Complete Genome Sequence of the Alfalfa Symbiont Sinorhizobium/ Ensifer meliloti Strain GR4

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).This work was supported by research grants BIO2011-24401 from the Spanish Ministerio de Ciencia e Innovación and CSD 2009-0006 of Programme Consolider-Ingenio, both including ERDF (European Regional Development Funds). L.M.-R. and J.A.L.-C. were supported by predoctoral fellowships (Programs JAE-Predoc and I3P from Consejo Superior de Investigaciones Científicas, respectively). We are particularly grateful to M. G. Claros and R. Bautista from Plataforma Andaluza de Bioinformática (Universidad de Málaga) Spain, to A. J. Fernández-González for bioinformatics support, and to the Estación Experimental del Zaidín—CSIC for DNA sequencing services.Peer reviewe

Dispersion of the RmInt1 group II intron in the Sinorhizobium meliloti genome upon acquisition by conjugative transfer

RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase–maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (ΔORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic ΔORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature

The Sinorhizobium meliloti RNA chaperone Hfq influences central carbon metabolism and the symbiotic interaction with alfalfa

<p>Abstract</p> <p>Background</p> <p>The bacterial Hfq protein is able to interact with diverse RNA molecules, including regulatory small non-coding RNAs (sRNAs), and thus it is recognized as a global post-transcriptional regulator of gene expression. Loss of Hfq has an extensive impact in bacterial physiology which in several animal pathogens influences virulence. <it>Sinorhizobium meliloti </it>is a model soil bacterium known for its ability to establish a beneficial nitrogen-fixing intracellular symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the establishment of successful bacteria-eukaryote interactions, its function in <it>S. meliloti </it>has remained unexplored.</p> <p>Results</p> <p>Two independent <it>S. meliloti </it>mutants, 2011-3.4 and 1021Δ<it>hfq</it>, were obtained by disruption and deletion of the <it>hfq </it>gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1021Δ<it>hfq </it>revealed a general down-regulation of genes of sugar transporters and some enzymes of the central carbon metabolism, whereas transcripts specifying the uptake and metabolism of nitrogen sources (mainly amino acids) were more abundant than in the wild-type strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these observations. Symbiotic tests showed that lack of Hfq led to a delayed nodulation, severely compromised bacterial competitiveness on alfalfa roots and impaired normal plant growth. Furthermore, a large proportion of nodules (55%-64%) elicited by the 1021Δ<it>hfq </it>mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and microoxic conditions revealed that Hfq contributes to regulation of <it>nifA </it>and <it>fixK1/K2</it>, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of <it>fixK </it>is only aerobiosis dependent. Finally, we found that some of the recently identified <it>S. meliloti </it>sRNAs co-inmunoprecipitate with a FLAG-epitope tagged Hfq protein.</p> <p>Conclusions</p> <p>Our results support that the <it>S. meliloti </it>RNA chaperone Hfq contributes to the control of central metabolic pathways in free-living bacteria and influences rhizospheric competence, survival of the microsymbiont within the nodule cells and nitrogen fixation during the symbiotic interaction with its legume host alfalfa. The identified <it>S. meliloti </it>Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network.</p

Structure and function of the alfa-rhizobia non-coding transcriptome investigated by RNAseq

Conferencia presentada en: I Spanish-Portuguese Congress on Beneficial Plant-Microbe Interactions (BeMiPlant) and XVIII National Meeting of the Spanish Society of Nitrogen Fixation (XVIII SEFIN). Oeiras, Portugal, 17-19 octubre (2022)This work was supported by grants BFU2017-82645-P and PID2020-114782GB-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” (BFU2017-82645-P), and grant P20_00185 funded by Junta de Andalucía PAIDI/FEDER/EU, awarded to J.I.J.-Z., and by grant US-1250546 funded by FEDER/Universidad de Sevilla to J.M.V

Bacteria-inducing legume nodules involved in the improvement of plant growth, health and nutrition

Bacteria-inducing legume nodules are known as rhizobia and belong to the class Alphaproteobacteria and Betaproteobacteria. They promote the growth and nutrition of their respective legume hosts through atmospheric nitrogen fixation which takes place in the nodules induced in their roots or stems. In addition, rhizobia have other plant growth-promoting mechanisms, mainly solubilization of phosphate and production of indoleacetic acid, ACC deaminase and siderophores. Some of these mechanisms have been reported for strains of rhizobia which are also able to promote the growth of several nonlegumes, such as cereals, oilseeds and vegetables. Less studied are the mechanisms that have the rhizobia to promote the plant health; however, these bacteria are able to exert biocontrol of some phytopathogens and to induce the plant resistance. In this chapter, we revised the available data about the ability of the legume nodule-inducing bacteria for improving the plant growth, health and nutrition of both legumes and nonlegumes. These data showed that rhizobia meet all the requirements of sustainable agriculture to be used as bio-inoculants allowing the total or partial replacement of chemicals used for fertilization or protection of crops

RNA silencing in plant symbiotic bacteria: Insights from a protein-centric view

Extensive work in model enterobacteria has evidenced that the RNA chaperone Hfq and several endoribonucleases, such as RNase E or RNase III, serve pivotal roles in small RNA-mediated post-transcriptional silencing of gene expression. Characterization of these protein hubs commonly provide global functional and mechanistic insights into complex sRNA regulatory networks. The legume endosymbiont Sinorhizobium meliloti is a non-classical model bacterium with a very complex lifestyle in which riboregulation is expected to play important adaptive functions. Here, we discuss current knowledge about RNA silencing in S. meliloti from the perspective of the activity of Hfq and a recently discovered endoribonuclease (YbeY) exhibiting unprecedented catalytic versatility for the cleavage of single- and double-stranded RNA molecules.This work was funded by the Spanish Ministerio de Economía y Competitividad ERDF-cofinanced grant BFU2013–48282-C2–2-P. M.R. was funded by a contract of the program of “Formacion Post-doctoral” from the same Ministry

Riboregulation in Nitrogen-Fixing Endosymbiotic Bacteria

Small non-coding RNAs (sRNAs) are ubiquitous components of bacterial adaptive regulatory networks underlying stress responses and chronic intracellular infection of eukaryotic hosts. Thus, sRNA-mediated regulation of gene expression is expected to play a major role in the establishment of mutualistic root nodule endosymbiosis between nitrogen-fixing rhizobia and legume plants. However, knowledge about this level of genetic regulation in this group of plant-interacting bacteria is still rather scarce. Here, we review insights into the rhizobial non-coding transcriptome and sRNA-mediated post-transcriptional regulation of symbiotic relevant traits such as nutrient uptake, cell cycle, quorum sensing, or nodule development. We provide details about the transcriptional control and protein-assisted activity mechanisms of the functionally characterized sRNAs involved in these processes. Finally, we discuss the forthcoming research on riboregulation in legume symbionts

Método para la insercción eficiente en sitios especificos de cualquier ADN diana de intrones del grupo II.

Fecha de solicitud: 24.01.2002.- Titular: Consejo Superior de Investigaciones Científicas (CSIC).[EN]The invention relates to a method for inserting a nucleic acid molecule into any specific target DNA sites. The method comprises the following steps: the introduction of the group II intron RNA which contains two nucleotide sequences capable of hybridising with two sequences contained in one of the strands of the DNA substrate; the modification of the intron RNA sequences, which hybridise with the substrate DNA, such that the specific sequences in one of the strands of a given DNA substrate can be identified; and the addition of a protein coded by the intron which, in its wild form, has no endonuclease domain. The reengineered group II intron is suitable for any in vivo or in vitro application and particularly for mutagenesis. [ES]El método permite la inserción de una molécula de ácido nucleico en sitios específicos de cualquier ADN diana. El método consiste en: la disposición del ARN de un intrón del grupo II que contenga dos secuencias nucleotídicas capaces de hibridar con dos secuencias contenidas en una de las hebras del ADN sustrato la modificación de las secuencias del ARN del intrón, que hibridan con el ADN sustrato, de tal manera que reconozcan secuencias específicas en una de las hebras de un sustrato dado de ADN y por último, aportar una proteína codificada por el intrón que en su forma silvestre carece del dominio endonucleasa. El intrón del grupo II rediseñado, puede emplearse en cualquier aplicación in vivo o in vitro, y en particular en mutagénesis.Peer reviewe

Insights into the non-coding RNome of nitrogen-fixing endosymbiotic α-proteobacteria

Symbiotic chronic infection of legumes by rhizobia involves transition of invading bacteria from a free-living environment in soil to an intracellular state as differentiated nitrogen-fixing bacteroids within the nodules elicited in the host plant. The adaptive flexibility demanded by this complex lifestyle is likely facilitated by the large set of regulatory proteins encoded by rhizobial genomes. However, proteins are not the only relevant players in the regulation of gene expression in bacteria. Large-scale high-throughput analysis of prokaryotic genomes is evidencing the expression of an unexpected plethora of small untranslated transcripts (sRNAs) with housekeeping or regulatory roles. sRNAs mostly act in response to environmental cues as post-transcriptional regulators of gene expression through protein-assisted base-pairing interactions with target mRNAs. Riboregulation contributes to fine-tune a wide range of bacterial processes which, in intracellular animal pathogens, largely compromise virulence traits. Here we summarize the incipient knowledge about the non-coding RNome structure of nitrogen-fixing endosymbiotic bacteria as inferred from genome-wide searches for sRNA genes in the alfalfa partner Sinorhizobium meliloti and further comparative genomics analysis. The biology of relevant S. meliloti RNA chaperones (e.g. Hfq) is also reviewed as a first global indicator of the impact of riboregulation in the establishment of the symbiotic interaction.Fil: Jiménez Zurdo, José I.. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; EspañaFil: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Becker, Anke. Philipps-Universität Marburg; Alemani