Deep sequencing of HetR-bound DNA reveals novel HetR targets in Anabaena sp. strain PCC 7120
Ontology highlight
ABSTRACT: We employed chromatin pull-down and deep sequencing to globally identify HetR DNA targets in vivo at 6 hours after fixed-nitrogen deprivation. We identified novel DNA binding targets of tagged HetR-6xHis and defined a consensus HetR binding site from these HetR target sequences. Chromatin pull down of hetR mutant strain UHM103 carrying pAM4375, which expresses HetR-6xHis, and a wild-type control. One dataset was collected.
Project description:Protein phosphorylation via serine/threonine protein kinases (Spk) is a widespread mechanism to adjust cellular processes toward changing environmental conditions. To study their role(s) in cyanobacteria, we established a collection of 11 completely segregated spk mutants among the 12 annotated Spk’s in the model cyanobacterium Synechocystis sp. PCC 6803. Screening of the mutant collection revealed that especially the mutant defective in SpkB encoded by slr1697 showed clear deviations compared to wild type (WT) regarding carbon metabolism, i.e., a reduced growth rate at low CO2 and in the presence of glucose, different glycogen accumulation patterns, and a higher tolerance to external H2O2 than the WT. The proteome of ∆spkB showed several distinct differences compared to WT, which indicate changes of the cell surface but also metabolic functions. A phospho-proteome analysis revealed decreased phosphorylation of the carboxysome-associated protein CcmM and the regulatory PII protein in the mutant compared to WT, whereas the allophycocyanin alpha subunit was stronger phosphorylated. The decreased phosphorylation of PII was verified in Western-blot experiments, indicating a clearly delayed PII phosphorylation in cells shifted from nitrate-containing to nitrate-free medium. Furthermore, the mutant ∆spkB showed differences in the state transition consistent with the changed phosphorylation of allophycocyanin. Collectively our results indicate that SpkB is an important regulator under different environmental conditions in Synechocystis and seems to interact in the PII phosphorylation and probably with further substrates in a kinase network.
Project description:Mass spectrometry analysis of ZNF827 Halotag pulldown ZNF827 pull-downs were performed in HEK 293T cells overexpressing HaloTag ZNF827 or empty vector control using the HaloTag® Protein Pull-Down and Labelling System (Promega) according to the manufacturer’s protocol. Pull-downs were subjected mass spectrometry (MS) for purity validation.
Project description:We found that assassin bugs from the earliest-diverging subfamily of higher Reduviidae (Peiratinae), as well as a subfamily closely related to Triatominae (Stenopodainae) have venom that is highly similar in composition to that produced by previously examined reduviids from Harpactorinae and Reduviinae. This finding suggests that venom composition has been largely stable due to purifying selection among the higher Reduviidae, which is consistent with the ancient origin of venom in the ancestors of Heteroptera 250–300 million years ago (Sunagar and Moran 2015; Walker et al. 2018a). This near homogeneity of venom composition is perhaps surprising considering that reduviid predators have evolved numerous instances of prey specialization and specialized hunting strategies that might be expected to co-evolve with venom. Possibly, further studies focussing on species with more specialized hunting strategies, or different kinds of venom bioactivities, will uncover more nuanced venom adaptations. Alternatively, it is possible that the protease-rich venoms of predatory reduviids are simply well-suited to myriad different hunting strategies. These data are consistent with other examples where venoms are surprisingly similar despite great differences in biology, for example between solitary and eusocial bees. A more detailed picture of venom evolution in Reduviidae would examine venom produced by the early-diverging Phymatine complex as well as venoms of non-reduviid cimicomorphs, prey specialists such as the arachnophagous Emesinae and the myrmecophagous Holoptilinae, and some of the many groups that employ hunting specializations, such as the use of plant resins to catch prey (Hwang and Weirauch 2012). Within Triatominae, examination of saliva produced by additional species from multiple lineages (especially those that switched to blood-feeding independently, if the subfamily is shown to be polyphyletic) and including generalists and specialists on different host taxa and species associated especially with nests and burrows will be informative. The venoms of predatory reduviids such as Zelurus spp. and Opisthacidius spp. that are most closely related to Triatominae, and share some behaviours such as habitation of bird nests by Opisthacidius spp. may also provide more information about the evolution of triatomine saliva.
Project description:In a recent study, we showed that a T-DNA insertional mutation in a mitochondrial PPR protein, POCO1, led to the earlier floral transition (Emami and Kempken 2019). We used RNA-seq analysis to provide an overview of the global transcriptome changes in poco1 mutant during different developmental stages.
Project description:Here, we report the transcriptome of Anabaena sp. strain 7120, a cyanobacterium that forms specialized nitrogen-fixing cells called heterocysts. Our data suggests that cyanobacteria frequently have more complex transcripts than thought, with large 5' UTRs, numerous antisense transcripts, and multiple transcriptional start sites or processing sites. Four samples of total filament RNA were sequenced with Illumina 40bp reads using directional RNA sequencing (see the Illumina small RNA prep protocol). The samples are 0hr (vegetative cells grown in the presence of ammonia) and 6hr, 12hr, and 21hr cells (after nitrogen step down).
Project description:Nucleoid-associated proteins (NAPs) are critical during the process of chromatin compaction in Streptomyces soil bacteria. HupS is one of the two NAPs encoded in the Streptomyces genome. Its unique C-terminal domain, rich in lysine repeats (LR domain), is alike to the H2B histone found in eukaryotic cells or the HupB protein found in Mycobacterium. Project study aim was to identify post-translationally acetylated lysine residues of HupS via bottom-up LC-MS. Two approaches were uptaken. One was based on HupS enrichment from a strain expressing a recombinant HupS with a C-terminal FLAG tag (TM015) via an antiFLAG pull-down, and the other relied on a proteome-wide search of acetylated peptides in a WT strain lysate. Controls for spectral false-positives were also carried out in parallel, which were an antiFLAG pull-down with a WT strain, and a proteome-wide search of acetylated peptides in a HupS deletion mutant strain (ΔhupS) lysate. 5 lysine acetylation sites were confidently determined for the HupS protein within this study, them being Lys51, Lys85, Lys104, Lys119, and either Lys193 or Lys194.
Project description:Among the flaviviral proteins, NS5 is the largest and most conserved. NS5 contains major enzymatic components of the viral replication complex. Disruption of the common key NS5-host protein-protein interactions critical for viral replication could aid in the development of broad-spectrum anti-flaviviral therapeutics. To this end, we investigated the JEV- and ZIKV-NS5 interactomes in human cells using GFP pull-downs with mass spectrometry analysis in a label-free fashion. A total of 138 cellular proteins interacting with NS5 from JEV, ZIKV, or both were identified as Protein classification analysis of identified cellular targets revealed the enrichment of RNA binding, processing and splicing including spliceosomal and spliceosome-associated proteins in both datasets. Comparison of our data with literature not only revealed several cellular NS5 interacting proteins shared among flaviviruses, but also identified proteins that have no known function in flavivirus biology such as RNA polymerase II-associated Paf1 complex, protein phosphatase 6, and s-adenosylmethionine synthetase. Our study generates the first landscape of the JEV and ZIKV NS5 interactome in human cells and identifies cellular proteins that are potentially targetable for broad-spectrum anti-flaviviral therapy.
Project description:Glycosylation is an abundant post-translational modification of both intracellular and extracellular proteins [1]. The majority of glycans are classified as N-linked chains, where the carbohydrate moiety is attached to asparagine residues, or O-linked chains, most commonly linked to a serine or threonine. N-linked glycosylation is initiated by the oligosaccharyltransferase complex with only two paralogs of the catalytic subunit, whereas O-glycan initiation is more complex. There are several types of O-linked glycosylation, but among the most diverse is the mucin or GalNAc type (hereafter referred to as O-glycosylation). O-glycosylation is initiated by 20 evolutionarily conserved polypeptide GalNAc-transferases (GalNAc-Ts), which catalyze the first step in the O-glycosylation of proteins by adding GalNAc residues to threonine, serine, and tyrosine amino acids (Fig 1A). Each of the GalNAc-Ts are differentially expressed in various tissues and have both distinct and overlapping peptide substrate specificities [2-12]. Thus, the repertoire of GalNAc-Ts expressed in a given cell determines the subset and O-glycosite pattern of glycosylated proteins [13]. Substantial efforts have been made to characterize and predict the substrate specificities of GalNAc-Ts in vitro, but understanding of the in vivo specificities of the individual GalNAc-Ts or their biological functions is limited [13-15]. This lack of insight prevents an understanding of how site-specific O-linked glycosylation affects diseases, such as metabolic disorders, cardiovascular disease, and various malignancies, that have been associated with GalNAc-Ts through genome-wide association studies and other linkage studies [16-26]. Therefore, it is imperative that we establish how O-glycosylation at specific sites in proteins affects protein function. A major task in achieving this goal is to identify the non-redundant biological functions of site-specific O-glycosylation. We and others recently developed new strategies for identifying specific sites on proteins that undergo O-glycosylation in different cell types and tissues [27-31]. Characterization of the O-glycoproteomic landscape in isolated human cells and multiple human cell lines suggests that more than 80 % of all proteins that traffic through the secretory pathway are O-glycoproteins [28, 30]. Probing the non-redundant contributions of individual GalNAc-Ts in cells with and without specific GalNAc-Ts [32-34] has revealed broad substrate specificities for some of the individual isoforms, whereas others seem to have very restricted substrate specificities [33-35]. Assessing all of the mapped O-glycosylation sites to identify associations between O-glycosites and protein annotations, we recently found that O-glycans are over-represented close to tandem repeat regions, protease cleavage sites, within propeptides, and on a select group of protein domains [28, 30, 36]. Although such general associations between the location of O-glycans and protein functions may direct future investigations, the strategy does not define the function of site-specific glycosylation. Further progress in discovering and defining novel functions of site-specific glycosylation events requires direct quantitative analysis of potential biological responses induced by the loss of distinct GalNAc-T isoforms, and such biological responses are not easily observed in single cell culture systems. Instead, more complex model systems can be used to examine and dissect the molecular mechanisms underlying the important biological functions of site-specific glycosylation. We previously used an organotypic tissue model equipped with genetically engineered cells to decipher the function of elongated O-glycans [29]. In the present study, we use the model combined with quantitative O-glycoproteomics and phosphoproteomics to perform open-ended discovery of the biological functions of site-specific glycosylation governed by GalNAc-Ts (Fig 1B). With this combinatorial strategy, we demonstrate that loss of individual GalNAc-T isoforms has distinct phenotypic consequences through their effect on distinct biological pathways, suggesting specific roles during epithelial formation.
Project description:Here, we report the comparison of transcriptomes of Anabaena sp. PCC7120 and the FurB(Zur) deletion derivative strain (MN38). Anabaena sp PCC7120 is a cyanobacterium that differentiates specialized nitrogen-fixing cells called heterocysts and that is capable of forming biofilms. Our data showed that the deletion of FurB negativily affected the heterocyst development and the biofilm formation. In addition, the RNA-seq data together with gel retardation assays unveiled that FurB is directly involved in the regulation of several genes related to heterocyst development and biofilm formation and other novel functions different from the ones related to the canonical Zur regulon.
Project description:H. seropedicae is a diazotrophic and endophytic bacterium that associates with economically important grasses promoting plant growth and increasing productivity. To identify genes related to bacterial ability to colonize and promote plant growth wheat seedlings growing hydroponically in Hoaglandâs medium were inoculated with H. seropedicae the bacteria and incubated for 3 days. mRNA from the bacteria present in the root surface and in the plant medium were purified, depleted from rRNA and used for RNA-seq profiling. RT-qPCR analyses were conducted to confirm regulation of selected genes. Comparison of RNA profile of bacteria attached to the root and planktonic revealed an extensive metabolic adaptation to the epiphytic life style.