Rhodobactersphaeroides methionine sulfoxide reductase P reduces R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates.

Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S-diastereomer and R-diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram-negative bacteria, was identified as a key actor for the reduction of oxidized periplasmic proteins. The haem-binding membrane protein MsrQ transmits reducing power from the electron transport chains to the molybdoenzyme MsrP, which acts as a protein-MetO reductase. The MsrQ/MsrP function was well established genetically, but the identity and biochemical properties of MsrP substrates remain unknown. In this work, using the purified MsrP enzyme from the photosynthetic bacteria Rhodobacter sphaeroides as a model, we show that it can reduce a broad spectrum of protein substrates. The most efficiently reduced MetO is found in clusters, in amino acid sequences devoid of threonine and proline on the C-terminal side. Moreover, R. sphaeroides MsrP lacks stereospecificity as it can reduce both R- and S-diastereomers of MetO, similarly to its Escherichia coli homolog, and preferentially acts on unfolded oxidized proteins. Overall, these results provide important insights into the function of a bacterial envelop protecting system, which should help understand how bacteria cope in harmful environments.

Genomic study of a novel magnetotactic Alphaproteobacteria uncovers the multiple ancestry of magnetotaxis.

Ecological and evolutionary processes involved in magnetotactic bacteria (MTB) adaptation to their environment have been a matter of debate for many years. Ongoing efforts for their characterization are progressively contributing to understand these processes, including the genetic and molecular mechanisms responsible for biomineralization. Despite numerous culture-independent MTB characterizations, essentially within the Proteobacteria phylum, only few species have been isolated in culture because of their complex growth conditions. Here, we report a newly cultivated magnetotactic, microaerophilic and chemoorganoheterotrophic bacterium isolated from the Mediterranean Sea in Marseille, France: Candidatus Terasakiella magnetica strain PR-1 that belongs to an Alphaproteobacteria genus with no magnetotactic relative. By comparing the morphology and the whole genome shotgun sequence of this MTB with those of closer relatives, we brought further evidence that the apparent vertical ancestry of magnetosome genes suggested by previous studies within Alphaproteobacteria hides a more complex evolutionary history involving horizontal gene transfers and/or duplication events before and after the emergence of Magnetospirillum, Magnetovibrio and Magnetospira genera. A genome-scale comparative genomics analysis identified several additional candidate functions and genes that could be specifically associated to MTB lifestyle in this class of bacteria.

Subcellular localization and interaction network of the mRNA decay activator Pat1 upon UV stress.

To identify nucleo-cytoplasmic shuttle proteins that relocate to the nucleus upon UV stress, we selected 18 targets on the basis of their conservation amongst eukaryotes and their relatively poor functional description. Their relocation was assayed using quantitative nuclear relocation assay (QNR). We focused on Pat1, a component of the cytoplasmic foci called processing bodies (p-bodies), because it had the strongest response to the stress. We verified that Pat1 accumulates in the nucleus after GFP tagging and fluorescence microscopy. Using tandem affinity purification coupled to a mass spectrometry shotgun detection and quantitation approach, we explored the dynamics of Pat1 protein-protein interaction network after UV stress. We have shown that Pat1 co-purifies with Dhh1 specifically upon UV stress. We observed that the nuclear accumulation of Pat1 upon UV stress is abolished in a dhh1∆ strain. These data provide the first evidence that Dhh1 is required for Pat1 nuclear relocation after UV stress.

The gammaproteobacterium Achromatium forms intracellular amorphous calcium carbonate and not (crystalline) calcite.

Achromatium is a long known uncultured giant gammaproteobacterium forming intracellular CaCO3 that impacts C and S geochemical cycles functioning in some anoxic sediments and at oxic-anoxic boundaries. While intracellular CaCO3 granules have first been described as Ca oxalate then colloidal CaCO3 more than one century ago, they have often been referred to as crystalline solids and more specifically calcite over the last 25 years. Such a crystallographic distinction is important since the respective chemical reactivities of amorphous calcium carbonate (ACC) and calcite, hence their potential physiological role and conditions of formation, are significantly different. Here, we analyzed the intracellular CaCO3 granules of Achromatium cells from Lake Pavin using a combination of Raman microspectroscopy and scanning electron microscopy. Granules in intact Achromatium cells were unequivocally composed of ACC. Moreover, ACC spontaneously transformed into calcite when irradiated at high laser irradiance during Raman analyses. Few ACC granules also transformed spontaneously into calcite in lysed cells upon cell death and/or sample preparation. Overall, the present study supports the original claims that intracellular Ca-carbonates in Achromatium are amorphous and not crystalline. In that sense, Achromatium is similar to a diverse group of Cyanobacteria and a recently discovered magnetotactic alphaproteobacterium, which all form intracellular ACC. The implications for the physiology and ecology of Achromatium are discussed. Whether the mechanisms responsible for the preservation of such unstable compounds in these bacteria are similar to those involved in numerous ACC-forming eukaryotes remains to be discovered. Last, we recommend to future studies addressing the crystallinity of CaCO3 granules in Achromatium cells recovered from diverse environments all over the world to take care of the potential pitfalls evidenced by the present study.

Intracellular amorphous Ca-carbonate and magnetite biomineralization by a magnetotactic bacterium affiliated to the Alphaproteobacteria.

Bacteria synthesize a wide range of intracellular submicrometer-sized inorganic precipitates of diverse chemical compositions and structures, called biominerals. Their occurrences, functions and ultrastructures are not yet fully described despite great advances in our knowledge of microbial diversity. Here, we report bacteria inhabiting the sediments and water column of the permanently stratified ferruginous Lake Pavin, that have the peculiarity to biomineralize both intracellular magnetic particles and calcium carbonate granules. Based on an ultrastructural characterization using transmission electron microscopy (TEM) and synchrotron-based scanning transmission X-ray microscopy (STXM), we showed that the calcium carbonate granules are amorphous and contained within membrane-delimited vesicles. Single-cell sorting, correlative fluorescent in situ hybridization (FISH), scanning electron microscopy (SEM) and molecular typing of populations inhabiting sediments affiliated these bacteria to a new genus of the Alphaproteobacteria. The partially assembled genome sequence of a representative isolate revealed an atypical structure of the magnetosome gene cluster while geochemical analyses indicate that calcium carbonate production is an active process that costs energy to the cell to maintain an environment suitable for their formation. This discovery further expands the diversity of organisms capable of intracellular Ca-carbonate biomineralization. If the role of such biomineralization is still unclear, cell behaviour suggests that it may participate to cell motility in aquatic habitats as magnetite biomineralization does.

Repeated horizontal gene transfers triggered parallel evolution of magnetotaxis in two evolutionary divergent lineages of magnetotactic bacteria.

Under the same selection pressures, two genetically divergent populations may evolve in parallel toward the same adaptive solutions. Here, we hypothesized that magnetotaxis (i.e., magnetically guided chemotaxis) represents a key adaptation to micro-oxic habitats in aquatic sediments and that its parallel evolution homogenized the phenotypes of two evolutionary divergent clusters of freshwater spirilla. All magnetotactic bacteria affiliated to the Magnetospirillum genus (Alphaproteobacteria class) biomineralize the same magnetic particle chains and share highly similar physiological and ultrastructural features. We looked for the processes that could have contributed at shaping such an evolutionary pattern by reconciling species and gene trees using newly sequenced genomes of Magnetospirillum related bacteria. We showed that repeated horizontal gene transfers and homologous recombination of entire operons contributed to the parallel evolution of magnetotaxis. We propose that such processes could represent a more parsimonious and rapid solution for adaptation compared with independent and repeated de novo mutations, especially in the case of traits as complex as magnetotaxis involving tens of interacting proteins. Besides strengthening the idea about the importance of such a function in micro-oxic habitats, these results reinforce previous observations in experimental evolution suggesting that gene flow could alleviate clonal interference and speed up adaptation under some circumstances.

Crystal structures of an Extracytoplasmic Solute Receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimer requiring a cation for alpha-keto acid binding.

BACKGROUND: The import of solutes into the bacterial cytoplasm involves several types of membrane transporters, which may be driven by ATP hydrolysis (ABC transporters) or by an ion or H+ electrochemical membrane potential, as in the tripartite ATP-independent periplasmic system (TRAP). In both the ABC and TRAP systems, a specific periplasmic protein from the ESR family (Extracytoplasmic Solute Receptors) is often involved for the recruitment of the solute and its presentation to the membrane complex. In Rhodobacter sphaeroides, TakP (previously named SmoM) is an ESR from a TRAP transporter and binds alpha-keto acids in vitro. RESULTS: We describe the high-resolution crystal structures of TakP in its unliganded form and as a complex with sodium-pyruvate. The results show a limited "Venus flytrap" conformational change induced by substrate binding. In the liganded structure, a cation (most probably a sodium ion) is present and plays a key role in the association of the pyruvate to the protein. The structure of the binding pocket gives a rationale for the relative affinities of various ligands that were tested from a fluorescence assay. The protein appears to be dimeric in solution and in the crystals, with a helix-swapping structure largely participating in the dimer formation. A 30 A-long water channel buried at the dimer interface connects the two ligand binding cavities of the dimer. CONCLUSION: The concerted recruitment by TakP of the substrate group with a cation could represent a first step in the coupled transport of both partners, providing the driving force for solute import. Furthermore, the unexpected dimeric structure of TakP suggests a molecular mechanism of solute uptake by the dimeric ESR via a channel that connects the binding sites of the two monomers.

Quantitation of p53 nuclear relocation in response to stress using a yeast functional assay: effects of irradiation and modulation by heavy metal ions.

Many regulatory proteins undergo transient nuclear relocation under physical or chemical stress. This phenomenon is, however, difficult to assess due to the lack of sensitive and standardized biological assays. Here, we describe a new quantitative nuclear relocation assay (QNR), based on expression in yeasts of chimeric proteins in which an artificial transcription factor is fused to a target protein acting as driver for relocation. This assay combines the experimental versatility of yeast with quantitation of nuclear relocation at low levels of protein expression. We have assessed the nuclear relocation of yeast Yap1 and human p53, two transcription factors that relocate to the nucleus in response to oxidative-stress and DNA damage, respectively. We show that p53 efficiently drives the relocation of the chimeric reporter in response to irradiation and that this process requires the C-terminal nuclear export signal (NES). Cd2+ and Hg2+, two metal ions inducing DNA damage as well as conformational changes in p53, have opposite effects on p53 relocation in response to DNA damage. Whereas Hg2+ effects are synergistic to DNA damage, Cd2+ inhibits relocation and sequesters p53 into the cytoplasm. These results demonstrate the effectiveness of QNR to investigate the regulation of p53 shuttling in response to stress signals including suspected environmental carcinogens.

RNA-binding proteins are a major target of silica nanoparticles in cell extracts.

Upon contact with biological fluids, nanoparticles (NPs) are readily coated by cellular compounds, particularly proteins, which are determining factors for the localization and toxicity of NPs in the organism. Here, we improved a methodological approach to identify proteins that adsorb on silica NPs with high affinity. Using large-scale proteomics and mixtures of soluble proteins prepared either from yeast cells or from alveolar human cells, we observed that proteins with large unstructured region(s) are more prone to bind on silica NPs. These disordered regions provide flexibility to proteins, a property that promotes their adsorption. The statistical analyses also pointed to a marked overrepresentation of RNA-binding proteins (RBPs) and of translation initiation factors among the adsorbed proteins. We propose that silica surfaces, which are mainly composed of Si-O- and Si-OH groups, mimic ribose-phosphate molecules (rich in -O- and -OH) and trap the proteins able to interact with ribose-phosphate containing molecules. Finally, using an in vitro assay, we showed that the sequestration of translation initiation factors by silica NPs results in an inhibition of the in vitro translational activity. This result demonstrates that characterizing the protein corona of various NPs would be a relevant approach to predict their potential toxicological effects.

Eukaryotic GPN-loop GTPases paralogs use a dimeric assembly reminiscent of archeal GPN.

GTPases are molecular switches that regulate a wide-range of cellular processes. The GPN-loop GTPase (GPN) is a sub-family of P-loop NTPase that evolved from a single gene copy in archaea to triplicate paralog genes in eukaryotes, each having a non-redundant essential function in cell. In Saccharomyces cerevisiae, yGPN1 and yGPN2 are involved in sister chromatid cohesion mechanism, whereas nothing is known regarding yGPN3 function. Previous high-throughput experiments suggested that GPN paralogs interaction may occur. In this work, GPN|GPN contact was analyzed in details using TAP-Tag approach, yeast two-hybrid assay, in silico energy computation and site-directed mutagenesis of a conserved Glu residue located at the center of the interaction interface. It is demonstrated that this residue is essential for cell viability. A chromatid cohesion assay revealed that, like yGPN1 and yGPN2, yGPN3 also plays a role in sister chromatid cohesion. These results suggest that all three GPN proteins act at the molecular level in sister chromatid cohesion mechanism as a GPN|GPN complex reminiscent of the homodimeric structure of PAB0955, an archaeal member of GPN-loop GTPase.

A role for GPN-loop GTPase yGPN1 in sister chromatid cohesion.

Sister chromatid cohesion and separation, involving the cohesin complex, are crucial for accurate inheritance of genetic information. This complex is also fundamental for efficient post-replicative repair of DNA double-strand breaks and has a key role in the mechanisms of gene transcription control. Cohesin is subjected to many post-translational modifications but the regulators implicated in the control of its activity have been poorly described. Here, we show that the conserved and essential GPN loop GTPase yGPN1 in Saccharomyces cerevisiae is involved in sister chromatid cohesion mechanisms. Based on a sister chromatid cohesion assay, we found that over-expression of the yGPN1 gene promotes sister chromatid separation during anaphase. The sharp slowdown in progression of the S phase observed in cells where yGPN1 expression is down-regulated strongly suggests that yGPN1 is necessary for DNA replication. Moreover, analysis of yGPN1 protein-protein interaction network highlights the yGPN1 links with DNA replication, sister chromatid cohesion/separation and the gene expression process.

Stress induces the expression of AtNADK-1, a gene encoding a NAD(H) kinase in Arabidopsis thaliana.

A novel Arabidopsis thaliana gene (AtNADK-1) was identified based on its response to radiation and oxidative stress. Levels of AtNADK-1 mRNA increase eight-fold following exposure to ionising radiation and are enhanced three-fold by treatment with hydrogen peroxide. The gene also appears to be differentially regulated during compatible and incompatible plant-pathogen interactions in response to Pseudomonas syringae pv. tomato. The full-length AtNADK-1 cDNA encodes a 58-kDa protein that shows high sequence homology to the recently defined family of NAD(H) kinases. Recombinant AtNADK-1 utilises ATP to phosphorylate both NAD and NADH, showing a two-fold preference for NADH. Using reverse genetics, we demonstrate that AtNADK-1 deficient plants display enhanced sensitivity to gamma irradiation and to paraquat-induced oxidative stress. Our results indicate that this novel NAD(H) kinase may contribute to the maintenance of redox status in Arabidopsis thaliana.