RESUMO
Genes annotated as ygfE and yiiU in the genome of Salmonella enterica serovar Typhimurium encode proteins homologous to Escherichia coli cell division factors ZapA and ZapB, respectively. ZapA- and ZapB- mutants of S. enterica are bile-sensitive. The amount of zapB mRNA increases in the presence of a sublethal concentration of sodium deoxycholate (DOC) while zapA mRNA remains unaffected. Increased zapB mRNA level in the presence of DOC is not caused by upregulation of zapB transcription but by increased stability of zapB mRNA. This increase is suppressed by an hfq mutation, suggesting the involvement of a small regulatory RNA. We provide evidence that such sRNA is MicA. The ZapB protein is degraded in the presence of DOC, and degradation appears to involve the Lon protease. We propose that increased stability of zapB mRNA in the presence of DOC may counter degradation of bile-damaged ZapB, thereby providing sufficient level of functional ZapB protein to permit Z-ring assembly in the presence of bile.
RESUMO
The taxonomic concept of species has received continuous attention. A microbial species as a discrete box contains a limited number of highly similar microorganisms assigned to that taxon, following a polyphasic approach. In the 21st Century, with the advancements of sequencing technologies and genomics, the existence of a huge prokaryotic diversity has become well known. At present, the prokaryotic species might no longer have to be understood as discrete values (such as 1 or 2, by homology to Natural numbers); rather, it is expected that some microorganisms could be potentially distributed (according to their genome features and phenotypes) in between others (such as decimal numbers between 1 and 2; real numbers). We propose a continuous species concept for microorganisms, which adapts to the current knowledge on the huge diversity, variability and heterogeneity existing among bacteria and archaea. Likely, this concept could be extended to eukaryotic microorganisms. The continuous species concept considers a species to be delimited by the distance between a range of variable features following a Gaussian-type distribution around a reference organism (i.e., its type strain). Some potential pros and cons of a continuous concept are commented on, offering novel perspectives on our understanding of the highly diversified prokaryotic world, thus promoting discussion and further investigation in the field.
RESUMO
Bistable expression of the Salmonella enterica std operon is controlled by an AND logic gate involving three transcriptional activators: the LysR-type factor HdfR and the StdE and StdF regulators encoded by the std operon itself. StdE activates transcription of the hdfR gene, and StdF activates std transcription together with HdfR. Binding of HdfR upstream of the std promoter is hindered by methylation of GATC sites located within the upstream activating sequence (UAS). Epigenetic control by Dam methylation thus antagonizes formation of the StdE-StdF-HdfR loop and tilts the std switch toward the StdOFF state. In turn, HdfR binding hinders methylation of the UAS, permitting activation of the StdE-StdF-HdfR loop and concomitant formation of StdON cells. Bistability is thus the outcome of competition between DNA adenine methylation and the StdE-StdF-HdfR activator loop.
Assuntos
Metilação de DNA , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Salmonella enterica/genética , Fatores de Transcrição/genética , Adenina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Salmonella enterica/metabolismo , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Cell-to-cell differences in bacterial gene expression can merely reflect the occurrence of noise. In certain cases, however, heterogeneous gene expression is a programmed event that results in bistable expression. If bistability is heritable, bacterial lineages are formed. When programmed bistability is reversible, the phenomenon is known as phase variation. In certain cases, bistability is controlled by genetic mechanisms (e. g., DNA rearrangement). In other cases, bistability has epigenetic origin. A robust epigenetic mechanism for the formation of bacterial lineages is the formation of heritable DNA methylation patterns. However, bistability can also arise upon propagation of gene expression patterns by feedback loops that are stable upon cell division. This review describes examples of bistability and phase variation in Salmonella enterica and discusses their adaptive value, sometimes in a speculative manner.
Assuntos
Proteínas de Bactérias/genética , Salmonella enterica/genética , Divisão Celular , Metilação de DNA , Epigênese Genética , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica , Rearranjo GênicoRESUMO
The std locus of Salmonella enterica, an operon acquired by horizontal transfer, encodes fimbriae that permit adhesion to epithelial cells in the large intestine. Expression of the std operon is bistable, yielding a major subpopulation of StdOFF cells (99.7%) and a minor subpopulation of StdON cells (0.3%). In addition to fimbrial proteins, the std operon encodes two proteins, StdE and StdF, that have DNA binding capacity and control transcription of loci involved in flagellar synthesis, chemotaxis, virulence, conjugal transfer, biofilm formation, and other cellular functions. As a consequence of StdEF pleiotropic transcriptional control, StdON and StdOFF subpopulations may differ not only in the presence or absence of Std fimbriae but also in additional phenotypic traits. Separation of StdOFF and StdON lineages by cell sorting confirms the occurrence of lineage-specific features. Formation of StdOFF and StdON lineages may thus be viewed as a rudimentary bacterial differentiation program.
Assuntos
Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Salmonella enterica/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Citometria de Fluxo , Transferência Genética Horizontal , Fenótipo , Análise de Célula ÚnicaRESUMO
Invasion of the intestinal epithelium is a critical step in Salmonella enterica infection and requires functions encoded in the gene cluster known as Salmonella Pathogenicity Island 1 (SPI-1). Expression of SPI-1 genes is repressed by L-arabinose, and not by other pentoses. Transport of L-arabinose is necessary to repress SPI-1; however, repression is independent of L-arabinose metabolism and of the L-arabinose-responsive regulator AraC. SPI-1 repression by L-arabinose is exerted at a single target, HilD, and the mechanism appears to be post-translational. As a consequence of SPI-1 repression, l-arabinose reduces translocation of SPI-1 effectors to epithelial cells and decreases Salmonella invasion in vitro. These observations reveal a hitherto unknown role of L-arabinose in gene expression control and raise the possibility that Salmonella may use L-arabinose as an environmental signal.
Assuntos
Arabinose/metabolismo , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Fator de Transcrição AraC/metabolismo , Salmonella enterica/metabolismo , Virulência/genéticaRESUMO
Long 3' untranslated regions (3'UTRs) are common in eukaryotic mRNAs. In contrast, long 3'UTRs are rare in bacteria, and have not been characterized in detail. We describe a 3'UTR of 310 nucleotides in hilD mRNA, a transcript that encodes a transcriptional activator of Salmonella enterica pathogenicity island 1 (SPI-1). Deletion of the hilD 3'UTR increases the hilD mRNA level, suggesting that the hilD 3'UTR may play a role in hilD mRNA turnover. Cloning of the hilD 3'UTR downstream of the green fluorescent protein (gfp) gene decreases green fluorescent protein (GFP) activity in both Escherichia coli and S. enterica, indicating that the hilD 3'UTR can act as an independent module. S. enterica mutants lacking either ribonuclease E or polynucleotide phosphorylase contain similar amounts of hilD and hilD Δ3'UTR mRNAs, suggesting that the hilD 3'UTR is a target for hilD mRNA degradation by the degradosome. The hilD 3'UTR is also necessary for modulation of hilD and SPI-1 expression by the RNA chaperone Hfq. Overexpression of SPI-1 in the absence of the hilD 3'UTR retards Salmonella growth and causes uncontrolled invasion of epithelial cells. Based on these observations, we propose that the S. enterica hilD 3'UTR is a cis-acting element that contributes to cellular homeostasis by promoting hilD mRNA turnover.
Assuntos
Proteínas de Bactérias/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Salmonella typhimurium/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas , Proteínas de Bactérias/metabolismo , Sequência de Bases , Endorribonucleases/fisiologia , Regulação Bacteriana da Expressão Gênica , Sequências Repetidas Invertidas , Dados de Sequência Molecular , Complexos Multienzimáticos/fisiologia , Polirribonucleotídeo Nucleotidiltransferase/fisiologia , RNA Helicases/fisiologia , Estabilidade de RNA , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Hfq is an RNA binding protein involved in posttranscriptional regulation of gene expression in bacteria. It acts by binding to regulatory small RNAs (sRNAs), which confer specificity for the regulation. Recently, orthologues of the Hfq protein were annotated in cyanobacterial genomes, although its capacity to regulate gene expression by interacting with sRNAs has not been yet demonstrated. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that, in the absence of combined nitrogen, is able to fix atmospheric nitrogen by differentiating specialized cells called heterocysts. We have generated an hfq knockout mutant of Anabaena sp. PCC 7120. Deletion of this gene results in differentiation of heterocysts in the presence of nitrate, suggesting a defect in nitrate assimilation. We show that hfq mutant cells are affected in transport and use of nitrate and nitrite. An analysis of the expression of several genes in the nir operon, encoding different elements of the nitrate assimilation pathway, demonstrates a downregulation of their transcription in mutant cells. We also observed that genes ntcB and cnaT, involved in the regulation of the nir operon, show a lower expression in cells lacking Hfq. Finally, when hfq was reintroduced in the mutant, heterocyst differentiation was no longer observed in the presence of nitrate. Therefore, our results indicate that the RNA chaperone Hfq is involved in the regulation of the nir operon, although the mechanism for this regulation is still unknown.
Assuntos
Anabaena/metabolismo , Proteínas de Bactérias/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Nitratos/metabolismo , Sequência de Aminoácidos , Anabaena/química , Anabaena/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/química , Fator Proteico 1 do Hospedeiro/genética , Dados de Sequência Molecular , Nitritos/metabolismo , Óperon , Alinhamento de SequênciaRESUMO
Ornate, large, extremophilic (OLE) RNAs are large, non-coding transcripts characterized by their ornate secondary structure and presence predominantly in Gram-positive, extremophilic bacteria. A gene for an OLE-associated protein (OAP) is almost always located immediately downstream of the OLE gene. OAP has no extensive homology to other proteins and is predicted to form multiple transmembrane domains. We show that this protein forms a ribonucleoprotein complex with OLE RNA using at least 2:1 protein : RNA stoichiometry. A series of truncated OLE RNA constructs was used to establish that most of the RNA can be deleted without eliminating protein binding. Two primary binding sites are present within the RNA, although additional binding determinants exist and extensive structural stabilization is induced by OAP. RNA fluorescence in situ hybridization (FISH) was used in Escherichia coli to demonstrate that ribonucleoprotein complex formation localizes the RNA near cell membranes of this heterologous system. Therefore, the majority of the complex structure formed by OLE RNA may perform a biochemical function that requires membrane localization.
Assuntos
Bactérias Anaeróbias/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , RNA não Traduzido/metabolismo , Ribonucleoproteínas/metabolismo , Bactérias Anaeróbias/química , Bactérias Anaeróbias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Membrana Celular/química , Membrana Celular/genética , Clonagem Molecular , Escherichia coli/química , Escherichia coli/genética , Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA não Traduzido/química , RNA não Traduzido/genética , Ribonucleoproteínas/química , Deleção de SequênciaRESUMO
The 5'-untranslated region (5'UTR) of the HIV-1 RNA is an attractive target for engineered ribozymes due to its high sequence and structural conservation. This region encodes several conserved structural RNA domains essential in key processes of the viral replication and infection cycles. This paper reports the inhibitory effects of catalytic antisense RNAs composed of two inhibitory RNA domains: an engineered ribozyme targeting the 5' UTR and a decoy or antisense domain of the dimerization initiation site (DIS). These chimeric molecules are able to cleave the HIV-1 5'UTR efficiently and prevent viral genome dimerization in vitro. Furthermore, catalytic antisense RNAs inhibited viral production up to 90% measured as p24 antigen levels in ex vivo assays. The use of chimeric RNA molecules targeting different domains represents an attractive antiviral strategy to be explored for the prevention of side effects from current drugs and of the rapid emergence of escape variants of HIV-1.
Assuntos
Fármacos Anti-HIV/síntese química , HIV-1/efeitos dos fármacos , RNA Catalítico/síntese química , Replicação Viral/efeitos dos fármacos , Regiões 5' não Traduzidas , Fármacos Anti-HIV/farmacologia , Dimerização , HIV-1/genética , HIV-1/fisiologia , Humanos , RNA Catalítico/farmacologia , RNA Catalítico/uso terapêutico , RNA Viral/efeitos dos fármacosRESUMO
We have discovered a large and highly conserved RNA motif that typically resides in a noncoding section of a multigene messenger RNA in extremophilic Gram-positive eubacteria. RNAs of this class adopt an ornate secondary structure, are large compared with most other noncoding RNAs, and have been identified only in certain extremophilic bacteria. These ornate, large, extremophilic (OLE) RNAs have a length of approximately 610 nucleotides, and the 35 representatives examined exhibit extraordinary conservation of nucleotide sequence and base pairing. Structural probing of the OLE RNA from Bacillus halodurans corroborates a complex secondary structure model predicted from comparative sequence analysis. The patterns of structural conservation, and its unique phylogenetic distribution, suggest that OLE RNA carries out a complex and critical function only in certain extremophilic bacteria.
Assuntos
Bactérias/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Sequência de Bases , Clonagem Molecular , Biologia Computacional , Primers do DNA , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
OBJECTIVE: The use of small RNA molecules able to effect gene inactivation has emerged as a powerful method of gene therapy. These small inhibitory RNAs are widely used for silencing malignant cellular and viral genes. We have assayed a series of inhibitory RNAs named catalytic antisense RNAs, consisting of a catalytic domain, hairpin or hammerhead ribozyme, and an antisense domain. The aim of the present study was to evaluate the effect of these inhibitory RNAs on HIV-1 replication. METHODS: A series of expression vectors has been constructed for the intracellular synthesis of inhibitory RNAs, differing in the promoter that drives their synthesis. These inhibitory RNAs were designed to act at two possible cleavage sites in the long terminal repeat (LTR) region and the TAR domain was chosen as a target for the antisense domain. We have evaluated the effects of different inhibitory RNAs in HIV replication via changes in p24 antigen levels. Mobility shift assays have been used to check the binding capacity of inhibitory RNAs. RESULTS: Catalytic antisense RNA designed to target the LTR region of HIV-1 inhibited viral replication in an eukaryotic cell environment by more than 90%. The conventional hairpin and hammerhead ribozymes, however, failed to inhibit viral replication. CONCLUSIONS: The data provide preliminary evidence of a new class of inhibitory RNAs that can be used to block HIV replication. The results clearly show the importance of the ex vivo antisense effect in the inhibition achieved. A good correlation was found between the in vitro binding efficiency of the inhibitor RNA to the HIV-1 LTR and the inhibition of viral replication.
Assuntos
Marcação de Genes/métodos , Repetição Terminal Longa de HIV/genética , HIV-1/fisiologia , Replicação Viral/genética , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Inativação Gênica , Vetores Genéticos , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/genética , Humanos , Regiões Promotoras Genéticas/genética , RNA Antissenso/biossíntese , RNA Antissenso/genética , RNA Catalítico/biossíntese , RNA Catalítico/genética , Células Tumorais CultivadasRESUMO
Hepatitis C virus (HCV) infection is one of the world's major health problems, and the identification of efficient HCV inhibitors is a major goal. Here we report the isolation of efficient anti-HCV internal ribosome entry site (IRES) RNA molecules identified by a new in vitro selection method. The newly developed procedure consists of two sequential steps that use distinct criteria for selection: selection for binding and selection for cleaving. The selection protocol was applied to a population of more than 10(15) variants of an anti-hepatitis C virus ribozyme covalently linked to an aptamer motif. The ribozyme was directed against positions 357 to 369 of the HCV IRES, and the cleavage substrate was a 691-nucleotide-long RNA fragment that comprises the entire HCV IRES domain. After six selection cycles, seven groups of RNA variants were identified. A representative of each group was tested for its capacity to inhibit IRES activity using in vitro translation assays. All selected RNAs promoted significant inhibition, some by as much as 95%.
Assuntos
Regiões 5' não Traduzidas , Hepacivirus/genética , RNA Catalítico/metabolismo , RNA Viral/metabolismo , Ribossomos/metabolismo , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Hepacivirus/efeitos dos fármacos , Hepacivirus/metabolismo , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , Conformação de Ácido Nucleico , Oligorribonucleotídeos/isolamento & purificação , Oligorribonucleotídeos/farmacologia , Biossíntese de Proteínas , RNA Catalítico/isolamento & purificação , RNA Viral/isolamento & purificaçãoRESUMO
An anti-Tat hairpin ribozyme and a TAR RNA decoy were combined in one molecule. The chimeric molecule strongly inhibited HIV-1 replication (measured as changes in p24 levels in viral replication assays). The inhibitory action of the ribodecozyme (85%) was significantly greater than that shown by ribozyme and a non-catalytic variant carrying the functional decoy RNA domain (55% and 35%, respectively). This represents a significant improvement of the inhibitory efficiency of the ribozyme, suggesting there is an additive inhibitory effect on HIV-1 replication by the catalytic and decoy domains. This strategy could be used to create new inhibitor RNAs with enhanced in vivo performance.
Assuntos
HIV-1/efeitos dos fármacos , HIV-1/fisiologia , RNA Catalítico/farmacologia , RNA Viral/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Sequência de Bases , Células Cultivadas , Desenho de Fármacos , Produtos do Gene tat/antagonistas & inibidores , Humanos , Dados de Sequência Molecular , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The proper selection of target sites and the correct design of specific ribozymes are decisive initial steps in any attempt to perform ribozyme-mediated gene silencing. Combinatorial methodologies can be used to improve ribozyme targeting and design. The in vitro selection strategy described in this chapter uses a combinatorial library of potentially self-cleaving RNA molecules. The hairpin ribozyme is attached to the target mRNA, and is adequately randomized to generate a population representing all possible substrate specificities. The selection procedure yields information on the best target sites, and provides information about optimal ribozyme sequences. Thus, this method helps in the rational design of efficient hairpin ribozymes for targeting purposes, and avoids trial-and-error assays usually associated with theoretical ribozyme design.
Assuntos
RNA Catalítico/biossíntese , Sequência de Bases , Sítios de Ligação , Inativação Gênica , Indicadores e Reagentes , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , RNA Catalítico/química , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
The hairpin ribozyme belongs to a group of small catalytic RNAs that have been extensively used to trans-cleave RNA molecules. Many efforts have been made to elucidate its reaction mechanism, and there is great interest in designing hairpin ribozymes with improved catalytic activity for use in the development of agents that specifically inactivate RNA molecules. This chapter summarizes the general principles in the design of hairpin ribozymes for targeting purposes, and provides a brief overview of the well-characterized modifications of the ribozyme sequences and structural domains that are necessary for optimal activity. The main features of the target sequence are also examined and other procedures or modifications of interest are also discussed.
Assuntos
RNA Catalítico/biossíntese , RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Indicadores e Reagentes , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , RNA/química , RNA Catalítico/química , Especificidade por Substrato , Transcrição GênicaRESUMO
The discovery 20 years ago that some RNA molecules, called ribozymes, are able to catalyze chemical reactions was a breakthrough in biology. Over the last two decades numerous natural RNA motifs endowed with catalytic activity have been described. They all fit within a few well-defined types that respond to a specific RNA structure. The prototype catalytic domain of each one has been engineered to generate trans-acting ribozymes that catalyze the site-specific cleavage of other RNA molecules. On the 20th anniversary of ribozyme discovery we briefly summarize the main features of the different natural catalytic RNAs. We also describe progress towards developing strategies to ensure an efficient ribozyme-based technology, dedicating special attention to the ones aimed to achieve a new generation of therapeutic agents.
Assuntos
RNA Catalítico/química , RNA Catalítico/metabolismo , Animais , Sequência de Bases , Terapia Genética/tendências , Genômica , Humanos , Íntrons/genética , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/classificação , RNA/fisiologia , RNA/uso terapêutico , Splicing de RNA , RNA Catalítico/uso terapêuticoRESUMO
Ribozymes have a great potential for developing specific gene silencing molecules. One of the main limitations to ensure the efficient application of ribozymes is to achieve effective binding to the target. Stem-loop domains support efficient formation of the kissing complex between natural antisense molecules and their target sequence. We have characterized catalytic antisense RNA hybrid molecules composed of a hammerhead ribozyme and a stem-loop antisense domain. A series of artificial RNA substrates containing the TAR-RNA stem-loop and a target for the hammerhead ribozyme were constructed and challenged with a catalytic antisense RNA carrying the TAR complementary stem-loop. The catalytic antisense RNA cleaves each of these substrates significantly more efficiently than the parental hammerhead ribozyme. Deletion of the TAR domain in the substrate abolishes the positive effect. These results suggest that the enhancement is due to the interaction of both complementary stem-loop motifs. A similar improvement was corroborated when targeting the LTR region of HIV-1 with either hammerhead- and hairpin-based catalytic antisense RNAs. Our results indicate that the TAR domain can be used as an anchoring site to facilitate the access of ribozymes to their specific target sequences within TAR-containing RNAs. Finally, we propose the addition of stable stem-loop motifs to the ribozyme domain as a rational way for constructing catalytic antisense RNAs.