O-linked N-acetylglucosamine (O-GlcNAc) transferase modifying a variety of proteins involved in different cellular processes including transcription. Here, we confirm OGT as a HOXA1 interactor, we characterise which domains of HOXA1 and OGT are required for the interaction, and we provide evidence that OGT post-translationally modifies HOXA1. Mass spectrometry experiments indeed reveal that HOXA1 can be phosphorylated on the AGGTVGSPQYIHHSY peptide and that upon OGT expression, the phosphate adduct is replaced by an O-GlcNAc group.

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F1FO-ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three- dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical alpha, beta, gamma, delta, epsilon and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase.

Symbiotic bacteria are common in insects and can affect various aspects of their hosts’ biology. Although the effects of insect symbionts have been clarified for various insect symbiosis models, due to the difficulty of cultivating them in vitro, there is still limited knowledge available on the molecular features that drive symbiosis. Serratia symbiotica is one of the most common symbionts found in aphids. The recent findings of free-living strains that are considered as nascent partners of aphids provide the opportunity to examine the molecular mechanisms that a symbiont can deployed at the early stages of the symbiosis (i.e., symbiotic factors). In this work, a proteomic approach was used to establish a comprehensive proteome map of the free-living S. symbiotica strain CWBI-2.3T. Most of the 720 proteins identified are related to housekeeping or primary metabolism. Of these, 76 were identified as candidate proteins possibly promoting host colonization. Our results provide strong evidence that S. symbiotica CWBI-2.3T is well-armed for invading insect host tissues, and suggest that certain molecular features usually harbored by pathogenic bacteria are no longer present. This comprehensive proteome map provides a series of candidate genes for further studies to understand the molecular cross-talk between insects and symbiotic bacteria.

Yeast cells, to be able to grow on a wide variety of nitrogen sources, regulate the set of nitrogen transporters present at their plasma membrane. Such regulation relies on both transcriptional and post-translational events. While microarray studies have identified most nitrogen-sensitive genes, nitrogen-induced post-translational regulation has only been studied for very few proteins among which the general amino acid permease Gap1. Adding a preferred nitrogen source to proline-grown cells triggers Gap1 endocytosis and vacuolar degradation in an Rsp5-Bul1/2-dependent manner. Here, we used a proteomic approach to follow the dynamics of the plasma membrane proteome after addition of a preferred nitrogen source. We identified new targets of the nitrogen regulation and four transporters of poor nitrogen sources - Put4, Opt2, Dal5 and Ptr2 - that rapidly decrease in abundance. Although the kinetics is different for each transporter, we found that three of them - Put4, Dal5 and Ptr2 - are endocytosed, like Gap1, in an Rsp5-dependent manner and degraded in the vacuole. Finally, we showed that Gap1 stabilization at the plasma membrane, through deletion of Bul proteins, regulates the abundance of Put4, Dal5 and Ptr2.