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Theranostics 2016; 6(10):1672-1682

RNA was extracted from the same cultures used for the nucleotide analysis detailed above, and gene expression measurements obtained by hybridization to Affymetrix diS_div712a GeneChips containing oligo probes for 97% of the 7,825 protein-encoding genes in S. coelicolor. Data analysis revealed a total of 752 genes whose expression profiles were significantly influenced by the induction (Additional data file 1). Genes in this list include not only those affected as a result of induction of ppGpp synthesis, but also those changed in abundance as a result of thiostrepton addition. Using strain M667 [ΔrelA tipAp::] it was possible to identify those genes altered by the addition only of thiostrepton (see Materials and methods), resulting in a final list of 589 genes that had been significantly affected by induction of ppGpp synthesis alone. To reduce the number of genes for consideration and to focus in on only the largest changes, the data for these 589 genes were subjected to further tests (see Materials and methods). These were based on analysing fold-change ratios between induced and non-induced samples to identify those that are clearly induced or repressed by ppGpp synthesis, and gave lists of 98 and 189 genes, respectively (Additional data file 2). These lists of genes were analysed further to identify over-represented (P

AB - Chloroplasts possess common biosynthetic pathways for generating guanosine 3′,5′-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5′-(β,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.

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Here we demonstrate the ability of bacterialstringent factor to be stimulated by eukaryotic ribosomal preparationsto synthesise both ppGpp and pppGpp.

Regulation of production of the translational apparatus via the stringent factor ppGpp in response to amino acid starvation is conserved in many bacteria. However, in addition to this core function, it is clear that ppGpp also exhibits genus-specific regulatory effects. In this study we used Affymetrix GeneChips to more fully characterize the regulatory influence of ppGpp synthesis on the biology of Streptomyces coelicolor A3(2), with emphasis on the control of antibiotic biosynthesis and morphological differentiation.

Induction of ppGpp synthesis repressed transcription of the major sigma factor hrdB, genes with functions associated with active growth, and six of the thirteen conservons present in the S. coelicolor genome. Genes induced following ppGpp synthesis included the alternative sigma factor SCO4005, many for production of the antibiotics CDA and actinorhodin, the regulatory genes SCO4198 and SCO4336, and two alternative ribosomal proteins. Induction of the CDA and actinorhodin clusters was accompanied by an increase in transcription of the pathway regulators cdaR and actII-ORF4, respectively. Comparison of transcriptome profiles of a relA null strain, M570, incapable of ppGpp synthesis with its parent M600 suggested the occurrence of metabolic stress in the mutant. The failure of M570 to sporulate was associated with a stalling between production of the surfactant peptide SapB, and of the hydrophobins: it overproduced SapB but failed to express the chaplin and rodlin genes.

Professor Jonathan Jones - The Sainsbury Laboratory

In S. coelicolor, ppGpp synthesis influences the expression of several genomic elements that are particularly characteristic of streptomycete biology, notably antibiotic gene clusters, conservons, and morphogenetic proteins.

One important system for sensing nutrient starvation and triggering adaptive responses in bacteria involves the highly phosphorylated guanine nucleotide ppGpp, also known as stringent factor. This has long been known to effect a rapid response to amino acid starvation in Escherichia coli, down-regulating both rRNA biosynthesis and ribosome production [,]. Under amino acid limiting conditions, the RelA protein associated with ribosomes synthesises ppGpp in response to occupancy of the ribosomal A-site by uncharged tRNAs. The mode of action of ppGpp has been studied extensively in E. coli, and involves reorienting gene transcription via binding to RNA polymerase (reviewed in []).

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Professor Jonathan Jones. The Sainsbury Laboratory, Norwich, UK.


Homepage - Harvard University - Department of Molecular & …

Mutants which fail toexhibit the stringent response to amino acid starvation with thesynthesis of ppGpp pppGpp are known as relaxed mutants4 andin Escherichia coli are mapped at three loci; relA (ref.

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Chloroplasts possess common biosynthetic pathways for generating guanosine 3′,5′-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5′-(β,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.

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The purpose of this study was to use methods for the genome-wide analysis of gene transcription to more fully characterize the regulatory influence of ppGpp synthesis on the biology of S. coelicolor, with particular emphasis on the processes of morphological differentiation and secondary metabolite production. Classically, the effects of ppGpp have been analysed following induction of ppGpp production via starvation for one or more amino acids. This complicates interpretation of the results since the changes observed include responses both to the increase in ppGpp concentration, and to the ppGpp-independent effects of starvation. The levels of ppGpp produced in this way are also often artificially high in comparison to those observed when starvation occurs naturally. In this work we utilize a system that enables controlled induction of more physiologically relevant levels of ppGpp in the absence of amino acid starvation, allowing the effects of ppGpp synthesis to be viewed in isolation. This is supplemented by a comparison of relA+ (ppGpp+) and relA- (ppGpp-) strains to observe the longer term differences in gene expression resulting from an absence of ppGpp synthesis, and how this affects the transition to antibiotic production and morphological differentiation during growth. The results extend the known involvement of ppGpp synthesis in the regulation of antibiotic and secondary metabolite production, and paint a picture of a global regulatory mechanism with inhibitory and stimulatory effects on the transcription of a broad range of genes with diverse cellular functions. Although the direct regulatory routes remain unclear, it appears that, at least under certain growth conditions, ppGpp synthesis is required for correctly redirecting and coordinating gene transcription in S. coelicolor to allow it to progress normally through its developmental life-cycle.

RGD Peptide Cell-Surface Display Enhances the Targeting …

typhimurium that are defective in the synthesis of ppGpp (strain ΔppGpp) suppress tumor growth by activating the immune system via the release of TNF-α and IL-1β[, ].

2017 Poster Authors and Abstracts – CSU Annual …

N2 - Chloroplasts possess common biosynthetic pathways for generating guanosine 3′,5′-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5′-(β,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.

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