Membrane lipid adaptation of soil Bacteroidetes isolates to temperature and pH.

3-hydroxy fatty acids (3-OH FAs) are characteristic components of the Gram-negative bacterial membrane, recently proposed as promising temperature and pH (paleo) proxies in soil. Nevertheless, to date, the relationships between the 3-OH FA distribution and temperature/pH are only based on empirical studies, with no ground truthing work at the microbial level. This work investigated the influence of growth temperature and pH on the lipid composition of three strains of soil Gram-negative bacteria belonging to the Bacteroidetes phylum. Even though non-hydroxy FAs were more abundant than 3-OH FAs in the investigated strains, our results suggest that 3-OH FAs are involved in the membrane adaptation of these bacteria to temperature. The strains shared a common adaptation mechanism to temperature, with a significant increase in the ratio of anteiso vs. iso or normal 3-OH FAs at lower temperature. In contrast with temperature, no common adaptation mechanism to pH was observed, as the variations in the FA lipid profiles differed from one strain to another. We suggest that models reconstructing environmental changes in soils should include the whole suite of 3-OH FAs present in the membrane of Gram-negative bacteria, as all of them could be influenced by temperature or pH at the microbial level.

DIACYLGLYCEROL KINASE 5 participates in flagellin-induced signaling in Arabidopsis.

Flagellin perception is a keystone of pattern-triggered immunity in plants. The recognition of this protein by a plasma membrane (PM) receptor complex is the beginning of a signaling cascade that includes protein phosphorylation and the production of reactive oxygen species (ROS). In both Arabidopsis (Arabidopsis thaliana) seedlings and suspension cells, we found that treatment with flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, caused a rapid and transient decrease in the level of phosphatidylinositol (PI) 4,5-bisphosphate along with a parallel increase in phosphatidic acid (PA). In suspension cells, inhibitors of either phosphoinositide-dependent phospholipases C (PLC) or diacylglycerol kinases (DGKs) inhibited flg22-triggered PA production and the oxidative burst. In response to flg22, receptor-like kinase-deficient fls2, bak1, and bik1 mutants (FLAGELLIN SENSITIVE 2, BRASSINOSTEROID INSENSITIVE 1-associated kinase 1, and BOTRYTIS-INDUCED KINASE 1, respectively) produced less PA than wild-type (WT) plants, whereas this response did not differ in NADPH oxidase-deficient rbohD (RESPIRATORY BURST OXIDASE HOMOLOG D) plants. Among the DGK-deficient lines tested, the dgk5.1 mutant produced less PA and less ROS after flg22 treatment compared with WT seedlings. In response to flg22, dgk5.1 plants showed lower callose accumulation and impaired resistance to Pseudomonas syringae pv. tomato DC3000 hrcC-. Transcriptomics revealed that the basal expression of defense-related genes was altered in dgk5.1 seedlings compared with the WT. A GFP-DGK5 fusion protein localized to the PM, where RBOHD and PLC2 (proteins involved in plant immunity) are also located. The role of DGK5 and its enzymatic activity in flagellin signaling and fine-tuning of early immune responses in plant-microbe interactions is discussed.

Urbanization Constrains Skin Bacterial Phylogenetic Diversity in Wild Fish Populations and Correlates with the Proliferation of Aeromonads.

Changes in the state of rivers resulting from the activity and expansion of urban areas are likely to affect aquatic populations by increasing stress and disease, with the microbiota playing a potentially important intermediary role. Unraveling the dynamics of microbial flora is therefore essential to better apprehend the impact of anthropogenic disturbances on the health of host populations and the ecological integrity of hydrosystems. In this context, the present study simultaneously examined changes in the microbial communities associated with mucosal skin and gut tissues of eight fish species along an urbanization gradient in the Orge River (France). 16S rRNA gene metabarcoding revealed that the structure and composition of the skin microbiota varied substantially along the disturbance gradient and to a lesser extent according to fish taxonomy. Sequences affiliated with the Gammaproteobacteria, in particular the genus Aeromonas, prevailed on fish caught in the most urbanized areas, whereas they were nearly absent upstream. This rise of opportunistic taxa was concomitant with a decline in phylogenetic diversity, suggesting more constraining environmental pressures. In comparison, fish gut microbiota varied much more moderately with the degree of urbanization, possibly because this niche might be less directly exposed to environmental stressors. Co-occurrence networks further identified pairs of associated bacterial taxa, co-existing more or less often than expected at random. Few correlations could be identified between skin and gut bacterial taxa, supporting the assumption that these two microbial niches are disconnected and do not suffer from the same vulnerability to anthropic pressures.

topIb, a phylogenetic hallmark gene of Thaumarchaeota encodes a functional eukaryote-like topoisomerase IB.

Type IB DNA topoisomerases can eliminate torsional stresses produced during replication and transcription. These enzymes are found in all eukaryotes and a short version is present in some bacteria and viruses. Among prokaryotes, the long eukaryotic version is only observed in archaea of the phylum Thaumarchaeota. However, the activities and the roles of these topoisomerases have remained an open question. Here, we demonstrate that all available thaumarchaeal genomes contain a topoisomerase IB gene that defines a monophyletic group closely related to the eukaryotic enzymes. We show that the topIB gene is expressed in the model thaumarchaeon Nitrososphaera viennensis and we purified the recombinant enzyme from the uncultivated thaumarchaeon Candidatus Caldiarchaeum subterraneum. This enzyme is active in vitro at high temperature, making it the first thermophilic topoisomerase IB characterized so far. We have compared this archaeal type IB enzyme to its human mitochondrial and nuclear counterparts. The archaeal enzyme relaxes both negatively and positively supercoiled DNA like the eukaryotic enzymes. However, its pattern of DNA cleavage specificity is different and it is resistant to camptothecins (CPTs) and non-CPT Top1 inhibitors, LMP744 and lamellarin D. This newly described thermostable topoisomerases IB should be a promising new model for evolutionary, mechanistic and structural studies.

The Arabidopsis DREB2 genetic pathway is constitutively repressed by basal phosphoinositide-dependent phospholipase C coupled to diacylglycerol kinase.

Phosphoinositide-dependent phospholipases C (PI-PLCs) are activated in response to various stimuli. They utilize substrates provided by type III-Phosphatidylinositol-4 kinases (PI4KIII) to produce inositol triphosphate and diacylglycerol (DAG) that is phosphorylated into phosphatidic acid (PA) by DAG-kinases (DGKs). The roles of PI4KIIIs, PI-PLCs, and DGKs in basal signaling are poorly understood. We investigated the control of gene expression by basal PI-PLC pathway in Arabidopsis thaliana suspension cells. A transcriptome-wide analysis allowed the identification of genes whose expression was altered by edelfosine, 30 µM wortmannin, or R59022, inhibitors of PI-PLCs, PI4KIIIs, and DGKs, respectively. We found that a gene responsive to one of these molecules is more likely to be similarly regulated by the other two inhibitors. The common action of these agents is to inhibit PA formation, showing that basal PI-PLCs act, in part, on gene expression through their coupling to DGKs. Amongst the genes up-regulated in presence of the inhibitors, were some DREB2 genes, in suspension cells and in seedlings. The DREB2 genes encode transcription factors with major roles in responses to environmental stresses, including dehydration. They bind to C-repeat motifs, known as Drought-Responsive Elements that are indeed enriched in the promoters of genes up-regulated by PI-PLC pathway inhibitors. PA can also be produced by phospholipases D (PLDs). We show that the DREB2 genes that are up-regulated by PI-PLC inhibitors are positively or negatively regulated, or indifferent, to PLD basal activity. Our data show that the DREB2 genetic pathway is constitutively repressed in resting conditions and that DGK coupled to PI-PLC is active in this process, in suspension cells and seedlings. We discuss how this basal negative regulation of DREB2 genes is compatible with their stress-triggered positive regulation.

Phosphatidylinositol synthase activity from plant membrane fractions and from E. coli-expressed recombinant peptides.

Phosphatidylinositol (PtdIns) synthase is a lipid-synthesizing enzyme responsible for the synthesis of the phospholipid, PtdIns. Its enzymatic properties have been studied in in vitro assays using either membrane-enriched fractions or the purified protein in reconstituted lipid vesicles as a source of enzyme. More recently the specificities in terms of substrate preferences have also been studied using the recombinant protein expressed in Escherichia coli. This chapter deals with the purification of membranes as a source of PtdIns synthase before focusing on the in vitro assays of the enzymatic activities of the protein and, briefly, on the analysis of the product.

Acyl chains of phospholipase D transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry.

BACKGROUND: Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor. METHODOLOGY/PRINCIPAL FINDINGS: Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18:2- and 16:0/18:3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates. CONCLUSIONS: MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18:2- and 16:0/18:3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.

Assessment of mitochondria as a compartment for phosphatidylinositol synthesis in Solanum tuberosum.

The outer mitochondrial membrane is particularly rich in phosphatidylinositol (PtdIns), a phospholipid found in different amounts in all eukaryotic membranes, but not synthesized in situ by all. PtdIns is therefore subjected to traffic from the synthesizing membranes to the non-synthesizing ones. The contribution of mitochondria to the cell PtdIns pool has never been the focus of a specific study in plants, whereas in yeast, the presence of the enzyme responsible for synthesis, PtdIns synthase (PIS, cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11), has clearly been demonstrated in mitochondria. As these organelles have now been shown to be responsible for the synthesis of several lipids, the present work aimed at evaluating mitochondria as a compartment for the synthesis of PtdIns in plants. The sub-cellular localization of PIS was studied in Solanum tuberosum L. by membrane fractionation, enzymatic analysis and by confocal microscopy in living cells. In potato, beside the endoplasmic reticulum, the activity of PIS was found to be tightly associated to mitochondria. Using a fluorescent reporter fusion, the enzyme was also found to be associated to these organelles. The enzyme was not present at the plasma membrane. A comparison of the localization in other cell systems suggests that the mitochondrial localization could be regulated.

Phospholipase D activation is an early component of the salicylic acid signaling pathway in Arabidopsis cell suspensions.

Salicylic acid (SA) plays a central role in defense against pathogen attack, as well as in germination, flowering, senescence, and the acquisition of thermotolerance. In this report we investigate the involvement of phospholipase D (PLD) in the SA signaling pathway. In presence of exogenous primary alcohols, the production of phosphatidic acid by PLD is diverted toward the formation of phosphatidylalcohols through a reaction called transphosphatidylation. By in vivo metabolic phospholipid labeling with (33)P(i), PLD activity was found to be induced 45 min after addition of SA. We show that incubation of Arabidopsis (Arabidopsis thaliana) cell suspensions with primary alcohols inhibited the induction of two SA-responsive genes, PATHOGENESIS-RELATED1 and WRKY38, in a dose-dependent manner. This inhibitory effect was more pronounced when the primary alcohols were more hydrophobic. Secondary or tertiary alcohols had no inhibitory effect. These results provide compelling arguments for PLD activity being upstream of the induction of these genes by SA. A subsequent study of n-butanol effects on the SA-responsive transcriptome identified 1,327 genes differentially expressed upon SA treatment. Strikingly, the SA response of 380 of these genes was inhibited by n-butanol but not by tert-butanol. A detailed analysis of the regulation of these genes showed that PLD could act both positively and negatively, either on gene induction or gene repression. The overlap with the previously described phosphatidylinositol-4-kinase pathway is discussed.

Synthetic capacity of Arabidopsis phosphatidylinositol synthase 1 expressed in Escherichia coli.

Phosphatidylinositol (PtdIns) synthase 1 from the plant Arabidopsis thaliana has been expressed in Escherichia coli in order to study the synthetic capacities of the enzyme. Analysis of the total fatty acid content of the bacteria shows that PtdIns synthase activity does not have a profound effect on the proportions of the different fatty acids produced, even if the presence of an extra acidic phospholipid leads to a global reduction of the lipid content. A closer analysis carried out on individual phospholipids reveals a global fatty acid composition almost unchanged in the two major bacterial lipids phosphatidylethanolamine (PtdEtn) and phosphatidylglycerol (PtdGro). Phosphatidylinositol has a very unusual composition that shows the ability of the plant enzyme to use CDP-diacylglycerol molecular species absent from plants. We identified the various PtdIns molecular species. They represent a pool of the major molecular species of PtdEtn and PtdGro. These results, together with the determination of the apparent affinity constants of AtPIS1 for myo-inositol and CDP-diacylglycerol, allow us to discuss some of the constraints of PtdIns synthesis in plants in terms of specificity, which will depend on the subcellular localization of the protein.

Regulation of MSV and WDV virion-sense promoters by WDV nonstructural proteins: a role for their retinoblastoma protein-binding motifs.

In this work we demonstrate that wheat dwarf virus (WDV) RepA can activate WDV and maize streak virus (MSV) virion (V)-sense expression in plant tissues. Rep alone does not have any effect on the silent WDV promoter and it represses the basal MSV promoter activity. MSV promoter activation by RepA depends on an intact RepA retinoblastoma protein (RB)-binding domain. Promoter repression by Rep also depends on this domain to some extent. Mutation of the RepA RB-binding domain has no effect on WDV promoter activation. The WDV promoter contains two sites that fit the consensus E2F-binding site. One, WDV1, binds human E2F-1 in one-hybrid assays in yeast. It also binds specifically to maize and wheat proteins in vitro and, when fused to a minimal 35S promoter, it confers responsiveness to RepA only when the RepA RB-binding domain and the WDV1 site are intact. In the whole WDV V-sense promoter context, mutations of this sequence have no effect, suggesting that additional sequences are important for RepA-mediated promoter activation.

Phosphatidylinositol synthesis and exchange of the inositol head are catalysed by the single phosphatidylinositol synthase 1 from Arabidopsis.

In order to study some of its enzymatic properties, phosphatidylinositol synthase 1 (AtPIS1) from the plant Arabidopsis thaliana was expressed in Escherichia coli, a host naturally devoid of phosphatidylinositol (PtdIns). In the context of the bacterial membrane and in addition to de novo synthesis, the plant enzyme is capable of catalysing the exchange of the inositol polar head for another inositol. Our data clearly show that the CDP-diacylglycerol-independent exchange reaction can occur using endogenous PtdIns molecular species or PtdIns molecular species from soybean added exogenously. Exchange has been observed in the absence of cytidine monophosphate (CMP), but is greatly enhanced in the presence of 4 microm CMP. Our data also show that AtPIS1 catalyses the removal of the polar head in the presence of much higher concentrations of CMP, in a manner that suggests a reverse of synthesis. All of the PtdIns metabolizing activities require free manganese ions. EDTA, in the presence of low Mn2+ concentrations, also has an enhancing effect.