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1.
Nat Struct Mol Biol ; 30(5): 600-607, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36997761

RESUMO

Second messenger (p)ppGpp (collectively guanosine tetraphosphate and guanosine pentaphosphate) mediates bacterial adaptation to nutritional stress by modulating transcription initiation. More recently, ppGpp has been implicated in coupling transcription and DNA repair; however, the mechanism of ppGpp engagement remained elusive. Here we present structural, biochemical and genetic evidence that ppGpp controls Escherichia coli RNA polymerase (RNAP) during elongation via a specific site that is nonfunctional during initiation. Structure-guided mutagenesis renders the elongation (but not initiation) complex unresponsive to ppGpp and increases bacterial sensitivity to genotoxic agents and ultraviolet radiation. Thus, ppGpp binds RNAP at sites with distinct functions in initiation and elongation, with the latter being important for promoting DNA repair. Our data provide insights on the molecular mechanism of ppGpp-mediated adaptation during stress, and further highlight the intricate relationships between genome stability, stress responses and transcription.


Assuntos
Proteínas de Escherichia coli , Guanosina Tetrafosfato , Guanosina Tetrafosfato/química , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Proteínas de Escherichia coli/metabolismo , Raios Ultravioleta , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Reparo do DNA , Transcrição Gênica , Regulação Bacteriana da Expressão Gênica
2.
J Hazard Mater ; 448: 130941, 2023 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-36758433

RESUMO

Pseudomonas aeruginosa causes public health problems in drinking water systems. This study investigated the potential role of the stringent response in regulating the adaptive physiological metabolic behaviors of P. aeruginosa to low nitrogen stress and bacterial competition in drinking water systems. The results indicated that guanosine tetraphosphate (ppGpp) concentrations in P. aeruginosa increased to 135.5 pmol/g SS under short-term nitrogen deficiency. Meanwhile, the expression levels of the ppGpp synthesis genes (ppx, relA) and degradation gene (spoT) were upregulated by 37.0% and downregulated by 26.8%, respectively, indicating that the stringent response was triggered. The triggered stringent response inhibited the growth of P. aeruginosa and enhanced the metabolic activity of P. aeruginosa to adapt to nutrient deprivation. The interspecific competition significantly affected the regulation of the stringent response in P. aeruginosa. During short-term nitrogen deficiency, the extracellular polymeric substances concentration of P. aeruginosa decreased significantly, leading to desorption and diffusion of attached bacteria and increased ecological risks. The regulatory effect of stringent response on P. aeruginosa gradually weakened under long-term nitrogen deficiency. However, the expression of pathogenic genes (nalD/PA3310) and flagellar assembly genes (fliC) in P. aeruginosa was upregulated by the stringent response, which increased the risk of disease.


Assuntos
Água Potável , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Nitrogênio/metabolismo , Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica
3.
Plant Cell Physiol ; 63(7): 919-931, 2022 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-35428891

RESUMO

Guanosine tetraphosphate (ppGpp) is known as an alarmone that mediates bacterial stress responses. In plants, ppGpp is synthesized in chloroplasts from GTP and ATP and functions as a regulator of chloroplast gene expression to affect photosynthesis and plant growth. This observation indicates that ppGpp metabolism is closely related to chloroplast function, but the regulation of ppGpp and its role in chloroplast differentiation are not well understood. In rice, ppGpp directly inhibits plastidial guanylate kinase (GKpm), a key enzyme in GTP biosynthesis. GKpm is highly expressed during early leaf development in rice, and the GKpm-deficient mutant, virescent-2 (v2), develops chloroplast-deficient chlorotic leaves under low-temperature conditions. To examine the relationship between GTP synthesis and ppGpp homeostasis, we generated transgenic rice plants over-expressing RSH3, a protein known to act as a ppGpp synthase. When RSH3 was overexpressed in v2, the leaf chlorosis was more severe. Although the RSH3 overexpression in the wild type caused no visible effects, pulse amplitude modulation fluorometer measurements indicated that photosynthetic rates were reduced in this line. This finding implies that the regulation of ppGpp synthesis in rice is involved in the maintenance of the GTP pool required to regulate plastid gene expression during early chloroplast biogenesis. We further investigated changes in the expressions of RelA/SpoT Homolog (RSH) genes encoding ppGpp synthases and hydrolases during the same period. Comparing the expression of these genes with the cellular ppGpp content suggests that the basal ppGpp level is determined by the antagonistic action of multiple RSH enzymatic activities during early leaf development in rice.


Assuntos
Guanosina Tetrafosfato , Oryza , Cloroplastos/metabolismo , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligases/metabolismo , Oryza/genética , Oryza/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo
4.
J Genet Genomics ; 49(5): 469-480, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35189402

RESUMO

Nitrogen is an essential macronutrient for all living organisms and is critical for crop productivity and quality. In higher plants, inorganic nitrogen is absorbed through roots and then assimilated into amino acids by the highly conserved glutamine synthetase/glutamine:2-oxoglutarate aminotransferase (GS/GOGAT) cycle. How nitrogen metabolism and nitrogen starvation responses of plants are regulated remains largely unknown. Previous studies revealed that mutations in the rice ABNORMAL CYTOKININ RESPONSE1 (ABC1) gene encoding Fd-GOGAT cause a typical nitrogen deficiency syndrome. Here, we show that ARE2 (for ABC1 REPRESSOR2) is a key regulator of nitrogen starvation responses in rice. The are2 mutations partially rescue the nitrogen-deficient phenotype of abc1 and the are2 mutants show enhanced tolerance to nitrogen deficiency, suggesting that ARE2 genetically interacts with ABC1/Fd-GOGAT. ARE2 encodes a chloroplast-localized RelA/SpoT homolog protein that catalyzes the hydrolysis of guanosine pentaphosphate or tetraphosphate (p)ppGpp, an alarmone regulating the stringent response in bacteria under nutritional stress conditions. The are2 mutants accumulate excessive amounts of (p)ppGpp, which correlate with lower levels of photosynthetic proteins and higher amino acid levels. Collectively, these observations suggest that the alarmone (p)ppGpp mediates nitrogen stress responses and may constitute a highly conserved mechanism from bacteria to plants.


Assuntos
Guanosina Pentafosfato , Oryza , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cloroplastos/metabolismo , Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Nitrogênio/metabolismo , Oryza/genética , Oryza/metabolismo , Plantas/metabolismo
5.
RNA Biol ; 18(11): 2028-2037, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33573428

RESUMO

The bacterial σ factor plays the central role in promoter recognition by RNA polymerase (RNAP). The primary σ factor, involved in transcription of housekeeping genes, was also shown to participate in the initiation of RNA synthesis and promoter escape by RNAP. In the open promoter complex, the σ finger formed by σ region 3.2 directly interacts with the template DNA strand upstream of the transcription start site. Here, we analysed the role of the σ finger in transcription initiation by four alternative σ factors in Escherichia coli, σ38, σ32, σ28 and σ24. We found that deletions of the σ finger to various extent compromise the activity of RNAP holoenzymes containing alternative σ factors, especially at low NTP concentrations. All four σs are able to utilize NADH as a noncanonical priming substrate but it has only mild effects on the efficiency of transcription initiation. The mediators of the stringent response, transcription factor DksA and the alarmone ppGpp decrease RNAP activity and promoter complex stability for all four σ factors on tested promoters. For all σs except σ38, deletions of the σ finger conversely increase the stability of promoter complexes and decrease their sensitivity to DksA and ppGpp. The result suggests that the σ finger plays a universal role in transcription initiation by alternative σ factors and sensitizes promoter complexes to the action of global transcription regulators DksA and ppGpp by modulating promoter complex stability.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , Fator sigma/metabolismo , Sítio de Iniciação de Transcrição , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Regiões Promotoras Genéticas , RNA Bacteriano/genética , Fator sigma/genética , Transcrição Gênica
6.
FEBS J ; 288(7): 2377-2397, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33067840

RESUMO

The stringent response, regulated by the bifunctional (p)ppGpp synthetase/hydrolase Rel in mycobacteria, is critical for long-term survival of the drug-tolerant dormant state of Mycobacterium tuberculosis. During amino acid starvation, MtRel senses a drop in amino acid concentration and synthesizes the messengers pppGpp and ppGpp, collectively called (p)ppGpp. Here, we investigate the role of the regulatory 'Aspartokinase, Chorismate mutase and TyrA' (ACT) domain in MtRel. Using NMR spectroscopy approaches, we report the high-resolution structure of dimeric MtRel ACT which selectively binds to valine out of all other branched-chain amino acids tested. A set of MtRel ACT mutants were generated to identify the residues required for maintaining the head-to-tail dimer. Through NMR titrations, we determined the crucial residues for binding of valine and show structural rearrangement of the MtRel ACT dimer in the presence of valine. This study suggests the direct involvement of amino acids in (p)ppGpp accumulation mediated by MtRel independent to interactions with stalled ribosomes. Database Structural data are available in the PDB database under the accession number 6LXG.


Assuntos
Aspartato Quinase/genética , Corismato Mutase/genética , Ligases/genética , Mycobacterium tuberculosis/genética , Aspartato Quinase/química , Aspartato Quinase/ultraestrutura , Corismato Mutase/química , Corismato Mutase/ultraestrutura , Guanosina Tetrafosfato/genética , Hidrolases/genética , Ligases/química , Ligases/ultraestrutura , Espectroscopia de Ressonância Magnética , Mycobacterium tuberculosis/patogenicidade , Domínios Proteicos/genética , Multimerização Proteica , Fatores de Transcrição/genética
7.
J Mol Biol ; 432(24): 166716, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33220263

RESUMO

The non-protein amino acid meta-Tyrosine (m-Tyr) is produced in cells under conditions of oxidative stress, and m-Tyr has been shown to be toxic to a broad range of biological systems. However, the mechanism by which m-Tyr damages cells is unclear. In E. coli, the quality control (QC) function of phenyalanyl-tRNA synthetase (PheRS) is required for resistantce to m-Tyr. To determine the mechanism of m-Tyr toxicity, we utilitized a strain of E. coli that expresses a QC-defective PheRS. The global responses of E. coli cells to m-Tyr were assessed by RNA-seq, and >500 genes were differentially expressed after the addition of m-Tyr. The most strongly up-regulated genes are involved in unfolded-protein stress response, and cells exposed to m-Tyr contained large, electron-dense protein aggregates, indicating that m-Tyr destabilized a large fraction of the proteome. Additionally, we observed that amino acid biosynthesis and transport regulons, controlled by ArgR, TrpR, and TyrR, and the stringent-response regulon, controlled by DksA/ppGpp, were differentially expressed. m-Tyr resistant mutants were isolated and found to have altered a promoter to increase expression of the enzymes for Phe production or to have altered transporters, which likely result in less uptake or increased efflux of m-Tyr. These findings indicate that when m-Tyr has passed the QC checkpoint by the PheRS, this toxicity of m-Tyr may result from interfering with amino acid metabolism, destabalizing a large number of proteins, and the formation of protein aggregates.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Proteínas Repressoras/genética , Tirosina/metabolismo , Aminoacil-tRNA Sintetases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Guanosina Tetrafosfato/genética , Estresse Oxidativo/genética , Fenilalanina/genética , Agregados Proteicos/genética , Proteoma/genética , Proteoma/metabolismo , Tirosina/genética , Tirosina/toxicidade
8.
Microbiology (Reading) ; 166(8): 735-750, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32496187

RESUMO

Pseudomonas aeruginosa, a versatile bacterium present in terrestrial and aquatic environments and a relevant opportunistic human pathogen, is largely known for the production of robust biofilms. The unique properties of these structures complicate biofilm eradication, because they make the biofilms very resistant to diverse antibacterial agents. Biofilm development and establishment is a complex process regulated by multiple regulatory genetic systems, among them is quorum sensing (QS), a mechanism employed by bacteria to regulate gene transcription in response to population density. In addition, environmental factors such as UVA radiation (400-315 nm) have been linked to biofilm formation. In this work, we further investigate the mechanism underlying the induction of biofilm formation by UVA, analysing the role of QS in this phenomenon. We demonstrate that UVA induces key genes of the Las and Rhl QS systems at the transcriptional level. We also report that pelA and pslA genes, which are essential for biofilm formation and whose transcription depends in part on QS, are significantly induced under UVA exposure. Finally, the results demonstrate that in a relA strain (impaired for ppGpp production), the UVA treatment does not induce biofilm formation or QS genes, suggesting that the increase of biofilm formation due to exposure to UVA in P. aeruginosa could rely on a ppGpp-dependent QS induction.


Assuntos
Biofilmes/efeitos da radiação , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/fisiologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/genética , 4-Butirolactona/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Genes Bacterianos/genética , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Mutação , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Pseudomonas aeruginosa/efeitos da radiação , Percepção de Quorum/genética , Percepção de Quorum/efeitos da radiação , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta
9.
J Mol Biol ; 432(14): 4108-4126, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32446804

RESUMO

The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacteria. In the bacterial phyla Firmicutes and Bacteroidetes, xanthine phosphoribosyltransferase (XPRT) is a purine salvage enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical, and genetic analyses, we show that pppGpp and ppGpp, as well as a third newly identified alarmone pGpp, all directly interact with XPRT from the Gram-positive bacterium Bacillus subtilis and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine salvage enzyme HPRT, suggesting evolutionary conservation in different enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. (p)ppGpp's interaction with an XPRT bridging loop across the interface results in XPRT cooperatively binding (p)ppGpp. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in B. subtilis, suggesting that regulation of XPRT is key for regulating the purine salvage pathway.


Assuntos
Guanosina Pentafosfato/genética , Guanosina Tetrafosfato/genética , Pentosiltransferases/genética , Purinas/metabolismo , Bacillus subtilis/enzimologia , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Nucleotídeos/genética , Ligação Proteica/genética
10.
mBio ; 11(1)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098823

RESUMO

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.


Assuntos
Proteínas de Bactérias/metabolismo , Ligases/metabolismo , Fagossomos/metabolismo , Pirofosfatases/metabolismo , Salmonella/metabolismo , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Guanosina Pentafosfato/genética , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Imunidade Inata , Ligases/genética , Camundongos , Pirofosfatases/genética , Salmonella/genética , Fator de Transcrição RelA/metabolismo , Virulência/genética
11.
Proc Natl Acad Sci U S A ; 116(17): 8310-8319, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30971496

RESUMO

The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) in Escherichia coli, but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.


Assuntos
Sítios de Ligação/genética , RNA Polimerases Dirigidas por DNA , Escherichia coli/genética , Guanosina Tetrafosfato , Transcrição Gênica/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genoma Bacteriano/genética , Guanosina Tetrafosfato/química , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Regiões Promotoras Genéticas/genética , Transcriptoma
12.
Nucleic Acids Res ; 47(9): 4684-4693, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30916318

RESUMO

Understanding how bacteria coordinate gene expression with biomass growth to adapt to various stress conditions remains a grand challenge in biology. Stress response is often associated with dramatic accumulation of cellular guanosine tetra- or penta-phosphate (p)ppGpp (also known as 'magic spot'), which is a key second messenger participating in regulating various biochemical and physiological processes of bacteria. Despite of the extensive studies on the mechanism of gene regulation by (p)ppGpp during stringent response, the connection between (p)ppGpp and bacterial steady-state exponential growth remains elusive. Here, we establish a versatile genetic approach to systematically perturb the (p)ppGpp level of Escherichia coli through titrating either the single-function (p)ppGpp synthetase or the singe-function (p)ppGpp hydrolase and quantitatively characterize cell growth and gene expression. Strikingly, increased and decreased (p)ppGpp levels both cause remarkable growth suppression of E. coli. From a coarse-grained insight, we demonstrate that increased (p)ppGpp levels limit ribosome synthesis while decreased (p)ppGpp levels limit the expression of metabolic proteins, both resulting in non-optimal resource allocation. Our study reveals a profound role of (p)ppGpp in regulating bacterial growth through governing global resource allocation. Moreover, we highlight the Mesh1 (p)ppGpp hydrolase from Drosophila melanogaster as a powerful genetic tool for interrogating bacterial (p)ppGpp physiology.


Assuntos
Escherichia coli/genética , Guanosina Tetrafosfato/genética , Ligases/genética , Ribossomos/genética , Animais , Biomassa , Proliferação de Células/genética , Drosophila melanogaster/enzimologia , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/genética , Guanosina Pentafosfato/genética , Hidrolases/genética , Estresse Fisiológico/genética
13.
Proc Natl Acad Sci U S A ; 115(23): 6052-6057, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29784782

RESUMO

Riboswitches are noncoding portions of certain mRNAs that bind metabolite, coenzyme, signaling molecule, or inorganic ion ligands and regulate gene expression. Most known riboswitches sense derivatives of RNA monomers. This bias in ligand chemical composition is consistent with the hypothesis that widespread riboswitch classes first emerged during the RNA World, which is proposed to have existed before proteins were present. Here we report the discovery and biochemical validation of a natural riboswitch class that selectively binds guanosine tetraphosphate (ppGpp), a widespread signaling molecule and bacterial "alarmone" derived from the ribonucleotide GTP. Riboswitches for ppGpp are predicted to regulate genes involved in branched-chain amino acid biosynthesis and transport, as well as other gene classes that previously had not been implicated to be part of its signaling network. This newfound riboswitch-alarmone partnership supports the hypothesis that prominent RNA World signaling pathways have been retained by modern cells to control key biological processes.


Assuntos
Guanosina Tetrafosfato/fisiologia , Riboswitch/fisiologia , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Ligantes , Conformação de Ácido Nucleico , RNA Bacteriano , Ribonucleotídeos/metabolismo , Riboswitch/genética , Transdução de Sinais
14.
Genes Dev ; 31(15): 1549-1560, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28864445

RESUMO

Francisella tularensis, the etiological agent of tularemia, is one of the most infectious bacteria known. Because of its extreme pathogenicity, F. tularensis is classified as a category A bioweapon by the US government. F. tularensis virulence stems from genes encoded on the Francisella pathogenicity island (FPI). An unusual set of Francisella regulators-the heteromeric macrophage growth locus protein A (MglA)-stringent starvation protein A (SspA) complex and the DNA-binding protein pathogenicity island gene regulator (PigR)-activates FPI transcription and thus is essential for virulence. Intriguingly, the second messenger, guanosine-tetraphosphate (ppGpp), which is produced during infection, is also involved in coordinating Francisella virulence; however, its role has been unclear. Here we identify MglA-SspA as a novel ppGpp-binding complex and describe structures of apo- and ppGpp-bound MglA-SspA. We demonstrate that MglA-SspA, which binds RNA polymerase (RNAP), also interacts with the C-terminal domain of PigR, thus anchoring the (MglA-SspA)-RNAP complex to the FPI promoter. Furthermore, we show that MglA-SspA must be bound to ppGpp to mediate high-affinity interactions with PigR. Thus, these studies unveil a novel pathway different from those described previously for regulation of transcription by ppGpp. The data also indicate that F. tularensis pathogenesis is controlled by a highly interconnected molecular circuitry in which the virulence machinery directly senses infection via a small molecule stress signal.


Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Francisella tularensis/patogenicidade , Ilhas Genômicas/genética , Guanosina Tetrafosfato/metabolismo , Tularemia/microbiologia , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Bioterrorismo/prevenção & controle , Células Cultivadas , Cristalografia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/genética , Humanos , Macrófagos/metabolismo , Conformação Proteica , Transcrição Gênica , Virulência/genética
15.
Cell Syst ; 4(6): 611-621.e6, 2017 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-28624614

RESUMO

The Bartonella gene transfer agent (BaGTA) is an archetypical example for domestication of a phage-derived element to permit high-frequency genetic exchange in bacterial populations. Here we used multiplexed transposon sequencing (TnSeq) and single-cell reporters to globally define the core components and transfer dynamics of BaGTA. Our systems-level analysis has identified inner- and outer-circle components of the BaGTA system, including 55 regulatory components, as well as an additional 74 and 107 components mediating donor transfer and recipient uptake functions. We show that the stringent response signal guanosine-tetraphosphate (ppGpp) restricts BaGTA induction to a subset of fast-growing cells, whereas BaGTA particle uptake depends on a functional Tol-Pal trans-envelope complex that mediates outer-membrane invagination upon cell division. Our findings suggest that Bartonella evolved an efficient strategy to promote genetic exchange within the fittest subpopulation while disfavoring exchange of deleterious genetic information, thereby facilitating genome integrity and rapid host adaptation.


Assuntos
Bartonella/genética , Transferência Genética Horizontal/genética , Proteínas de Bactérias/genética , Bacteriófagos/genética , Divisão Celular/genética , Genética , Guanosina Tetrafosfato/genética
16.
Plant Signal Behav ; 11(2): e1132966, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26825398

RESUMO

In bacteria a second messenger, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), synthesized upon nutrient starvation, controls many gene expressions and enzyme activities, which is necessary for growth under changeable environments. Recent studies have shown that ppGpp synthase and hydrolase are also conserved in eukaryotes, although their functions are not well understood. We recently showed that ppGpp-overaccumulation in Arabidopsis chloroplasts results in robust growth under nutrient-limited conditions, demonstrating that the bacterial-like stringent response at least functions in plastids. To test if ppGpp also functions in the cytosol, we constructed the transgenic Arabidopsis expressing Bacillus subtilis ppGpp synthase gene yjbM. Upon induction of the gene, the mutant synthesizes ∼10-20-fold higher levels of ppGpp, and its fresh weight was reduced to ˜80% that of the wild type. These results indicate that cytosolic ppGpp negatively regulates plant growth and development.


Assuntos
Arabidopsis/genética , Guanosina Tetrafosfato/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Citosol/metabolismo , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Plantas Geneticamente Modificadas
17.
FEMS Microbiol Lett ; 362(23): fnv187, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26449557

RESUMO

FtsZ, a protein essential for prokaryotic cell division, forms a ring structure known as the Z-ring at the division site. FtsZ has a GTP binding site and is assembled into linear structures in a GTP-dependent manner in vitro. We assessed whether guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a global regulator of gene expression in starved bacteria, affects cell division in Salmonella Paratyphi A. Elevation of intracellular ppGpp levels by using the relA expression vector induced repression of bacterial growth and incorrect FtsZ assembly. We found that FtsZ forms helical structures in the presence of ppGpp by using the GTP binding site; however, ppGpp levels required to form helical structures were at least 20-fold higher than the required GTP levels in vitro. Furthermore, once formed, helical structures did not change to the straight form even after GTP addition. Our data indicate that elevation of the ppGpp level leads to inhibition of bacterial growth and interferes with FtsZ assembly.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Guanosina Tetrafosfato/metabolismo , Salmonella paratyphi A/crescimento & desenvolvimento , Arabinose/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Divisão Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/isolamento & purificação , Vetores Genéticos , Guanosina Tetrafosfato/genética , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Ligação Proteica , Salmonella paratyphi A/efeitos dos fármacos , Salmonella paratyphi A/genética , Salmonella paratyphi A/ultraestrutura
18.
Nat Rev Microbiol ; 13(5): 298-309, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25853779

RESUMO

The alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively referred to as (p)ppGpp) are involved in regulating growth and several different stress responses in bacteria. In recent years, substantial progress has been made in our understanding of the molecular mechanisms of (p)ppGpp metabolism and (p)ppGpp-mediated regulation. In this Review, we summarize these recent insights, with a focus on the molecular mechanisms governing the activity of the RelA/SpoT homologue (RSH) proteins, which are key players that regulate the cellular levels of (p)ppGpp. We also discuss the structural basis of transcriptional regulation by (p)ppGpp and the role of (p)ppGpp in GTP metabolism and in the emergence of bacterial persisters.


Assuntos
Bactérias/metabolismo , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Bactérias/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Guanosina Pentafosfato/genética , Guanosina Tetrafosfato/genética , Ligases/metabolismo , Pirofosfatases/metabolismo
19.
Environ Microbiol ; 17(11): 4253-70, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25626964

RESUMO

The stringent response, mediated by second messenger (p)ppGpp, results in swift and massive transcriptional reprogramming under nutrient limited conditions. In this study, the role of (p)ppGpp on virulence of Pseudomonas syringae pv. syringae B728a (PssB728a) was investigated. The virulence of the relA/spoT (ppGpp(0) ) double mutant was completely impaired on bean, and bacterial growth was significantly reduced, suggesting that (p)ppGpp is required for full virulence of P. syringae. Expression of T3SS and other virulence genes was reduced in ppGpp(0) mutants. In addition, ppGpp deficiency resulted in loss of swarming motility, reduction of pyoverdine production, increased sensitivity to oxidative stress and antibiotic tolerance, as well as reduced ability to utilize γ-amino butyric acid. Increased levels of ppGpp resulted in reduced cell size of PssB728a when grown in a minimal medium and on plant surfaces, while most ppGpp(0) mutant cells were not viable on plant surfaces 24 h after spray inoculation, suggesting that ppGpp-mediated stringent response temporarily limits cell growth, and might control cell survival on plants by limiting their growth. These results demonstrated that ppGpp-mediated stringent response plays a central role in P. syringae virulence and survival and indicated that ppGpp serves as a global signal for regulating various virulence traits in PssB728a.


Assuntos
Proteínas de Bactérias/genética , Guanosina Pentafosfato/fisiologia , Guanosina Tetrafosfato/fisiologia , Doenças das Plantas/microbiologia , Plantas/microbiologia , Pseudomonas syringae/patogenicidade , Farmacorresistência Bacteriana , Guanosina Pentafosfato/genética , Guanosina Tetrafosfato/genética , Oligopeptídeos/biossíntese , Estresse Oxidativo/genética , Folhas de Planta/microbiologia , Pseudomonas syringae/genética , Sistemas do Segundo Mensageiro/genética , Virulência , Fatores de Virulência/genética
20.
Mol Microbiol ; 90(3): 457-71, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23957692

RESUMO

Pectobacterium atrosepticum (Pca) is a Gram-negative phytopathogen which causes disease by secreting plant cell wall degrading exoenzymes (PCWDEs). Previous studies have shown that PCWDE production is regulated by (i) the intercellular quorum sensing (QS) signal molecule, 3-oxo-hexanoyl-l-homoserine lactone (OHHL), and (ii) the intracellular 'alarmone', (p)ppGpp, which reports on nutrient limitation. Here we show that these two signals form an integrated coincidence circuit which ensures that metabolically costly PCWDE synthesis does not occur unless the population is simultaneously quorate and nutrient limited. A (p)ppGpp null ΔrelAΔspoT mutant was defective in both OHHL and PCWDE production, and nutritional supplementation of wild type cultures (which suppresses (p)ppGpp production) also suppressed OHHL and PCWDE production. There was a substantial overlap in the transcriptome of a (p)ppGpp deficient relA mutant and of a QS defective expI (OHHL synthase) mutant, especially with regards to virulence-associated genes. Random transposon mutagenesis revealed that disruption of rsmA was sufficient to restore PCWDE production in the (p)ppGpp null strain. We found that the ratio of RsmA protein to its RNA antagonist, rsmB, was modulated independently by (p)ppGpp and QS. While QS predominantly controlled virulence by modulating RsmA levels, (p)ppGpp exerted regulation through the modulation of the RsmA antagonist, rsmB.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Guanosina Tetrafosfato/metabolismo , Pectobacterium/genética , Pectobacterium/patogenicidade , 4-Butirolactona/análogos & derivados , 4-Butirolactona/genética , 4-Butirolactona/metabolismo , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/genética , Dados de Sequência Molecular , Mutação , Pectobacterium/classificação , Pectobacterium/enzimologia , Percepção de Quorum , Virulência
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