A Workflow for the Functional Characterization of Noncoding RNAs in Legume Symbiotic Bacteria.

Computational comparative genomics and, later, high-throughput transcriptome profiling (RNAseq) have uncovered a plethora of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. A large fraction of sRNAs are differentially regulated in response to different biotic and abiotic stimuli and have the ability to fine-tune posttranscriptional reprogramming of gene expression through protein-assisted antisense interactions with trans-encoded target mRNAs. However, this level of gene regulation is still understudied in most non-model bacteria.

An Integrated Affinity Chromatography-Based Approach to Unravel the sRNA Interactome in Nitrogen-Fixing Rhizobia.

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.

A double-negative feedback loop between NtrBC and a small RNA rewires nitrogen metabolism in legume symbionts.

Root nodule endosymbioses between diazotrophic rhizobia and legumes provide the largest input of combined N to the biosphere, thus representing an alternative to harmful chemical fertilizers for sustainable crop production. Rhizobia have evolved intricate strategies to coordinate N assimilation for their own benefit with N fixation to sustain plant growth. The rhizobial N status is transduced by the NtrBC two-component system, the seemingly ubiquitous form of N signal transduction in Proteobacteria.

Pervasive RNA Regulation of Metabolism Enhances the Root Colonization Ability of Nitrogen-Fixing Symbiotic α-Rhizobia.

The rhizosphere and rhizoplane are nutrient-rich but selective environments for the root microbiome. Here, we deciphered a posttranscriptional network regulated by the homologous -small RNAs (sRNAs) AbcR1 and AbcR2, which rewire the metabolism of the nitrogen-fixing α-rhizobium Sinorhizobium meliloti during preinfection stages of symbiosis with its legume host alfalfa. The LysR-type regulator LsrB, which transduces the cell redox state, is indispensable for AbcR1 expression in actively dividing bacteria, whereas the stress-induced transcription of AbcR2 depends on the alternative σ factor RpoH1.

Synthetase of the methyl donor S-adenosylmethionine from nitrogen-fixing α-rhizobia can bind functionally diverse RNA species.

Function of bacterial small non-coding RNAs (sRNAs) and overall RNA metabolism is largely shaped by a vast diversity of RNA-protein interactions. However, in non-model bacteria with defined non-coding transcriptomes the sRNA interactome remains almost unexplored. We used affinity chromatography to capture proteins associated with MS2-tagged -sRNAs that regulate nutrient uptake (AbcR2 and NfeR1) and cell cycle (EcpR1) mRNAs by antisense-based translational inhibition in the nitrogen-fixing α-rhizobia .

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.

Complete Genome Sequence of Sinorhizobium meliloti Strain AK21, a Salt-Tolerant Isolate from the Aral Sea Region.

We report here the complete genome sequence of the salt-tolerant strain AK21, isolated from nodules of L. subsp. inhabiting the northern Aral Sea Region.

RNase III: Catalytic Features and Impact on Symbiosis.

Members of the ribonuclease (RNase) III family of enzymes are metal-dependent double-strand specific endoribonucleases. They are ubiquitously found and eukaryotic RNase III-like enzymes include Dicer and Drosha, involved in RNA processing and RNA interference. In this work, we have addressed the primary characterization of RNase III from the symbiotic nitrogen-fixing α-proteobacterium .

An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA.

Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation.

Identification of Small RNA-Protein Partners in Plant Symbiotic Bacteria.

The identification of the protein partners of bacterial small noncoding RNAs (sRNAs) is essential to understand the mechanistic principles and functions of riboregulation in prokaryotic cells. Here, we describe an optimized affinity chromatography protocol that enables purification of in vivo formed sRNA-protein complexes in Sinorhizobium meliloti, a genetically tractable nitrogen-fixing plant symbiotic bacterium. The procedure requires the tagging of the desired sRNA with the MS2 aptamer, which is affinity-captured by the MS2-MBP protein conjugated to an amylose resin.

Heterologous Expression of Rhizobial CelC2 Cellulase Impairs Symbiotic Signaling and Nodulation in Medicago truncatula.

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.

Primary Characterization of Small RNAs in Symbiotic Nitrogen-Fixing Bacteria.

High-throughput transcriptome profiling (RNAseq) has uncovered large and heterogeneous populations of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. A large fraction of sRNAs are differentially regulated and rely on protein-assisted antisense interactions to trans-encoded target mRNAs to fine-tune posttranscriptional reprogramming of gene expression in response to external cues. However, annotation and function of sRNAs are still largely overlooked in nonmodel bacteria with complex lifestyles.

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.

A conserved α-proteobacterial small RNA contributes to osmoadaptation and symbiotic efficiency of rhizobia on legume roots.

Small non-coding RNAs (sRNAs) are expected to have pivotal roles in the adaptive responses underlying symbiosis of nitrogen-fixing rhizobia with legumes. Here, we provide primary insights into the function and activity mechanism of the Sinorhizobium meliloti trans-sRNA NfeR1 (Nodule Formation Efficiency RNA). Northern blot probing and transcription tracking with fluorescent promoter-reporter fusions unveiled high nfeR1 expression in response to salt stress and throughout the symbiotic interaction.

Sinorhizobium meliloti YbeY is an endoribonuclease with unprecedented catalytic features, acting as silencing enzyme in riboregulation.

Structural and biochemical features suggest that the almost ubiquitous bacterial YbeY protein may serve catalytic and/or Hfq-like protective functions central to small RNA (sRNA)-mediated regulation and RNA metabolism. We have biochemically and genetically characterized the YbeY ortholog of the legume symbiont Sinorhizobium meliloti (SmYbeY). Co-immunoprecipitation (CoIP) with a FLAG-tagged SmYbeY yielded a poor enrichment in RNA species, compared to Hfq CoIP-RNA uncovered previously by a similar experimental setup.

Unraveling the effect of arsenic on the model Medicago-Ensifer interaction: a transcriptomic meta-analysis.

The genetic regulation underlying the effect of arsenic (As(III)) on the model symbiosis Medicago-Ensifer was investigated using a combination of physiological (split-roots), microscopy and genetic (microarrays, qRT-PCR and composite plants) tools. Nodulation was very sensitive to As(III) (median inhibitory dose (ID50) = 20 μM). The effect on root elongation and on nodulation was local (nonsystemic).

Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons in Sinorhizobium meliloti.

The RNA chaperone Hfq is a global post-transcriptional regulator in bacteria. Here, we used RNAseq to analyze RNA populations from the legume symbiont Sinorhizobium meliloti that were co-immunoprecipitated (CoIP-RNA) with a FLAG-tagged Hfq in five growth/stress conditions. Hfq-bound transcripts (1315) were largely identified in stressed bacteria and derived from small RNAs (sRNAs), both trans-encoded (6.

Rhizobial galactoglucan determines the predatory pattern of Myxococcus xanthus and protects Sinorhizobium meliloti from predation.

Myxococcus xanthus is a social bacterium that preys on prokaryotic and eukaryotic microorganisms. Co-culture of M. xanthus with reference laboratory strains and field isolates of the legume symbiont Sinorhizobium meliloti revealed two different predatory patterns that resemble frontal and wolf-pack attacks.

Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

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.

Independent activity of the homologous small regulatory RNAs AbcR1 and AbcR2 in the legume symbiont Sinorhizobium meliloti.

The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure.

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).

Insights into the noncoding 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.

Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and biofilm formation on plant roots and abiotic surfaces.

Background: The synthesis of cellulose is among the most important but poorly understood biochemical processes, especially in bacteria, due to its complexity and high degree of regulation. In this study, we analyzed both the production of cellulose by all known members of the Rhizobiaceae and the diversity of Rhizobium celABC operon predicted to be involved in cellulose biosynthesis. We also investigated the involvement in cellulose production and biofilm formation of celC gene encoding an endoglucanase (CelC2) that is required for canonical symbiotic root hair infection by Rhizobium leguminosarum bv.

A survey of sRNA families in α-proteobacteria.

We have performed a computational comparative analysis of six small non-coding RNA (sRNA) families in α-proteobacteria. Members of these families were first identified in the intergenic regions of the nitrogen-fixing endosymbiont S. meliloti by a combined bioinformatics screen followed by experimental verification.

Development of functional symbiotic white clover root hairs and nodules requires tightly regulated production of rhizobial cellulase CelC2.

The establishment of rhizobia as nitrogen-fixing endosymbionts within legume root nodules requires the disruption of the plant cell wall to breach the host barrier at strategic infection sites in the root hair tip and at points of bacterial release from infection threads (IT) within the root cortex. We previously found that Rhizobium leguminosarum bv. trifolii uses its chromosomally encoded CelC2 cellulase to erode the noncrystalline wall at the apex of root hairs, thereby creating the primary portal of its entry into white clover roots.