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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.

The rhizosphere and rhizoplane are nutrient-rich but selective environments for the root microbiome. Here, we deciphered a posttranscriptional network regulated by the homologous trans-small RNAs (sRNAs) AbcR1 and AbcR2, which rewire the metabolism of the nitrogen-fixing a-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 s factor RpoH1. MS2 affinity purification coupled with RNA sequencing unveiled exceptionally large and overlapping AbcR1/2 mRNA interactomes, jointly representing;6% of the S. meliloti protein-coding genes. Most mRNAs encode transport/metabolic proteins whose translation is silenced by base pairing to two distinct anti-Shine Dalgarno motifs that function independently in both sRNAs. A metabolic model-aided analysis of the targetomes predicted changes in AbcR1/2 expression driven by shifts in carbon/nitrogen sources, which were confirmed experimentally. Low AbcR1/2 levels in some defined media anticipated overexpression growth phenotypes linked to the silencing of specific mRNAs. As a proof of principle, we confirmed AbcR1/2-mediated downregulation of the L-amino acid AapQ permease. AbcR1/2 interactomes are well represented in rhizosphere-related S. meliloti transcriptomic signatures. Remarkably, a lack of AbcR1 specifically compromised the ability of S. meliloti to colonize the root rhizoplane. The AbcR1 regulon likely ranks the utilization of available substrates to optimize metabolism, thus conferring on S. meliloti an advantage for efficient rhizosphere/rhizoplane colonization. AbcR1 regulation is predicted to be conserved in related a-rhizobia, which opens unprecedented possibilities for engineering highly competitive biofertilizers. IMPORTANCE Nitrogen-fixing root nodule symbioses

We report here the complete genome sequence of the salt-tolerant Sinorhizobium meliloti strain AK21, isolated from nodules of Medicago sativa L. subsp. ambigua inhabiting the northern Aral Sea Region. This genome (7.36 Mb) consists of a chromosome and four accessory plasmids, two of which are the symbiotic megaplasmids pSymA and pSymB.

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.

Small non-coding RNAs (sRNAs) are ubiquitous components of regulatory netwoks underlying virtually any adaptive response in bacteria. As regulators of gene expression, sRNAs accumulate differentially in the cell in response to external cues and their main activity mechanism involves protein-assisted base-pairing interactions with short and discontinuous stretches of complementary nucleotides usually mapping to the translation initiation region of multiple trans-encoded target mRNAs. In most cases, this antisense interaction promotes post-transcriptional gene silencing by blocking of translation and degradation of the target mRNAs by cellular RNases (reviewed in Jiménez-Zurdo et al. 2013). In our laboratory, we are interested in the characterization of the non-coding transcriptome of the alfalfa symbiont Sinorhizobium meliloti. Deletion of one of the hundreds of sRNA genes identified in this bacterium alters nodule organogenesis and compromises nitrogen-fixation efficiency on alfalfa roots (Robledo et al, 2017). According to this symbiotic phenotype, this sRNA was named NfeR1 (Nodule Formation Efficiency RNA). Computational predictions, some of which have been verified experimentally, suggested that NfeR1 uses redundantly three identical anti-Shine Dalgarno (aSD) motifs for targeting and down-regulation of multiple mRNAs coding for the periplasmic component of ABC transport systems. Northern blot probing and transcription tracking with promoter-eGFP fusions uncovered maximum NfeR1 accumulation upon an osmotic upshift in free-living bacteria and during all steps of the symbiotic interaction. The strength and differential regulation of nfeR1 transcription is conferred by a motif that is conserved in the promoter regions of its ¿-proteobacterial homologs. Pull-down assays revealed that the NfeR1 promoter might bind the global nitrogen regulator NtrC and the LysR type transcriptional regulator LsrB (LysR Symbiotic Regulator). In the communication, we will provide novel i

Primary infection of legumes by rhizobia involves the controlled localized enzymatic breakdown of cell walls at root hair tips. Previous studies determined the role of rhizobial CelC2 cellulase in different steps of the symbiotic interaction Rhizobium leguminosarum-Trifolium repens. Recent findings also showed that CelC2 influences early signalling events in the Ensifer meliloti-Medicago truncatula interaction. Here, we have monitored the root hair phenotypes of two legume plants, T. repens and M. sativa, upon inoculation with strains of their cognate and non-cognate rhizobial species, R. leguminosarum bv trifolii and E. meliloti, (over)expressing the CelC2 coding gene, celC. Regardless of the host, CelC2 specifically elicited ‘hole-on-the-tip’ events (Hot phenotype) in the root hair apex, consistent with the role of this endoglucanase in eroding the noncrystalline cellulose found in polarly growing cell walls. Overproduction of CelC2 also increased root hair tip redirections (RaT phenotype) events in both cognate and non-cognate hosts. Interestingly, heterologous celC expression also induced non-canonical alterations in ROS (Reactive Oxygen Species) homeostasis at root hair tips of Trifolium and Medicago. These results suggest the concurrence of shared unspecific and host-related plant responses to CelC2 during early steps of symbiotic rhizobial infection. Our data thus identify CelC2 cellulase as an important determinant of events underlying early infection of the legume host by rhizobia.

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 a-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans-encoded sRNA (trans-sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving the

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 a-proteobacterium Sinorhizobium meliloti. The S. meliloti rnc gene does encode an RNase III-like protein (SmRNase III), with recognizable catalytic and double-stranded RNA (dsRNA)-binding domains that clusters in a branch with its a-proteobacterial counterparts. Purified SmRNase III dimerizes, is active at neutral to alkaline pH and behaves as a strict metal cofactor-dependent double-strand endoribonuclease, with catalytic features distinguishable from those of the prototypical member of the family, the Escherichia coli ortholog (EcRNase III). SmRNase III prefers Mn2+ rather than Mg as metal cofactor, cleaves the generic structured R1.1 substrate at a site atypical for RNase III cleavage, and requires higher cofactor concentrations and longer dsRNA substrates than EcRNase III for optimal activity. Furthermore, the ultraconserved E125 amino acid was shown to play a major role in the metal-dependent catalysis of SmRNase III. SmRNase III degrades endogenous RNA substrates of diverse biogenesis with different efficiency, and is involved in the maturation of the 23S rRNA. SmRNase III loss-of-function neither compromises viability nor alters morphology of S. meliloti cells, but influences growth, nodulation kinetics, the onset of nitrogen fixation and the overall symbiotic efficiency of this bacterium on the roots of its legume host, alfalfa, which ultimately affects plant growth. Our results support an impact of SmRNase III on nodulation and symbiotic nitrogen fixation in plants.

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 Ca 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.

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.

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).

[Background] 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. Sinorhizobium meliloti 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 S. meliloti has remained unexplored., [Results] Two independent S. meliloti mutants, 2011-3.4 and 1021Deltahfq, were obtained by disruption and deletion of the hfq gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1021Deltahfq 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 1021Deltahfq 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 nifA and fixK1/K2, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of fixK is only aerobiosis dependent. Finally, we found that some of the recently identified S. meliloti sRNAs co-inmunoprecipitate with a FLAG-epitope tagged Hfq protein., [Conclusions] Our results support that the S. meliloti RNA chaperone Hfq underlies 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 S. meliloti Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network.