RESUMO
The activity mechanism and function of bacterial base-pairing small non-coding RNA regulators (sRNAs) are largely shaped by their main interacting cellular partners, i.e., proteins and mRNAs. We describe here an MS2 affinity chromatography-based procedure adapted to unravel the sRNA interactome in nitrogen-fixing legume endosymbiotic bacteria. The method consists of tagging of the bait sRNA at its 5'-end with the MS2 aptamer followed by pulse overexpression and immobilization of the chimeric transcript from cell lysates by an MS2-MBP fusion protein conjugated to an amylose resin. The sRNA-binding proteins and target mRNAs are further profiled by mass spectrometry and RNAseq, respectively.
Assuntos
Bactérias Fixadoras de Nitrogênio , Pequeno RNA não Traduzido , Rhizobium , Pequeno RNA não Traduzido/genética , Rhizobium/genética , Rhizobium/metabolismo , Nitrogênio/metabolismo , Bactérias/genética , Bactérias Fixadoras de Nitrogênio/genética , Cromatografia de Afinidade/métodos , RNA Bacteriano/genética , Regulação Bacteriana da Expressão GênicaRESUMO
Bacteria are powerful models for understanding how cells divide and accomplish global regulatory programs. In Caulobacter crescentus, a cascade of essential master regulators supervises the correct and sequential activation of DNA replication, cell division, and development of different cell types. Among them, the response regulator CtrA plays a crucial role coordinating all those functions. Here, for the first time, we describe the role of a novel factor named CcnA (cell cycle noncoding RNA A), a cell cycle-regulated noncoding RNA (ncRNA) located at the origin of replication, presumably activated by CtrA, and responsible for the accumulation of CtrA itself. In addition, CcnA may be also involved in the inhibition of translation of the S-phase regulator, GcrA, by interacting with its 5' untranslated region (5' UTR). Performing in vitro experiments and mutagenesis, we propose a mechanism of action of CcnA based on liberation (ctrA) or sequestration (gcrA) of their ribosome-binding site (RBS). Finally, its role may be conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, representing indeed a potentially conserved process modulating cell cycle in Caulobacterales and Rhizobiales.
Assuntos
Caulobacter crescentus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Regiões Promotoras Genéticas , RNA não Traduzido/genética , Fatores de Transcrição/metabolismoRESUMO
The infection of legume plants by rhizobia is tightly regulated to ensure accurate bacterial penetration, infection, and development of functionally efficient nitrogen-fixing root nodules. Rhizobial Nod factors (NF) have key roles in the elicitation of nodulation signaling. Infection of white clover roots also involves the tightly regulated specific breakdown of the noncrystalline apex of cell walls in growing root hairs, which is mediated by Rhizobium leguminosarum bv. trifolii cellulase CelC2. Here, we have analyzed the impact of this endoglucanase on symbiotic signaling in the model legume Medicago truncatula. Ensifer meliloti constitutively expressing celC gene exhibited delayed nodulation and elicited aberrant ineffective nodules, hampering plant growth in the absence of nitrogen. Cotreatment of roots with NF and CelC2 altered Ca2+ spiking in root hairs and induction of the early nodulin gene ENOD11. Our data suggest that CelC2 alters early signaling between partners in the rhizobia-legume interaction.
Assuntos
Medicago truncatula/efeitos dos fármacos , Medicago truncatula/microbiologia , Nodulação/fisiologia , Rhizobiaceae/metabolismo , Transdução de Sinais/efeitos dos fármacos , beta-Glucosidase/metabolismo , Medicago truncatula/metabolismo , Nodulação/efeitos dos fármacos , SimbioseRESUMO
We present the complete nucleotide sequence of the multipartite genome of Sinorhizobium/Ensifer meliloti GR4, a predominant rhizobial strain in an agricultural field site. The genome (total size, 7.14 Mb) consists of five replicons: one chromosome, two expected symbiotic megaplasmids (pRmeGR4c and pRmeGR4d), and two accessory plasmids (pRmeGR4a and pRmeGR4b).
RESUMO
Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.
Assuntos
Arabidopsis/genética , Proteínas de Bactérias/metabolismo , Núcleo Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Íntrons/genética , Splicing de RNA/genética , Sinorhizobium meliloti/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/microbiologia , Proteínas de Bactérias/genética , Núcleo Celular/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mutação/genética , Protoplastos/metabolismo , Protoplastos/microbiologia , RNA Bacteriano/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinorhizobium meliloti/crescimento & desenvolvimento , Spliceossomos/genética , Frações SubcelularesRESUMO
Group II introns are both catalytic RNAs (ribozymes) and mobile retroelements that were discovered almost 14 years ago. It has been suggested that eukaryotic mRNA introns might have originated from the group II introns present in the alphaproteobacterial progenitor of the mitochondria. Bacterial group II introns are of considerable interest not only because of their evolutionary significance, but also because they could potentially be used as tools for genetic manipulation in biotechnology and for gene therapy. This review summarizes what is known about the splicing mechanisms and mobility of bacterial group II introns, and describes the recent development of group II intron-based gene-targetting methods. Bacterial group II intron diversity, evolutionary relationships, and behaviour in bacteria are also discussed.
