Ultrastructure of the denitrifying methanotroph "Candidatus Methylomirabilis oxyfera," a novel polygon-shaped bacterium

"Candidatus Methylomirabilis oxyfera" is a newly discovered denitrifying methanotroph that is unrelated to previously known methanotrophs. This bacterium is a member of the NC10 phylum and couples methane oxidation to denitrification through a newly discovered intra-aerobic pathway. In the present study, we report the first ultrastructural study of "Ca. Methylomirabilis oxyfera" using scanning electron microscopy, transmission electron microscopy, and electron tomography in combination with different sample preparation methods. We observed that "Ca. Methylomirabilis oxyfera" cells possess an atypical polygonal shape that is distinct from other bacterial shapes described so far. Also, an additional layer was observed as the outermost sheath, which might represent a (glyco)protein surface layer. Further, intracytoplasmic membranes, which are a common feature among proteobacterial methanotrophs, were never observed under the current growth conditions. Our results indicate that "Ca. Methylomirabilis oxyfera" is ultrastructurally distinct from other bacteria by its atypical cell shape and from the classical proteobacterial methanotrophs by its apparent lack of intracytoplasmic membranes.     

Indirect genetic effects and the evolution of aggression in a vertebrate system

Aggressive behaviours are necessarily expressed in a social context, such that individuals may be influenced by the phenotypes, and potentially the genotypes, of their social partners. Consequently, it has been hypothesized that indirect genetic effects (IGEs) arising from the social environment will provide a major source of heritable variation on which selection can act. However, there has been little empirical scrutiny of this to date. Here we test this hypothesis in an experimental population of deer mice (Peromyscus maniculatus). Using quantitative genetic models of five aggression traits, we find repeatable and heritable differences in agonistic behaviours of focal individuals when presented with an opponent mouse. For three of the traits, there is also support for the presence of IGEs, and estimated correlations between direct and indirect genetic (rAO,F) effects were high. As a consequence, any selection for aggression in the focal individuals should cause evolution of the social environment as a correlated response. In two traits, strong positive rAO,F will cause the rapid evolution of aggression, while in a third case changes in the phenotypic mean will be constrained by negative covariance between direct and IGEs. Our results illustrate how classical analyses may miss important components of heritable variation, and show that a full understanding of evolutionary dynamics requires explicit consideration of the genetic component of the social environment.     

Smoking Induces Long-Lasting Effects through a Monoamine-Oxidase Epigenetic Regulation

Background

Postulating that serotonin (5-HT), released from smoking-activated platelets could be involved in smoking-induced vascular modifications, we studied its catabolism in a series of 115 men distributed as current smokers (S), never smokers (NS) and former smokers (FS) who had stopped smoking for a mean of 13 years.

Methodology/Principal Findings

5-HT, monoamine oxidase (MAO-B) activities and amounts were measured in platelets, and 5-hydroxyindolacetic acid (5-HIAA)—the 5-HT/MAO catabolite—in plasma samples. Both platelet 5-HT and plasma 5-HIAA levels were correlated with the 10-year cardiovascular Framingham relative risk (P<0.01), but these correlations became non-significant after adjustment for smoking status, underlining that the determining risk factor among those taken into account in the Framingham risk calculation was smoking. Surprisingly, the platelet 5-HT content was similar in S and NS but lower in FS with a parallel higher plasma level of 5-HIAA in FS. This was unforeseen since MAO-B activity was inhibited during smoking (P<0.00001). It was, however, consistent with a higher enzyme protein concentration found in S and FS than in NS (P<0.001). It thus appears that MAO inhibition during smoking was compensated by a higher synthesis. To investigate the persistent increase in MAO-B protein concentration, a study of the methylation of its gene promoter was undertaken in a small supplementary cohort of similar subjects. We found that the methylation frequency of the MAOB gene promoter was markedly lower (P<0.0001) for S and FS vs. NS due to cigarette smoke-induced increase of nucleic acid demethylase activity.

Conclusions/Significance

This is one of the first reports that smoking induces an epigenetic modification. A better understanding of the epigenome may help to further elucidate the physiopathology and the development of new therapeutic approaches to tobacco addiction. The results could have a larger impact than cardiovascular damage, considering that MAO-dependent 5-HT catabolism is also involved in addiction, predisposition to cancer, behaviour and mental health.     

Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation: Sumoylation of pml and phospho-switch control of its sumo binding domain dissected in living cells*

Promyelocytic leukemia protein (PML) is a tumor suppressor acting as the organizer of subnuclear structures called PML nuclear bodies (NBs). Both covalent modification of PML by the small ubiquitin-like modifier (SUMO) and non-covalent binding of SUMO to the PML SUMO binding domain (SBD) are necessary for PML NB formation and maturation. PML sumoylation and proteasome-dependent degradation induced by the E3 ubiquitin ligase, RNF4, are enhanced by the acute promyelocytic leukemia therapeutic agent, arsenic trioxide (As₂O₃). Here, we established a novel bioluminescence resonance energy transfer (BRET) assay to dissect and monitor PML/SUMO interactions dynamically in living cells upon addition of therapeutic agents. Using this sensitive and quantitative SUMO BRET assay that distinguishes PML sumoylation from SBD-mediated PML/SUMO non-covalent interactions, we probed the respective roles of covalent and non-covalent PML/SUMO interactions in PML degradation and interaction with RNF4. We found that, although dispensable for As₂O₃-enhanced PML sumoylation and RNF4 interaction, PML SBD core sequence was required for As₂O₃- and RNF4-induced PML degradation. As confirmed with a phosphomimetic mutant, phosphorylation of a stretch of serine residues, contained within PML SBD was needed for PML interaction with SUMO-modified protein partners and thus for NB maturation. However, mutation of these serine residues did not impair As₂O₃- and RNF4-induced PML degradation, contrasting with the known role of these phosphoserine residues for casein kinase 2-promoted PML degradation. Altogether, these data suggest a model whereby sumoylation- and SBD-dependent PML oligomerization within NBs is sufficient for RNF4-mediated PML degradation and does not require the phosphorylation-dependent association of PML with other sumoylated partners.Promyelocytic leukemia protein (PML)⁵ is a tumor suppressor (1) whose gene is translocated in cases of acute promyelocytic leukemia (2). PML functions as the organizer of PML NBs, which are dynamic structures harboring numerous transiently and permanently localized proteins (3). The importance of PML NB structural integrity was first revealed in acute promyelocytic leukemia because, in this malignancy, the abnormal fusion protein PML/RARα leads to NB disruption. Patient treatment with As₂O₃ induces the reversion of the acute promyelocytic leukemia phenotype as well as PML/RARα degradation and PML NB reformation (4).PML is a target for the post-translational modification by SUMO, an ubiquitin-like protein that is covalently coupled to PML lysine residues 65, 160, and 490 via a process called sumoylation (5, 6). Among the four human SUMO paralogs identified, SUMO1, -2, and -3 were found to be conjugated to target proteins. It involves an enzymatic cascade for the transfer of the mature SUMO and the formation of an isopeptide bond between the COOH-terminal glycine of SUMO and a lysine from the target protein. Sumoylation is a reversible process due to the existence of several deconjugating enzymes.PML NB formation requires both the covalent linkage (sumoylation) (reviewed in Ref. 7) and the non-covalent interactions of SUMO with PML through a SUMO binding domain (SBD also named SIM for SUMO interacting motif) (8). Interestingly, PML SBD contains specific serines, acting as substrates for the caseine kinase-2 (CK2), which are implicated in PML ubiquitination and degradation (9) and which phosphorylation status could regulate the function of the SBD.Because sumoylation of proteins is dynamic and reversible, this post-translational modification is difficult to follow in vivo and its detection mainly relies on the identification of sumoylated protein species by Western blot following cell lysis. Recently, we used bioluminescence resonance energy transfer (BRET) to detect covalent linkage of ubiquitin (ubiquitination) in living mammalian cells and in real time (10). In brief, BRET monitors the interaction between a protein fused to a luciferase and a protein fused to yellow or green fluorescent protein (YFP or GFP), upon addition of a luciferase substrate; it is a proximity-based assay that requires that the donor of energy (luciferase fusion) and the acceptor (YFP or GFP fusions) are within 50 to 100 Å for an efficient energy transfer (11–13). However, a demonstration that BRET may provide a method of choice to follow the dynamics of protein sumoylation in living cells is lacking. Here, we developed a sensitive and quantitative SUMO BRET assay for the detection of PML interactions with SUMO in living cells. We proved that BRET can be used to detect both SUMO covalent and non-covalent interactions with PML (model, Fig. 1H). For this purpose, we used the PMLIII isoform in which sumoylation is induced by As₂O₃ and triggers a proteasome-dependent PML degradation (14); the degradation process involves the ubiquitination of poly-SUMO covalently coupled to PML by the poly-SUMO-specific E3 ubiquitin ligase RNF4 (15–17). Altogether, our BRET results indicate that, As₂O₃ and/or RNF4-induced PML degradation are dependent on the integrity of both PML sumoylation target sites and the PML SBD core sequence but not on the CK2 serine phosphorylation sites within the SBD. However, phosphorylation of these serines is required for most PML SBD-dependent non-covalent interactions. This phospho-regulation of PML SBD (“SBD phospho-switch”) establishes another link between the phosphorylation and SUMO, different from the phospho-sumoyl switch (18).Open in a separate windowFIGURE 1.BRET reveals both covalent and non-covalent PML/SUMO1 interactions as well as As₂O₃-induced PML sumoylation in living cells. A and B, detection of PML/SUMO1 interactions by BRET¹ (A) or BRET² (B) titration assays using HEK293T cells transfected for expression of increasing amounts of YFP-SUMO1 (BRET¹) or GFP-SUMO1 (BRET²) and a fixed amount of Luc fusion. Negative controls: BRET pairs including PML_C57,60A-Luc (a non-sumoylatable mutant with Cys⁵⁷ and Cys⁶⁰ mutated to Ala) or YFP-SUMO1G (a SUMO1 that cannot be processed) (dotted line) (A) and Luc fused to a NLS (B). C and D, detection of covalent and non-covalent PML/SUMO1 interactions by BRET¹ (C) or BRET² (D) titration assays in the presence (dotted lines, empty symbols) or absence (solid lines and symbols) of As₂O₃ in HEK293T cells transfected for expression of PML_WT-Luc or its sumoylation deficient mutant PML_3K-Luc in pairs with either YFP-SUMO1 (BRET¹) or GFP-SUMO1 (BRET²). Negative control: PML_WT-Luc in pairs with YFP-SUMO1G. E, kinetics of As₂O₃-induced PML_WT-Luc sumoylation revealed by BRET¹ (assay on attached cells) and BRET² (cells in suspension). F, dose-response curve to As₂O₃ treatment for PML_WT-Luc or PML_3KR-Luc/YFP-SUMO1 BRET¹ pairs. Negative control: PML_C57,60A-Luc/YFP-SUMO1. G, comparison of As₂O₃-induced sumoylation of PML_WT, PML_3KR, and its single lysine mutants at an identical YFP acceptor/Luc donor expression ratio as derived from titration curves. As₂O₃ treatment (C–G): 5 μm, 4 h exposure for BRET¹ and Western blot or 10 μm, 70-min exposure for BRET². H, model for the covalent (sumoylation) and non-covalent interactions between a tested protein fused to Luc and SUMO fused to a fluorescent protein (YFP) that generates a BRET signal. The black arrows indicate the bioluminescent transfer of energy (or BRET) that occurs between Luc and GFP fusion upon exposure to the cell-permeable luciferase substrate.     

Impact of peptidoglycan O-acetylation on autolytic activities of the Enterococcus faecalis N-acetylglucosaminidase AtlA and N-acetylmuramidase AtlB

Autolysins are potentially lethal enzymes that partially hydrolyze peptidoglycan for incorporation of new precursors and septum cleavage after cell division. Here, we explored the impact of peptidoglycan O-acetylation on the enzymatic activities of Enterococcus faecalis major autolysins, the N-acetylglucosaminidase AtlA and the N-acetylmuramidase AtlB. We constructed isogenic strains with various O-acetylation levels and used them as substrates to assay E. faecalis autolysin activities. Peptidoglycan O-acetylation had a marginal inhibitory impact on the activities of these enzymes. In contrast, removal of cell wall glycopolymers increased the AtlB activity (37-fold), suggesting that these polymers negatively control the activity of this enzyme.     

Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes

Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and beta-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40-GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.     

Regulation of Arabidopsis thaliana 14-3-3 gene expression by gamma-aminobutyric acid

The function in plants of the non-protein amino acid, gamma-aminobutyric acid (GABA) is poorly understood. In this study, we show that GABA down-regulates the expression of a large subset of 14-3-3 gene family members in Arabidopsis thaliana seedlings in a calcium, ethylene and abscisic acid (ABA)-dependent manner. Gene expression is not affected when seedlings are supplied with glutamate (GLU), a precursor of GABA. The repression of 14-3-3 gene expression by GABA is dependent on functional ethylene and ABA signalling pathways, because the response is lost in the etr1-1, abi1-1 and abi2-1 mutants. Calcium measurements show that in contrast to GLU, GABA does not elicit a cytoplasmic calcium elevation, suggesting that the GABA response is unlikely to be mediated by GLU receptors (GLRs), as has been suggested previously. We suggest that in addition to its role as a stress-related metabolite, GABA may regulate gene expression in A. thaliana, including members of the 14-3-3 gene family.     

Felid herpesvirus 1 glycoprotein G is a structural protein that mediates the binding of chemokines on the viral envelope

Glycoprotein G (gG) orthologues have been described in several alphaherpesviruses. gG is expressed both as a membrane-anchored form on infected cells and as a secreted form. Recently, we reported that both forms of gG encoded by alphaherpesviruses infecting large herbivores and by Felid herpesvirus 1 (FeHV-1) bind with high affinity to a broad range of CXC, CC and C-chemokines. Based on the viral species, gG has been reported either as a structural or a non-structural protein. To date, the incorporation of FeHV-1 gG into virions has never been tested, nor the property of alphaherpesvirus structural gG to bind chemokines on the virion surface. In the present study, to address these questions, various FeHV-1 gG recombinant strains were produced using an original technique based on an infectious FeHV-1 BAC clone and restriction endonuclease mediated recombination. Using the recombinants produced, we were able to determine that FeHV-1 gG is a structural protein that acts as a chemokine-binding protein on the virion surface. In the light of these results, putative roles of gG in alphaherpesvirus infections are discussed, and an evolutionary scenario is proposed to explain the structural versus non-structural property of gG amongst alphaherpesviruses.     

Expansion of the SOX gene family predated the emergence of the Bilateria

Members of the SOX gene family are involved in regulating many developmental processes including neuronal determination and differentiation, and in carcinogenesis. So far they have only been identified in species from the Bilateria (deuterostomes and protostomes). To understand the origins of the SOX family, we used a PCR-based strategy to obtain 28 new sequences of SOX gene HMG domains from four non-bilaterian Metazoa: two sponge species, one ctenophore and one cnidarian. One additional SOX sequence was retrieved from EST sequences of the cnidarian species Clytia hemisphaerica. Unexpected SOX gene diversity was found in these species, especially in the cnidarian and the ctenophore. The topology of gene relationships deduced by Maximum Likelihood analysis, although not supported by bootstrap values, suggested that the SOX family started to diversify in the metazoan stem branch prior to the divergence of demosponges, and that further diversification occurred in the eumetazoan branch, as well as later in calcisponges, ctenophores, cnidarians and vertebrates. In contrast, gene loss appears to have occurred in the nematode and probably in other protostome lineages, explaining their lower number of SOX genes.