Validation of the predicted EcpR1 binding sites in the <i>gcrA</i> and <i>dnaA</i> mRNAs.

<p>Morphological phenotype <b>(A)</b> and DNA content <b>(B)</b> of Rm4011<i>ecpR1</i> overexpressing <i>ecpR1-2</i> carrying 2 nt exchanges in the predicted interaction region. The bar represents 2 μm. <b>(C, D)</b> Predicted duplexes between EcpR1 and either <i>gcrA</i> or <i>dnaA</i> mRNAs. Numbers denote positions relative to the AUG start codon of the mRNA and the second 5’-end of EcpR1. The predicted energy score (E) is indicated in kcal/mol. The nucleotide exchanges in the mRNAs of <i>gcrA</i> (<i>gcrA</i>-BS-<i>egfp</i>) and <i>dnaA</i> (p<i>dnaA</i>-BSs-<i>egfp</i>) as well as in EcpR1 (EcpR1-2) are indicated in bold. <b>(E, F)</b> Fluorescence measurements of 4011<i>ecpR1</i> co-transformed with <i>ecpR1</i>, <i>ecpR1-2</i>, or control SmelC812 overexpression plasmids and the indicated reporter plasmids. Reporter constructs carried either native mRNA sequences derived from <i>gcrA</i> or <i>dnaA</i> or variants with mutations in predicted EcpR1 binding sites (BS). Fragments are delineated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005153#pgen.1005153.g004" target="_blank">Fig 4</a>. Reporter construct activities were determined as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005153#pgen.1005153.g004" target="_blank">Fig 4</a>.</p

GcrA depletion phenotype in <i>S</i>. <i>meliloti</i>.

<p>qRT-PCR analysis of <i>gcrA</i> transcript abundance and colony forming units <b>(A)</b>, growth rate <b>(B)</b>, morphology phenotypes <b>(C)</b> and DNA content <b>(D)</b> of Rm2011<i>gcrA</i>-P_lac<i>gcrA</i> subjected to different IPTG concentrations for 16 hours. qRT-PCR values were normalized to the SMc01852 transcript and <i>gcrA</i> levels in overnight cultures of Rm2011. 1C and 2C indicate one and two genome equivalents, respectively. Error bars indicate the standard deviation. Bars denote 2 μm.</p

qRT-PCR based verification of putative EcpR1 target genes displaying changes in transcript levels upon overproduction of EcpR1 as detected by global transcriptome profiling.

<p>Log₂ change in transcript amount normalized to levels of the SMc01852 mRNA. Errors represent the standard deviation of three replicates. Positions of microarray reporter oligonucleotides relative to the start codon are given in brackets for 5’-UTR regions.</p><p>*Description of gene product or associated gene product.</p><p>qRT-PCR based verification of putative EcpR1 target genes displaying changes in transcript levels upon overproduction of EcpR1 as detected by global transcriptome profiling.</p

A Stress-Induced Small RNA Modulates Alpha-Rhizobial Cell Cycle Progression

<div><p>Mechanisms adjusting replication initiation and cell cycle progression in response to environmental conditions are crucial for microbial survival. Functional characterization of the <i>trans</i>-encoded small non-coding RNA (<i>trans</i>-sRNA) EcpR1 in the plant-symbiotic alpha-proteobacterium <i>Sinorhizobium meliloti</i> revealed a role of this class of riboregulators in modulation of cell cycle regulation. EcpR1 is broadly conserved in at least five families of the Rhizobiales and is predicted to form a stable structure with two defined stem-loop domains. In <i>S</i>. <i>meliloti</i>, this <i>trans</i>-sRNA is encoded downstream of the <i>divK-pleD</i> operon. <i>ecpR1</i> belongs to the stringent response regulon, and its expression was induced by various stress factors and in stationary phase. Induced EcpR1 overproduction led to cell elongation and increased DNA content, while deletion of <i>ecpR1</i> resulted in reduced competitiveness. Computationally predicted EcpR1 targets were enriched with cell cycle-related mRNAs. Post-transcriptional repression of the cell cycle key regulatory genes <i>gcrA</i> and <i>dnaA</i> mediated by mRNA base-pairing with the strongly conserved loop 1 of EcpR1 was experimentally confirmed by two-plasmid differential gene expression assays and compensatory changes in sRNA and mRNA. Evidence is presented for EcpR1 promoting RNase E-dependent degradation of the <i>dnaA</i> mRNA. We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions.</p></div

Elongated cell phenotype induced by <i>ecpR1</i> overexpression.

<p><b>(A)</b> Northern blot detection of EcpR1 RNA variants in Rm4011 strains carrying either pSKControl⁺ (Control⁺), pSKEcpR1⁺ (EcpR1⁺), or pSKEcpR1-2⁺ (EcpR1-2⁺) 4 hours after induction with IPTG. Below, relative hybridization signals derived from the 101 nt EcpR1 species are plotted. The wild type level of EcpR1 in Control⁺ cells (OD₆₀₀ of ~0.9) has been normalized to 1 (dashed line) and the sRNA levels in other conditions are correlated to that value. Mean results from three experiments are shown. Error bars indicate the standard deviation. <b>(B)</b> Cell morphology, <b>(C)</b> motility assay, <b>(D)</b> cell length, and <b>(E)</b> DNA content of <i>S</i>. <i>meliloti</i> strains overexpressing <i>ecpR1</i> or the SmelC812 control antisense RNA gene. The 2011<i>visN</i> mutant was used as negative control for swimming motility. 1C and 2C indicate one and two genome equivalents, respectively. Bars correspond to 2 μm in <i>B</i> and 5 mm in <i>C</i>. Error bars in <i>D</i> represent standard errors (n = 100 cells).</p

<i>ecpR1</i> genomic locus and transcriptional regulation.

<p><b>(A)</b> Secondary structure of the dominant EcpR1 101 nt variant with a minimum free energy of -50.20 kcal/mol. Nucleotide positions relative to the second 5’-end are denoted. SL, stem loop domain. The 13 nt region predicted to bind the <i>gcrA</i> mRNA is boxed. Below, chromosomal region including the <i>ecpR1</i> gene and RNAseq coverage profile of the EcpR1 sRNA in <i>S</i>. <i>meliloti</i> Rm1021. Genome coordinates of the full length <i>ecpR1</i> variant are denoted. Black and grey areas represent coverages from samples enriched for processed and primary transcripts, respectively [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005153#pgen.1005153.ref021" target="_blank">21</a>]. Detected EcpR1 5’-ends are depicted by arrows and the dominant 101 nt EcpR1 variant used for structure prediction is marked by the bar. <b>(B)</b> Schematic representation of the fragments included in the <i>ecpR1</i> transcriptional fusions and fluorescence values of stationary phase Rm2011 wild type and derivative cells harbouring the indicated constructs: 5’1, pP<i>ecpR1</i>_5’1; 5’2, pP<i>ecpR1</i>_5’2; 5’2-Pσ70, pP<i>ecpR1</i>_5’2-Pσ70; 5’1–204, pP<i>ecpR1</i>_5’1–204. Specific activities were normalized to OD₆₀₀ to yield fluorescence units per unit of optical density (F/OD). Shown are means and standard deviation values of at least three independent measurements of three transconjugants grown in six independent cultures. <b>(C)</b> qRT-PCR analysis and Northern blot detection of EcpR1 transcript abundance in Rm2011 and the <i>relA</i> mutant under different growth and stress conditions in TY (left) and MOPS minimal and MOPSlim medium (MM, right). 40°C, heat stress; NaCl, 0.4 mM sodium chloride (osmotic stress); H₂O₂, 10mM hydrogen peroxide (oxidative stress); -O₂, microoxic conditions; 20°C, cold stress; -C and -N, growth in MM until OD₆₀₀ of 0.9 and then MM depleted for 1 hour for carbon or nitrogen. qRT-PCR values were normalized to the SMc01852 transcript and the levels of EcpR1 in Rm2011 growing in TY rich medium at OD₆₀₀ of 0.6 (left) or MOPS minimal medium at OD₆₀₀ of 0.9 (right, dashed line). Plots underneath the Northern blots represent relative hybridization signal intensities. The basal level of EcpR1 in Rm2011 growing in TY rich medium at OD₆₀₀ of 0.6 or MOPS minimal medium at OD₆₀₀ of 0.9 (right) has been normalized to 1 (dashed line) and the sRNA levels in other conditions have been correlated to this value. Mean results from three experiments are shown. Error bars indicate the standard deviation. Exposure times were optimized for each panel.</p

qRT-PCR based verification of putative EcpR1 target genes displaying expression changes in 2011<i>ecpR1</i> vs. Rm2011 wild type growing in MOPS or MOPSlim media.

<p>Log₂ change in transcript amount normalized to levels of the SMc01852 mRNA. Errors represent the standard deviation of three replicates. Positions of microarray reporter oligonucleotides relative to the start codon are given in brackets for 5’-UTR regions.</p><p>*Description of gene product or associated gene product.</p><p>qRT-PCR based verification of putative EcpR1 target genes displaying expression changes in 2011<i>ecpR1</i> vs. Rm2011 wild type growing in MOPS or MOPSlim media.</p

Hfq and RNase E activities are dispensable for EcpR1 overproduction-related cell elongation and post-transcriptional repression of <i>gcrA</i>.

<p><b>(A)</b> Northern blot analysis of EcpR1 stability in Rm2011 and <i>hfq</i> mutant strains grown to early stationary phase (OD₆₀₀ of 1.2, t = 0) and upon transcription arrest with Rf at indicated time points (in min). <b>(B)</b> Cell morphology of 2011<i>hfq</i> and 2011<i>rne</i>::<i>Tn5</i> mutants overexpressing either <i>ecpR1</i> (EcpR1⁺) or the control RNA gene SmelC812 (Control⁺) upon IPTG induction. Bars represent 2 μm. <b>(C)</b> Percentage of fluorescence in EcpR1 overproduction strains relative to the respective control strain overproducing SmelC812 in the Rm4011<i>ecpR1</i> or Rm4011<i>ecpR1 rne675</i> background co-transformed with plasmids carrying p<i>PgcrA-gcrA-egfp</i> or p<i>dnaA-154+162-egfp</i> translational fusions. <b>(D)</b> qRT-PCR analysis of <i>gcrA</i> and <i>dnaA</i> transcript abundance in Rm4011<i>ecpR1</i> EcpR1⁺ after transcription arrest with Rifampicin for 5 minutes. Values were normalized to the SMc01852 transcript and the levels in the IPTG induced control strain overexpressing the SmelC812 RNA gene. Results from three independent experiments are shown. Error bars indicate the standard deviation.</p

Lack of <i>ecpR1</i> reduces competitiveness of Rm2011.

<p>Mean percentage of <i>egfp</i>-labeled cells 1 and 4 weeks after mixing 2011<i>mCherry</i> with either 2011<i>egfp</i> or 2011<i>ecpR1 egfp</i> cells at a 1:1 ratio in MOPS (A) or MOPSlim media (B). Every week the mixed population was diluted 1000-fold in fresh media. The percentage of <i>egfp</i>-labeled cells was determined by microscopy. Error bars indicate the standard deviation of 3 biological replicates.</p

EcpR1 post-transcriptionally represses <i>gcrA</i> (A) and <i>dnaA</i> (B).

<p>Schematic representations of the genomic regions and the fragments (indicated by bars) translationally fused to <i>egfp</i>. Positions are denoted relative to the AUG; A is +1. Grey boxes indicate potential EcpR1-binding sites. Vertical arrows mark the regions covered by the oligonucleotide probes displaying altered signal intensities in the microarray hybridizations after <i>ecpR1</i> overexpression (see details in text). Means of relative fluorescence intensity values of Rm4011<i>ecpR1</i> co-transformed with the <i>ecpR1</i> or control SmelC812 overexpression plasmid, and the indicated reporter plasmid are shown below. The standard deviation represents at least three independent determinations of three double transconjugants grown in six independent cultures. Specific activities were normalized to the levels of the strain carrying the vector with the control RNA gene without IPTG added to yield percent relative fluorescence (% F).</p