Identification of differentially expressed small non-coding RNAs in the legume endosymbiont Sinorhizobium meliloti by comparative genomics

Bacterial small non-coding RNAs (sRNAs) are being recognized as novel widespread regulators of gene expression in response to environmental signals. Here, we present the first search for sRNA-encoding genes in the nitrogen-fixing endosymbiont Sinorhizobium meliloti, performed by a genome- wide computational analysis of its intergenic regions. Comparative sequence data from eight related alpha-proteobacteria were obtained, and the interspecies pairwise alignments were scored with the programs eQRNA and RNAz as complementary predictive tools to identify conserved and stable secondary structures corresponding to putative non-coding RNAs. Northern experiments confirmed that eight of the predicted loci, selected among the original 32 candidates as most probable sRNA genes, expressed small transcripts. This result supports the combined use of eQRNA and RNAz as a robust strategy to identify novel sRNAs in bacteria. Furthermore, seven of the transcripts accumulated differentially in free-living and symbiotic conditions. Experimental mapping of the 5 '-ends of the detected transcripts revealed that their encoding genes are organized in autonomous transcription units with recognizable promoter and, in most cases, termination signatures. These findings suggest novel regulatory functions for sRNAs related to the interactions of alpha-proteobacteria with their eukaryotic hosts.Spanish Ministerio de Educación y Ciencia (Project AGL2006-12466/AGR)Junta de Andalucía (Project CV1-01522)NIH Grant 1R01GM070538-02FPI Fellowship from the Spanish Ministerio de Educación y Cienci

A survey of sRNA families in alpha-proteobacteria

We have performed a computational comparative analysis of six small non-coding RNA (sRNA) families in alpha-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. Consensus secondary structures inferred from covariance models for each sRNA family evidenced in some cases conserved motifs putatively relevant to the function of trans-encoded base-pairing sRNAs i.e., Hfq-binding signatures and exposed anti Shine-Dalgarno sequences. Two particular family models, namely alpha r15 and alpha r35, shared own sub-structural modules with the Rfam model suhB (RF00519) and the uncharacterized sRNA family alpha r35b, respectively. A third sRNA family, termed alpha r45, has homology to the cis-acting regulatory element speF (RF00518). However, new experimental data further confirmed that the S. meliloti alpha r45 representative is an Hfq-binding sRNA processed from or expressed independently of speF, thus refining the Rfam speF model annotation. All the six families have members in phylogenetically related plant-interacting bacteria and animal pathogens of the order of the Rhizobiales, some occurring with high levels of paralogy in individual genomes. In silico and experimental evidences predict differential regulation of paralogous sRNAs in S. meliloti 1021. The distribution patterns of these sRNA families suggest major contributions of vertical inheritance and extensive ancestral duplication events to the evolution of sRNAs in plant-interacting bacteria.Janelia Farm Research Campus (HHMI)Ministry of Science and Innovation, Spain (MICINN) Instituto de Salud Carlos III Spanish Government TIN-2009-13950 AGL2009-07925Junta de Andalucia TIC-02788GENIL PYR-2010-28European Commission CSD2009-00006Spanish Ministerio de Ciencia e Innovacion (FPI)CSIC (JAE

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

<div><p>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. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in <i>Arabidopsis thaliana</i> protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.</p></div

Localization of the N-terminal IEP-EGFP fusion in bacteria by fluorescence microscopy.

<p>The constructs used in the assays are indicated above the panels. <i>E. coli</i> and <i>S. meliloti</i> were grown at 30°C. Panels <i>A</i> to <i>C</i> correspond to <i>E. coli</i> DH5α; Panels <i>D</i> to <i>F</i> correspond to <i>S. meliloti</i> RMO17. The numbers within the panels indicate the percentage of cells displaying the corresponding fluorescence pattern. Scale bar ≈ 2 µm.</p

Transient expression and subcellular localization in <i>A. thaliana</i> protoplasts of the IEP-EGFP N-terminal fusion.

<p>Images show fluorescence and bright-field microscopy. <i>A. thaliana</i> protoplasts were transfected as indicated in the Materials and Methods, with the constructs shown over the corresponding panels. The domains of the IEP are represented: in red, the reverse transcriptase; in blue, the maturase; and in orange, the C-terminal domain. The coordinates (nucleotides) of the different IEP subsegments cloned are indicated beneath the diagrams. RmInt1-ΔORF is indicated as a yellow box with flanking short exons E1(−20)/E2(+5). The IEP-EGFP fusion and derivatives were expressed under the control of the CaMV 35S promoter. The negative control used in the experiments was pK7WGF2. On the bright-field images, the nuclei are indicated by a dotted line with a black arrowhead; white arrowheads indicate nucleoli. (Scale bar ≈ 10 µm).</p

Determination of the localization patterns of various mutant IEP-EGFP fusions in <i>A. thaliana</i> protoplasts.

<p>Images show fluorescence and bright-field microscopy. <i>A. thaliana</i> protoplasts were transfected as indicated in the Materials and Methods, with the constructs indicated above the corresponding panels. The domains of the IEP are represented: in red, the reverse transcriptase; in blue, the maturase; and in orange, the C-terminal domain. For pK7-nΔC29, the coordinates (nucleotides) of the IEP subsegment cloned are indicated beneath the diagram. The wild-type IEP-EGFP fusion and derivatives were expressed under the control of the CaMV 35S promoter. In the colocalization experiments, we used the SRp34/31-RFP construct <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084056#pone.0084056-NisaMartinez1" target="_blank">[26]</a>, the product of which localizes to nuclear speckles. On the bright-field images, we indicate the nuclei by a dotted line and a black arrowhead; white arrowheads indicate nucleoli. Panels <i>C</i>, <i>F</i>, and <i>G</i> show confocal microscopy images. Images were merged with ImageJ software. The numbers within the EGFP panels indicate the percentage of transformed protoplasts displaying the corresponding fluorescence pattern. (Scale bar ≈ 10 µm).</p

Mobility assays for the IEP-EGFP fusions.

<p>(<i>A</i>) Schematic diagram of the constructs used in the retrohoming analysis of the WT (pKGEMA4), C-terminal (pKG4cGFP) and N-terminal (pKG4nGFP) fusions; pKm^R, promoter of the kanamycin resistance gene; IEP, intron-encoded protein of RmInt1; ΔORF; RmInt1 derivative in which domain IV of the ribozyme has been deleted between positions 611 and 1759; EGFP, enhanced green fluorescent protein. (<i>B</i>) Outline of the double-plasmid retrohoming assay: the target on the recipient plasmid could be invaded by an intron from the donor, resulting in the homing product. (<i>C</i>) Effect of the tagged IEPs on intron homing. For homing assays, plasmid pools from <i>S. meliloti</i> RMO17 cells harboring donor (pKGEMA4, pKG4cGFP or pKG4nGFP) and recipient plasmids (pJB0.6LAG) were analyzed by Southern hybridization with a DNA probe specific to the target (insertion sequence IS<i>Rm2011-2</i>). The recipient plasmid pJBΔ129 was used as a negative control in the assays (indicated as a minus sign in a circle above the blot). Target invasion rates in each homing assay were calculated as described in Materials and Methods and are plotted in the histogram shown below the blot.</p