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1.
World J Microbiol Biotechnol ; 35(8): 130, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31385043

RESUMO

Bacterial biofilms (BFs) are membrane-like structures formed by the secretion of extracellular polymeric substances (EPS) by bacteria. The formation of BFs contributes to bacterial survival and drug resistance. When bacteria proliferate, they produce secondary metabolites that act as signaling molecules in bacterial communities that regulate intracellular and cell-to-cell communication. This communication can directly affect the physiological behavior of bacteria, including the production and emission of light (bioluminescence), the expression of virulence factors, the resistance to antibiotics, and the shift between planktonic and biofilm lifestyles. We review the major signaling molecules that regulate BF formation, with a focus on quorum-sensing systems (QS), cyclic diguanylate (c-di-GMP), two-component systems (TCS), and small RNA (sRNA). Understanding these processes will lead to new approaches for treating chronic diseases and preventing bacterial resistance.


Assuntos
Bactérias/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Transdução de Sinais , Bactérias/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Percepção de Quorum , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo
2.
World J Microbiol Biotechnol ; 35(9): 140, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451938

RESUMO

Pseudomonas species are the most versatile of all known bacteria for metabolic flexibility and the extent of host range from plants to humans that remains unmatched. The evolution of diverse metabolic strategies in these species to adapt to the fluctuating environment guarantees high fitness as well as the ability to withstand stress at multiple levels. These abilities in Pseudomonas species are imprinted by an adaptable genetic repertoire through the integration of external and internal signals via complex regulatory networks. One of the main regulatory networks that lead to optimal growth, survival and cellular robustness is the phenomenon of carbon catabolite repression (CCR). Even though a large array of information is available, the molecular machinery and the mechanism of CCR in Pseudomonas are distinctly diverse from Escherichia coli and Bacillus subtilis. In Pseudomonas, the Crc and Hfq proteins, CbrAB two-component systems and the CrcZ/CrcY small RNA are key components of CCR. The main focus of this review is to elucidate the mechanism of CCR and the accessories involved in regulation of preferred carbon source utilisation over non-preferred ones and how CCR influences the virulence, antibiotic resistance, bioremediation and plant growth promotion pathways. Furthermore, we have also tried to shed some light on the "omics" approaches which can provide deep mechanistic insights into the regulation of CCR. Understanding the mechanistic picture of key regulatory entities and mechanism responsible for metabolic flexibility will create opportunities for exploitation of these versatile prokaryotes in several biotechnological processes.


Assuntos
Proteínas de Bactérias/metabolismo , Repressão Catabólica , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismo , Pseudomonas/metabolismo , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Carbono/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Pseudomonas/genética , RNA Bacteriano/metabolismo
3.
Nat Commun ; 10(1): 2544, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186424

RESUMO

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3'-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ribonucleases/metabolismo , Ruminococcus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Conformação de Ácido Nucleico , Domínios Proteicos , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Guia/genética , Ribonucleases/química , Ribonucleases/genética , Ruminococcus/enzimologia
4.
Int J Med Microbiol ; 309(3-4): 225-231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31054808

RESUMO

Brucella species are the causative agents of brucellosis, a worldwide zoonotic disease that affects a broad range of mammals and causes great economic losses. Small regulatory RNAs (sRNAs) are post-transcriptional regulatory molecules that participate in the stress adaptation and pathogenesis of Brucella. In this study, we characterized the role of a novel sRNA, BSR1141, in the intracellular survival and virulence of Brucella melitensis. The results show that BSR1141 was highly induced during host infections and under in vitro stress situations that simulated the conditions encountered within host phagocytes. In addition, a BSR1141 mutant showed reduced survival both under in vitro stress conditions and in mice, confirming the role of BSR1141 in Brucella intracellular survival. Bioinformatic and experimental approaches revealed that BSR1141 affects the expression of many target genes, including the Brucella virulence component virB2. These data indicate that BSR1141 could influence the expression of virB2, which is important for B. melitensis pathogenesis and intracellular survival. This work provides new insight into the mechanism of adaptation to environmental stress and into the pathogenesis of intracellular pathogens.


Assuntos
Brucella melitensis/fisiologia , Brucella melitensis/patogenicidade , Pequeno RNA não Traduzido/metabolismo , Fatores de Virulência/genética , Animais , Brucella melitensis/genética , Brucelose/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Mutação , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Baço/microbiologia , Estresse Fisiológico , Virulência/genética
5.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-31004423

RESUMO

Regulatory RNAs, present in many bacterial genomes and particularly in pathogenic bacteria such as Staphylococcus aureus, control the expression of genes encoding virulence factors or metabolic proteins. They are extremely diverse and include noncoding RNAs (sRNA), antisense RNAs, and some 5' or 3' untranslated regions of messenger RNAs that act as sensors for metabolites, tRNAs, or environmental conditions (e.g., temperature, pH). In this review we focus on specific examples of sRNAs of S. aureus that illustrate how numerous sRNAs and associated proteins are embedded in complex networks of regulation. In addition, we discuss the CRISPR-Cas systems defined as an RNA-interference-like mechanism, which also exist in staphylococcal strains.


Assuntos
RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Staphylococcus aureus/genética , Animais , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Humanos , RNA Bacteriano/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo
6.
Methods Mol Biol ; 1973: 251-260, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016707

RESUMO

A robust, fluorescence-based analysis and discovery platform is described for bacterial A-site binders. The assay relies on an incorporated isomorphic fluorescent uridine analog, which substitutes the A-site's U1406 and serves as a FRET donor to an A-site bound coumarin-labeled aminoglycoside that serves as the FRET acceptor. Binding efficiency of unlabeled A-site ligands can be determined by competition experiments, where the acceptor-labeled aminoglycoside is displaced. The replacement efficiency is gauged by the concentration-dependent loss of the sensitized FRET acceptor's signal with concomitant restoration of the donor's emission. Plotting the relative emission intensity of both the donor and acceptor as a function of ligand concentration followed by fitting of the data points to a dose-response curve yields IC50 values, one possible measure of the antibiotic potency of new A-site binders.


Assuntos
Aminoglicosídeos/metabolismo , Cumarínicos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Fluorescência , RNA Bacteriano/metabolismo , Uridina/metabolismo , Aminoglicosídeos/química , Cumarínicos/química , Ligantes , RNA Bacteriano/química , Uridina/química
7.
Mol Cell ; 74(1): 132-142.e5, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30872121

RESUMO

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/metabolismo , Mimetismo Molecular , Pseudomonas aeruginosa/enzimologia , RNA Bacteriano/metabolismo , RNA Guia/metabolismo , Proteínas Virais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Microscopia Crioeletrônica , DNA Bacteriano/química , DNA Bacteriano/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Guia/química , RNA Guia/genética , Relação Estrutura-Atividade , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologia
8.
Methods Mol Biol ; 1969: 33-49, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30877668

RESUMO

Deep sequencing technology has revolutionized transcriptome analyses of both prokaryotes and eukaryotes. RNA-sequencing (RNA-seq), which is based on massively parallel sequencing of cDNAs, has been used to annotate transcript boundaries and has revealed widespread antisense transcription as well as a wealth of novel noncoding transcripts in many bacterial pathogens. Moreover, RNA-seq is nowadays also widely used to comprehensively explore the interaction between RNA-binding proteins and their RNA targets on a genome-wide level in many human-pathogenic bacteria. In particular, immunoprecipitation of an RNA-binding protein (RBP) of interest followed by isolation and analysis of all bound RNAs (RNA immunoprecipitation (RIP)) allows rapid characterization of its RNA regulon. Here, we describe an experimental approach which employs co-immunoprecipitation (coIP) of the RNA-binding chaperone Hfq along with bound RNAs followed by deep-sequencing of co-purified RNAs (RIP-Seq) from a genetically modified strain of Neisseria meningitidis expressing a chromosomally encoded Hfq-3×FLAG protein. This approach allowed us to comprehensively identify both mRNAs and sRNAs as targets of Hfq and served as an excellent starting point for sRNA research in this human pathogenic bacterium.


Assuntos
Perfilação da Expressão Gênica/métodos , Fator Proteico 1 do Hospedeiro/metabolismo , Imunoprecipitação/métodos , Neisseria meningitidis/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Humanos , Neisseria meningitidis/genética , Neisseria meningitidis/isolamento & purificação , Ligação Proteica , RNA Bacteriano/genética , RNA Mensageiro/genética
9.
RNA ; 25(5): 573-589, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30792229

RESUMO

Identification and characterization of base-multiplets, which are essentially mediated by base-pairing interactions, can provide insights into the diversity in the structure and dynamics of complex functional RNAs, and thus facilitate hypothesis driven biological research. The necessary nomenclature scheme, an extension of the geometric classification scheme for base-pairs by Leontis and Westhof, is however available only for base-triplets. In the absence of information on topology, this scheme is not applicable to quartets and higher order multiplets. Here we propose a topology-based classification scheme which, in conjunction with a graph-based algorithm, can be used for the automated identification and characterization of higher order base-multiplets in RNA structures. Here, the RNA structure is represented as a graph, where nodes represent nucleotides and edges represent base-pairing connectivity. Sets of connected components (of n nodes) within these graphs constitute subgraphs representing multiplets of "n" nucleotides. The different topological variants of the RNA multiplets thus correspond to different nonisomorphic forms of these subgraphs. To annotate RNA base-multiplets unambiguously, we propose a set of topology-based nomenclature rules for quartets, which are extendable to higher multiplets. We also demonstrate the utility of our approach toward the identification and annotation of higher order RNA multiplets, by investigating the occurrence contexts of selected examples in order to gain insights regarding their probable functional roles.


Assuntos
Algoritmos , Conformação de Ácido Nucleico , Nucleotídeos/química , RNA Bacteriano/química , RNA Fúngico/química , Pareamento de Bases , Biologia Computacional/métodos , Ligações de Hidrogênio , Nucleotídeos/genética , Nucleotídeos/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Software , Thermus thermophilus/genética , Thermus thermophilus/metabolismo
10.
RNA ; 25(4): 472-480, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30705137

RESUMO

In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the Escherichia coli ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals were measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24, or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.


Assuntos
RNA Bacteriano/química , RNA Ribossômico 23S/química , Proteínas Ribossômicas/genética , Ribossomos/química , Pareamento de Bases , Sequência de Bases , Fenômenos Biomecânicos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Pinças Ópticas , Biogênese de Organelas , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
11.
Microb Pathog ; 129: 266-270, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30802490

RESUMO

OBJECTIVES: The non-coding RNA rprA can increase the resistance to ampicillin in Escherichia coli. METHODS: Bacterial DNA was extracted by boiling method and then amplified using polymerase chain reaction (PCR) with two different primer sets. Recombinant pET28a/rprA-sense and -antisense plasmids were separately transferred into the competent E. coli BL21 (DE3) by chemical methods using heat shock. The expression was analyzed at the RNA level using Semi quantitative RT PCR. The turbidity difference between the bacteria was checked by Broth Dilution method. RESULTS: The statistical analysis showed that the turbidity difference between the up regulated and control bacteria is significant (p value < 0.0001). The ANOVA test also showed the significant difference between the down regulated and control bacteria (p value < 0.0001). CONCLUSION: Considering this mechanism, there are some reports indicating the role of rprA in antibiotic resistance. However, the role of rprA in ampicillin resistance is remained to be unknown. The aim of this study was to analyze the up regulation and down regulation of rprA and check their effects on ampicillin resistance in Escherichia coli. It was found that the up regulation and down regulation of rprA can lead into more antibiotics resistance and susceptibility, respectively. Our results showed the potential role of rprA expression in the response to ampicillin stress in E. coli.


Assuntos
Resistência a Ampicilina , Ampicilina/farmacologia , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , RNA Bacteriano/metabolismo , RNA não Traduzido/metabolismo
12.
MBio ; 10(1)2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30723124

RESUMO

Small RNAs (sRNAs) remain an understudied class of regulatory molecules in bacteria in general and in Gram-positive bacteria in particular. In the major human pathogen Staphylococcus aureus, hundreds of sRNAs have been identified; however, only a few have been characterized in detail. In this study, we investigate the role of the sRNA Teg41 in S. aureus virulence. We demonstrate that Teg41, an sRNA divergently transcribed from the locus that encodes the cytolytic alpha phenol-soluble modulin (αPSM) peptides, plays a critical role in αPSM production. Overproduction of Teg41 leads to an increase in αPSM levels and a corresponding increase in hemolytic activity from S. aureus cells and cell-free culture supernatants. To identify regions of Teg41 important for its function, we performed an in silico RNA-RNA interaction analysis which predicted an interaction between the 3' end of Teg41 and the αPSM transcript. Deleting a 24-nucleotide region from the S. aureus genome, corresponding to the 3' end of Teg41, led to a 10-fold reduction in αPSM-dependent hemolytic activity and attenuation of virulence in a murine abscess model of infection. Restoration of hemolytic activity in the Teg41Δ3' strain was possible by expressing full-length Teg41 in trans Restoration of hemolytic activity was also possible by expressing the 3' end of Teg41, suggesting that this region of Teg41 is necessary and sufficient for αPSM-dependent hemolysis. Our results show that Teg41 is positively influencing αPSM production, demonstrating for the first time regulation of the αPSM peptides by an sRNA in S. aureus IMPORTANCE The alpha phenol-soluble modulins (αPSMs) are among the most potent toxins produced by Staphylococcus aureus Their biological role during infection has been studied in detail; however, the way they are produced by the bacterial cell is not well understood. In this work, we identify a small RNA molecule called Teg41 that plays an important role in αPSM production by S. aureus Teg41 positively influences αPSM production. The importance of Teg41 is highlighted by the fact that a strain containing a deletion in the 3' end of Teg41 produces significantly less αPSMs and is attenuated for virulence in a mouse abscess model of infection. As the search for new therapeutic strategies to combat S. aureus infection proceeds, Teg41 may represent a novel target.


Assuntos
Toxinas Bacterianas/biossíntese , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/genética , Fatores de Virulência/biossíntese , Abscesso/microbiologia , Abscesso/patologia , Animais , Modelos Animais de Doenças , Teste de Complementação Genética , Hemólise , Humanos , Camundongos , Deleção de Sequência , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Virulência
13.
Nat Commun ; 10(1): 930, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30804338

RESUMO

Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support cell proliferation in the absence of wild type ribosomes, Ribo-T cells grow slower than those with wild type ribosomes. Here, we show that cell growth defect is likely explained primarily by slow Ribo-T assembly rather than its imperfect functionality. Ribo-T maturation is stalled at a late assembly stage. Several post-transcriptional rRNA modifications and some ribosomal proteins are underrepresented in the accumulated assembly intermediates and rRNA ends are incompletely trimmed. Ribosome profiling of Ribo-T cells shows no defects in translation elongation but reveals somewhat higher occupancy by Ribo-T of the start codons and to a lesser extent stop codons, suggesting that subunit tethering mildly affects the initiation and termination stages of translation. Understanding limitations of Ribo-T system offers ways for its future development.


Assuntos
Subunidades Ribossômicas/química , Subunidades Ribossômicas/metabolismo , Códon de Iniciação/genética , Códon de Iniciação/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas/genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-30785097

RESUMO

In E. coli, a single oligomeric enzyme transcribes the genomic DNA, while multiple auxiliary proteins and regulatory RNA interact with the core RNA polymerase (RP) during different stages of the transcription cycle to influence its function. In this work, using fast protein isolation techniques combined with mass spectrometry (MS) and immuno-analyses, we studied growth phase-specific changes in the composition of E. coli transcription complexes. We show that RP isolated from actively growing cells is represented by prevalent double copy assemblies and single copy RP-RNA and RP-RNA-RapA complexes. We demonstrate that RpoD/σ70 obtained in fast purification protocols carries tightly associated RNA and show evidence pointing to a role of sigma-associated RNA in the formation of native RP-(RNA)-RpoD/σ70 (holoenzyme) complexes. We report that enzymes linked functionally to the metabolism of lipopolysaccharides co-purify with RP-RNA complexes and describe two classes of RP-associated molecules (phospholipids and putative phospholipid-rNT species). We hypothesize that these modifications could enable anchoring of RP-RNA and RNA in cell membranes. We also report that proteins loosely associated with ribosomes and degradosomes (S1, Hfq) co-purify with RP-RNA complexes isolated from actively growing cells - a result consistent with their proposed roles as adaptor-proteins. In contrast, GroEL, SecB, and SecA co-purified with RP obtained from cells harvested in early stationary phase. Our results demonstrate that fast, affinity chromatography-based isolation of large multi-protein assemblies in combination with MS can be used as a tool for analysis of their composition and the profiling of small protein-associated molecules (SPAM).


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli , RNA Bacteriano/metabolismo , Cromatografia Líquida de Alta Pressão , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Substâncias Macromoleculares/química , Substâncias Macromoleculares/isolamento & purificação , Substâncias Macromoleculares/metabolismo , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , Transcrição Genética
15.
Proc Natl Acad Sci U S A ; 116(3): 1043-1052, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30591570

RESUMO

Bacterial regulatory small RNAs act as crucial regulators in central carbon metabolism by modulating translation initiation and degradation of target mRNAs in metabolic pathways. Here, we demonstrate that a noncoding small RNA, SdhX, is produced by RNase E-dependent processing from the 3'UTR of the sdhCDAB-sucABCD operon, encoding enzymes of the tricarboxylic acid (TCA) cycle. In Escherichia coli, SdhX negatively regulates ackA, which encodes an enzyme critical for degradation of the signaling molecule acetyl phosphate, while the downstream pta gene, encoding the enzyme critical for acetyl phosphate synthesis, is not significantly affected. This discoordinate regulation of pta and ackA increases the accumulation of acetyl phosphate when SdhX is expressed. Mutations in sdhX that abolish regulation of ackA lead to more acetate in the medium (more overflow metabolism), as well as a strong growth defect in the presence of acetate as sole carbon source, when the AckA-Pta pathway runs in reverse. SdhX overproduction confers resistance to hydroxyurea, via regulation of ackA SdhX abundance is tightly coupled to the transcription signals of TCA cycle genes but escapes all known posttranscriptional regulation. Therefore, SdhX expression directly correlates with transcriptional input to the TCA cycle, providing an effective mechanism for the cell to link the TCA cycle with acetate metabolism pathways.


Assuntos
Acetatos/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Hidroxiureia/farmacologia , Óperon/fisiologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética
16.
Biochem Biophys Res Commun ; 509(1): 161-166, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30579600

RESUMO

Co-transcriptional RNA proofreading by RNA polymerase (RNAP) is essential for accurate mRNA synthesis and reactivation of stalled transcription complexes, which can otherwise compromise genome integrity. RNAP from the stress-resistant bacterium Deinococcus radiodurans exhibits high levels of RNA cleavage in comparison with RNAP from Escherichia coli, which allows it to remove misincorporated nucleotides with high efficiency. Here, we show that the rate of RNA cleavage by D. radiodurans RNAP depends on the structure of the (mis)matched RNA 3'-nucleotide and its contacts with the active site. These interactions likely position the reactive phosphodiester bond in the cleavage-competent conformation, thus facilitating its hydrolysis catalyzed by metal ions in the active center. The universal RNA cleavage factor GreA largely alleviates defects in RNA cleavage caused by modifications in the RNA 3'-nucleotide or in its binding pocket in RNAP, suggesting that GreA functionally substitutes for these contacts. The results demonstrate that various RNAPs rely on a conserved mechanism for RNA proofreading, which can be modulated by changes in accessory parts of the active center.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Deinococcus/metabolismo , Clivagem do RNA , RNA Bacteriano/metabolismo , Proteínas de Bactérias/química , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/química , Deinococcus/química , Deinococcus/enzimologia , Deinococcus/genética , Manganês/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética
17.
PLoS One ; 13(12): e0209191, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30557364

RESUMO

CbrAB is a high ranked global regulatory system exclusive of the Pseudomonads that responds to carbon limiting conditions. It has become necessary to define the particular regulon of CbrB and discriminate it from the downstream cascades through other regulatory components. We have performed in vivo binding analysis of CbrB in P. putida and determined that it directly controls the expression of at least 61 genes; 20% involved in regulatory functions, including the previously identified CrcZ and CrcY small regulatory RNAs. The remaining are porines or transporters (20%), metabolic enzymes (16%), activities related to protein translation (5%) and orfs of uncharacterised function (38%). Amongst the later, we have selected the operon PP2810-13 to make an exhaustive analysis of the CbrB binding sequences, together with those of crcZ and crcY. We describe the implication of three independent non-palindromic subsites with a variable spacing in three different targets; CrcZ, CrcY and operon PP2810-13 in the CbrAB activation. CbrB is a quite peculiar σN-dependent activator since it is barely dependent on phosphorylation for transcriptional activation. With the depiction of the precise contacts of CbrB with the DNA, the analysis of the multimerisation status and its dependence on other factors such as RpoN o IHF, we propose a model of transcriptional activation.


Assuntos
Proteínas de Bactérias/metabolismo , Regiões Promotoras Genéticas , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Imunoprecipitação da Cromatina , Mineração de Dados , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Mutagênese Sítio-Dirigida , Ligação Proteica , RNA Bacteriano/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Fatores de Transcrição/genética , Ativação Transcricional/fisiologia
18.
Mol Cell ; 72(6): 1013-1020.e6, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30576652

RESUMO

Expansion segments (ESs) are enigmatic insertions within the eukaryotic ribosome, the longest of which resemble tentacle-like extensions that vary in length and sequence across evolution, with a largely unknown function. By selectively engineering rRNA in yeast, we find that one of the largest ESs, ES27L, has an unexpected function in translation fidelity. Ribosomes harboring a deletion in the distal portion of ES27L have increased amino acid misincorporation, as well as readthrough and frameshifting errors. By employing quantitative mass spectrometry, we further find that ES27L acts as an RNA scaffold to facilitate binding of a conserved enzyme, methionine amino peptidase (MetAP). We show that MetAP unexpectedly controls the accuracy of ribosome decoding, which is coupled to an increase in its enzymatic function through its interaction with ES27L. These findings reveal that variable ESs of the ribosome serve important functional roles and act as platforms for the binding of proteins that modulate translation across evolution.


Assuntos
Caulobacter crescentus/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , RNA Bacteriano/metabolismo , RNA Fúngico/metabolismo , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminopeptidases/metabolismo , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sítios de Ligação , Caulobacter crescentus/genética , Linhagem Celular , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Camundongos , Conformação de Ácido Nucleico , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Fúngico/química , RNA Fúngico/genética , RNA Ribossômico/química , RNA Ribossômico/genética , Ribossomos/química , Ribossomos/genética , Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade
19.
Int J Mol Sci ; 20(1)2018 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-30583511

RESUMO

The genus Herbaspirillum includes several strains isolated from different grasses. The identification of non-coding RNAs (ncRNAs) in the genus Herbaspirillum is an important stage studying the interaction of these molecules and the way they modulate physiological responses of different mechanisms, through RNA⁻RNA interaction or RNA⁻protein interaction. This interaction with their target occurs through the perfect pairing of short sequences (cis-encoded ncRNAs) or by the partial pairing of short sequences (trans-encoded ncRNAs). However, the companion Hfq can stabilize interactions in the trans-acting class. In addition, there are Riboswitches, located at the 5' end of mRNA and less often at the 3' end, which respond to environmental signals, high temperatures, or small binder molecules. Recently, CRISPR (clustered regularly interspaced palindromic repeats), in prokaryotes, have been described that consist of serial repeats of base sequences (spacer DNA) resulting from a previous exposure to exogenous plasmids or bacteriophages. We identified 285 ncRNAs in Herbaspirillum seropedicae (H. seropedicae) SmR1, expressed in different experimental conditions of RNA-seq material, classified as cis-encoded ncRNAs or trans-encoded ncRNAs and detected RNA riboswitch domains and CRISPR sequences. The results provide a better understanding of the participation of this type of RNA in the regulation of the metabolism of bacteria of the genus Herbaspirillum spp.


Assuntos
Herbaspirillum/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Sequência de Bases/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Simulação por Computador , Perfilação da Expressão Gênica , Genes Bacterianos/genética , Genoma Bacteriano/genética , Herbaspirillum/fisiologia , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Riboswitch/genética
20.
Int J Mol Sci ; 19(12)2018 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-30562954

RESUMO

More than 90 different modified nucleosides have been identified in tRNA. Among the tRNA modifications, the 7-methylguanosine (m7G) modification is found widely in eubacteria, eukaryotes, and a few archaea. In most cases, the m7G modification occurs at position 46 in the variable region and is a product of tRNA (m7G46) methyltransferase. The m7G46 modification forms a tertiary base pair with C13-G22, and stabilizes the tRNA structure. A reaction mechanism for eubacterial tRNA m7G methyltransferase has been proposed based on the results of biochemical, bioinformatic, and structural studies. However, an experimentally determined mechanism of methyl-transfer remains to be ascertained. The physiological functions of m7G46 in tRNA have started to be determined over the past decade. For example, tRNA m7G46 or tRNA (m7G46) methyltransferase controls the amount of other tRNA modifications in thermophilic bacteria, contributes to the pathogenic infectivity, and is also associated with several diseases. In this review, information of tRNA m7G modifications and tRNA m7G methyltransferases is summarized and the differences in reaction mechanism between tRNA m7G methyltransferase and rRNA or mRNA m7G methylation enzyme are discussed.


Assuntos
Bactérias , Proteínas de Bactérias , Guanosina/análogos & derivados , Processamento Pós-Transcricional do RNA/fisiologia , RNA Bacteriano , RNA de Transferência , tRNA Metiltransferases , Animais , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Guanosina/genética , Guanosina/metabolismo , Humanos , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
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