In contrast to 5-methylcytosine (5-mC), which has been studied extensively1–3, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types4,5. Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC–containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized4. We also find a gene expression level–dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.

Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling pathways.

SUMMARYDrastic alteration in macronutrients causes large changes in gene expression in the photosynthetic unicellular alga Chlamydomonas reinhardtii. Preliminary data suggested that cells follow a biphasic response to this change hinging on the initiation of lipid accumulation, and we hypothesized that drastic repatterning of metabolism also followed this biphasic modality. To test this hypothesis, transcriptomic, proteomic, and metabolite changes that occur under nitrogen (N) deprivation were analyzed. Eight sampling times were selected covering the progressive slowing of growth and induction of oil synthesis between 4 and 6 h after N deprivation. Results of the combined, systems-level investigation indicated that C. reinhardtii cells sense and respond on a large scale within 30 min to a switch to N-deprived conditions turning on a largely gluconeogenic metabolic state, which then transitions to a glycolytic stage between 4 and 6 h after N depletion. This nitrogen-sensing system is transduced to carbon-and nitrogen-responsive pathways, leading to down-regulation of carbon assimilation and chlorophyll biosynthesis, and an increase in nitrogen metabolism and lipid biosynthesis. For example, the expression of nearly all the enzymes for assimilating nitrogen from ammonium, nitrate, nitrite, urea, formamide/acetamide, purines, pyrimidines, polyamines, amino acids and proteins increased significantly. Although arginine biosynthesis enzymes were also rapidly up-regulated, arginine pool size changes and isotopic labeling results indicated no increased flux through this pathway.

Mitogen-activated protein kinase (MAPK) cascades have been identified in various signaling pathways involved in plant development and stress responses. We identified a drought-hypersensitive mutant (drought-hypersensitive mutant1 [dsm1]) of a putative MAPK kinase kinase (MAPKKK) gene in rice (Oryza sativa). Two allelic dsm1 mutants were more sensitive than wildtype plants to drought stress at both seedling and panicle development stages. The dsm1 mutants lost water more rapidly than wild-type plants under drought stress, which was in agreement with the increased drought-sensitivity phenotype of the mutant plants. DSM1-RNA interference lines were also hypersensitive to drought stress. The predicted DSM1 protein belongs to a B3 subgroup of plant Raf-like MAPKKKs and was localized in the nucleus. By real-time PCR analysis, the DSM1 gene was induced by salt, drought, and abscisic acid, but not by cold. Microarray analysis revealed that two peroxidase (POX) genes, POX22.3 and POX8.1, were sharply down-regulated compared to wild type, suggesting that DSM1 may be involved in reactive oxygen species (ROS) signaling. Peroxidase activity, electrolyte leakage, chlorophyll content, and 3,3#-diaminobenzidine staining revealed that the dsm1 mutant was more sensitive to oxidative stress due to an increase in ROS damage caused by the reduced POX activity. Overexpression of DSM1 in rice increased the tolerance to dehydration stress at the seedling stage. Together, these results suggest that DSM1 might be a novel MAPKKK functioning as an early signaling component in regulating responses to drought stress by regulating scavenging of ROS in rice.

Calcium controls a number of critical events including motility, secretion, cell invasion, and egress by protozoan parasites 1. Compared to animal 2 and plant cells 3 , the molecular mechanisms that govern calcium signaling in parasites are poorly understood. Here we demonstrate that the production of the phytohormone abscisic acid (ABA) controls calcium signaling within the apicomplexan parasite Toxoplasma gondii, an important human pathogen. In plants, ABA controls a number of important events including environmental stress responses, embryo development, and seed dormancy 4 ,5 . ABA induces production of the second-messenger cyclic ADP ribose (cADPR), which controls release of intracellular calcium stores in plants 6 . cADPR also controls intracellular calcium release in the protozoan parasite T. gondii 7,8 ; however, previous studies have not revealed the molecular basis of this pathway 9 . Addition of exogenous ABA induced formation of cADPR in T. gondii, stimulated calcium-dependent protein secretion, and induced parasite egress from the infected host cell in a density-dependent manner. Production of endogenous ABA within the parasite was confirmed by HPLC purification and GC-MS analysis. Selective disruption of ABA synthesis by the inhibitor fluridone (FLU) delayed egress and induced development of the slow-growing, dormant cyst stage of the parasite. Thus, ABA-mediated calcium signaling controls the decision between lytic and chronic stage growth, a developmental switch that is central in pathogenesis and transmission. The pathway for ABA production was likely acquired with an algal endosymbiont that was retained as a non-photosynthetic plastid known as the apicoplast. The plant-like nature of this pathway may be exploited therapeutically as shown by the ability of a specific inhibitor of ABA synthesis to prevent toxoplasmosis in the mouse model.Calcium-mediated secretion in T. gondii controls both motility and cell invasion and previous studies have demonstrated that these processes utilize the second messenger cADPR, yet the signals triggering this pathway remain unresolved 7,8 . In plants 6 , hydra 10 , and sponges 11 , ABA stimulates release of intracellular calcium through elevation of the cyclic nucleotide cADPR. Addition of exogenous ABA proved to be a potent agonist of secretion in T. gondii as shown by the release of the protein MIC2, a parasite adhesin that is discharged into the supernatant in response to increases in intracellular calcium (Fig. 1A). Induction of MIC2 secretion by ABA was highly specific to (±) -ABA and was not induced by (−) -ABA, the precursor β-carotene, or retinoic acid (Fig. 1B). Treatment with ABA lead to a dose-dependent increase in the second messenger cADPR in T. gondii, suggesting ABA may be a natural agonist for calcium signaling in parasites (Fig. 1C). Finally, chelation of intracellular calcium in the parasite blocked secretion induced by ABA, confirming that it acts through release of an intracellular calcium pool (Fig. 1D). Collectively these results ind...

Protein posttranslational modifications (PTMs), particularly phosphorylation, dramatically expand the complexity of cellular regulatory networks. Although cysteine (Cys) in various proteins can be subject to multiple PTMs, its phosphorylation was previously considered a rare PTM with almost no regulatory role assigned. We report here that phosphorylation occurs to a reactive cysteine residue conserved in the staphylococcal accessary regulator A (SarA)/MarR family global transcriptional regulator A (MgrA) family of proteins, and is mediated by the eukaryotic-like kinase-phosphatase pair Stk1-Stp1 in Staphylococcus aureus. Cys-phosphorylation is crucial in regulating virulence determinant production and bacterial resistance to vancomycin. Cell wall-targeting antibiotics, such as vancomycin and ceftriaxone, inhibit the kinase activity of Stk1 and lead to decreased Cys-phosphorylation of SarA and MgrA. An in vivo mouse model of infection established that the absence of stp1, which results in elevated protein Cys-phosphorylation, significantly reduces staphylococcal virulence. Our data indicate that Cys-phosphorylation is a unique PTM that can play crucial roles in bacterial signaling and regulation.

Antimicrobial drug resistance is an urgent problem in control and treatment of many of the world's most serious infections, including Plasmodium falciparum malaria, tuberculosis, and healthcare-associated infections with Gram-negative bacteria. Because the non-mevalonate pathway of isoprenoid biosynthesis is essential in eubacteria and P. falciparum, and this pathway is not present in humans, there is great interest in targeting the enzymes of non-mevalonate metabolism for antibacterial and antiparasitic drug development. Fosmidomycin is a broad-spectrum antimicrobial agent currently in clinical trials of combination therapies to treat malaria. In vitro, fosmidomycin is known to inhibit the deoxyxylulose phosphate reductoisomerase (DXR) enzyme of isoprenoid biosynthesis from multiple pathogenic organisms. To define the in vivo metabolic response to fosmidomycin, we developed a novel mass spectrometry method to quantitate six metabolites of non-mevalonate isoprenoid metabolism from complex biological samples. Using this technique, we validate that the biological effects of fosmidomycin are mediated through blockade of de novo isoprenoid biosynthesis in both P. falciparum malaria parasites and E. coli bacteria: in both organisms, metabolic profiling demonstrated a block in isoprenoid metabolism following fosmidomycin treatment, and growth inhibition due to fosmidomycin was rescued by media supplemented with isoprenoid metabolites. Isoprenoid metabolism proceeded through DXR even in the presence of fosmidomycin, but was inhibited at the level of the downstream enzyme, methylerythritol phosphate cytidyltransferase (IspD). Overexpression of IspD in E. coli conferred fosmidomycin resistance, and fosmidomycin was found to inhibit IspD in vitro. This work has validated fosmidomycin as a biological reagent to block non-mevalonate isoprenoid metabolism, and suggests a second in vivo target for fosmidomycin within isoprenoid biosynthesis, in two evolutionarily diverse pathogens.

SummaryIn genetic hybrids displaying nucleolar dominance, acetylation of lysines 5, 8, 12 and 16 of histone H4 (H4K5, H4K8, H4K12, H4K16) and acetylation of histone H3 on lysines 9 and 14 (H3K9, H3K14) occurs at the promoters of active ribosomal RNA (rRNA) genes, whereas silenced rRNA genes are deacetylated. Likewise, histone hyperacetylation correlates with the active state of transgenes and of endogenous plant genes involved in physiological processes, including cold tolerance, light-responsiveness and flowering. To investigate histone hyperacetylation dynamics we used sodium butyrate, a histone deacetylase inhibitor known to switch silent rRNA genes on, in order to enrich the pool of acetylated histones. Mass spectrometric analyses revealed unique mono-(K16Ac), di-(K12Ac, K16Ac), tri-(K8Ac, K12Ac, K16Ac), and tetra-acetylated (K5Ac, K8Ac, K12Ac, K16Ac) histone H4 isoforms, suggesting that H4 hyperacetylation occurs in a processive fashion, beginning with lysine 16 and ending with lysine 5. Using a combination of molecular and mass spectrometric assays we then determined the specificities of seven of the nine functional co-activator type histone acetyltransferases (HATs) in Arabidopsis thaliana: specifically HATs of the CBP (HAC1, HAC5, HAC12), GNAT (HAG1, HAG2), and MYST families (HAM1, HAM2). Specific HATs acetylate histone H4K5 (HAM1, HAM2), H4K12 (HAG2), and H3K14 (HAG1), suggesting that acetylation of these lysines may have special regulatory significance. Other acetylation events, including histone H3K9 acetylation, are likely to result from the activities of the broad-specificity HAC1, HAC5, and HAC12 histone acetyltransferases.