The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA <i>sinI</i> in <i>Sinorhizobium meliloti</i>

<p>Quorum sensing is a cell density-dependent communication system of bacteria relying on autoinducer molecules. During the analysis of the post-transcriptional regulation of quorum sensing in the nitrogen fixing plant symbiont <i>Sinorhizobium meliloti,</i> we predicted and verified a direct interaction between the 5'-UTR of <i>sinI</i> mRNA encoding the autoinducer synthase and a small RNA (sRNA), which we named RcsR1. <i>In vitro</i>, RcsR1 prevented cleavage in the 5'-UTR of <i>sinI</i> by RNase E and impaired <i>sinI</i> translation. In line with low ribosomal occupancy and transcript destabilization upon binding of RcsR1 to <i>sinI</i>, overproduction of RcsR1 in <i>S. meliloti</i> resulted in lower level and shorter half-life of <i>sinI</i> mRNA, and in decreased autoinducer amount. Although RcsR1 can influence quorum sensing via <i>sinI</i>, its level did not vary at different cell densities, but decreased under salt stress and increased at low temperature. We found that RcsR1 and its stress-related expression pattern, but not the interaction with <i>sinI</i> homologs, are conserved in <i>Sinorhizobium</i>, <i>Rhizobium</i> and <i>Agrobacterium.</i> Consistently, overproduction of RcsR1 in <i>S. meliloti</i> and <i>Agrobacterium tumefaciens</i> inhibited growth at high salinity. We identified conserved targets of RcsR1 and showed that most conserved interactions and the effect on growth under salt stress are mediated by the first stem-loop of RcsR1, while its central part is responsible for the species-specific interaction with <i>sinI</i>. We conclude that RcsR1 is an ancient, stress-related riboregulator in rhizobia and propose that it links stress responses to quorum sensing in <i>S. meliloti.</i></p

TBLASTX analysis of <i>B</i>. <i>japonicum</i> sORFs showing the distribution of homologs among bacteria.

<p><b>A)</b> Analysis of 39 sORFs with proteomic evidence. Indicated is their presence only in <i>B</i>. <i>japonicum</i> strains (<i>B</i>. <i>japonicum</i>), in the genus <i>Bradyrhizobium</i>, or in Alphaproteobacteria (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.s021" target="_blank">S4 Table</a>). Other – the sORF blr0566_ISGA present in Alphaproteobacteria and outside Alphaproteobacteria. <b>B)</b> Analysis of all 1080 sORFs (with and without proteomic evidence) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.s023" target="_blank">S6 Table</a>). Alphaproteobacteria_Rare – sORFs found in less than five Alphaproteobacteria other than <i>Bradyrhizobium</i> spp.; Alphaproteobacteria_Conserved – sORFs found in five or more Alphaproteobacteria other than <i>Bradyrhizobium</i> spp.; Other – sORFs found in organisms outside Alphaproteobacteria; No homologs, – sORFs found only in <i>B</i>. <i>japonicum</i> USDA 110. <b>C)</b> Analysis of 47 sORFs with homologs outside Alphaproteobacteria (belonging to the category “Other”). Sporadic – sORFs found in less than 20 Alphaproteobacteria and less than 20 organisms outside Alphaproteobacteria; More in Alphaproteobacteria – sORFs found in at least 20 Alphaproteobacteria and less than 20 organisms outside Alphaproteobacteria; More outside Alphaproteobacteria – sORFs found in less than 20 Alphaproteobacteria and at least 20 organisms outside Alphaproteobacteria; Conserved – sORFs found in at least 20 Alphaproteobacteria and at least 20 organisms outside Alphaproteobacteria (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.s026" target="_blank">S9 Table</a>).</p

Transcription interference in blr1853.

<p><b>A)</b> Blr1853 locus and the structure of <i>lacZYA</i> reporter fusions used to analyze transcription interference in blr1853. Plasmid names are indicated. Blue straight line, DNA of the blr1853 locus; blue wave lines, asRNA AsR1-blr1853 (an abundant 65 nt form detected in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.g005" target="_blank">Fig 5C</a> and a long form previously detected by RT-PCR, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref015" target="_blank">15</a>]); orange line, <i>lacZYA</i> genes; thin black flexed arrows, active TSSs; thin gray flexed arrows, inactive TSSs; open boxes with promoter designations, promoters upstream of the TSSs; P_cyp, blr1853 promoter; P_int, internal promoter in the sense direction; P_as, internal promoter in the antisense direction. The genomic coordinates of the TSSs are given on top [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref015" target="_blank">15</a>]. Red stars indicate three mutations introduced in P_as (see B). The drawing is not to scale. <b>B)</b> Reporter fusions used to measure the activity of the wild type (wt) P_as and its mutated version P_as-mut3. Shown are parts of the cloned 63 nt sequence. The TSS of asRNA AsR1-blr1853 is indicated along with the –10 and –15 boxes of the P_as promoter. The mutated bases are in red. For other descriptions, see A). <b>C)</b> Beta-galactosidase activities of <i>B</i>. <i>japonicum</i> cells harboring the reporter constructs shown in A) and B). Measurements were performed with aerobic exponentially growing cultures. Shown are the results from three independent experiments with technical duplicates. Error bars indicate the standard deviation.</p

Non-annotated transcripts analyzed by Northern blot hybridization.

<p><b>A)</b> New sRNAs detected in liquid cultures and in nodules. <b>B)</b> New sRNAs detected only in nodules. <b>C)</b> mRNA-associated small transcripts with previously verified TSSs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref015" target="_blank">15</a>]. Total RNA from the exponential growth phase (E) and the stationary phase (S) of a liquid culture and from nodules (N) was used. For sRNAs detected in N only, control RNA from roots (R) was also included. After hybridization with sRNA-specific probes (indicated above the panels), the membranes were re-hybridized with probes specific for 5S rRNA from <i>B</i>. <i>japonicum</i> (5S <i>B</i>.<i>j</i>.) and, when root RNA was included, from <i>G</i>. <i>max</i> (5S <i>G</i>.<i>m</i>). In A) and B), the positions of the marker RNAs on the membranes (160 nt, 120 nt and 60 nt corresponding to 6S rRNA, 5S rRNA and a fragment detected by the 6S RNA-specific probe, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref029" target="_blank">29</a>]) are given on the right side (in nt). In C), the approximate lengths of the indicated bands are given (in nt), as calculated from the migration of the marker RNAs mentioned above. BjsR5a,b* and BjsR6a,b,c* – no discrimination between homologs in the Northern blot hybridization; BjsR8** – processing product according to dRNA-seq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.s012" target="_blank">S12 Fig</a>); E# – RNA isolated by TRIzol resulting in enrichment of sRNAs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref053" target="_blank">53</a>]; all other RNA samples were isolated by hot phenol.</p

Stabilizing selection for sORFs with proteomic evidence and bll1319_ISGA.

<p><b>A)</b> Distribution of the dN/dS ratio for homologs of 39 sORFs with proteomic evidence. <b>B)</b> Distribution of the dN/dS ratio for othologs of bll1319_ISGA. All pairs of homologs were considered to construct the histograms.</p

Characteristics of ten sRNAs analyzed by Northern blot hybridization.

<p>Characteristics of ten sRNAs analyzed by Northern blot hybridization.</p

New repetitive element Br-REP1.

<p><b>A)</b> cDNA reads at the blr0385, bll0386 locus. Purple box, repetitive element. For other descriptions see the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.g007" target="_blank">Fig 7</a>. <b>B)</b> Predicted secondary structure of Br-REP1 and flanking palindromes. The corresponding alignment is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.s017" target="_blank">S17 Fig</a>. For the color code see [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref042" target="_blank">42</a>].</p

Length distribution of the recently annotated ORFs of <i>B</i>. <i>japonicum</i> USDA 110.

<p><b>A)</b> Length distribution of 107 ORFs with proteomic evidence. <b>B</b>) Length distribution of all 1391 new ORFs.</p

Down-regulation of BjsR4 under tellurite and iron stresses, and impact of this down-regulation on survival at high iron concentration.

<p><b>A)</b> The steady-state level of BjsR4 is decreased upon addition of tellurite. Total RNA of three independent cultures was isolated before (control) and after the following stresses: 5 min exposure to 2 mM H₂O₂ or 30 min exposure to 50 mM NaCl (salt), 1 mM tellurite (K₂TeO₂), 39°C (heat) or 20°C (cold). After hybridization with the BjsR4-specific probe (top panel) the membrane was re-hybridized with the probe specific for 5S rRNA as loading control (bottom panel). <b>B)</b> The level of BjsR4 is decreased upon treatment with 1 mM tellurite and 10 mM Fe₂SO₄ but not 5 mM Zn₂SO₄ or 5mM Mn₂SO₄. Cultures were exposed for 30 min to the indicated metal ion solutions and then Northern blot analysis of total RNA was performed with probes specific to BjsR4 and 5S rRNA. <b>C)</b> Overexpression of the sRNA BjsR4 in <i>B</i>. <i>japonicum</i>. A representative Northern blot hybridization of the empty vector control (EVC) and the overexpressing strain (OE) is shown. The detected RNAs are indicated. The fold increase in the BjsR4 amount (Fold OE) given below the panel was calculated from four independent biological replicates. <b>D)</b> Increased sensitivity of the BjsR4 overexpressing strain to iron. Cells of the EVC and OE strains were grown to an OD₆₀₀ of 0.2 and 10 mM Mn₂SO_4, 5 mM K₂TeO₂ or 10 mM Fe₂SO₄ was added. Two hours later the cells were washed, serially diluted, spotted onto PSY plates with spectinomycine and tetracycline and grown for seven days at 30°C Shown is a representative example of six biologically independent experiments with similar results. <b>E)</b> Quantitative analysis of percentage colony forming units (c.f.u.) of the EVC and OE strains that survived after exposure to 10 mM Fe₂SO₄. Shown are the means and standard deviations from six biologically independent replicates.</p

Analysis of two putative toxin genes in the symbiotic island.

<p><b>A)</b> cDNA reads at the blr0229_ISGA (<i>higB</i>), blr1638 (<i>higA</i>) locus. <b>B)</b> cDNA reads at the bll1687 (<i>yhaV</i>), blr1688 (<i>prlF</i>) locus. RNA was isolated from exponentially growing, free-living cells (F) in liquid cultures and from nodules (N). RNA samples were treated (+) or not treated (–) with terminal exonuclease (TEX), which degrades 5’-monophosphorylated (processed) transcripts. The scale of each library is indicated (reads). Grey arrows with indicated gene designations, annotated transcripts; Blue arrows, non-annotated transcript; flexed thin black arrow, mapped TSS; dark grey boxes with P and T, mapped putative promoter and terminator, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref015" target="_blank">15</a>]. <b>C)</b> Growth curves of <i>E</i>. <i>coli</i> containing the empty vector pBAD_Cm (empty vector control, EVC), pBAD_Cm::blr0229_ISGA (blr0229_ISGA) or pBAD::_Cm::bll1687 (bll1687) after induction with arabinose.</p