Transcriptome of Sinorhizobium meliloti RNase mutants
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ABSTRACT: The ribonucleases (RNases) E and J are essential in Escherichia coli and Bacillus subtilis, respectively. Sinorhizobium meliloti contains both, the rne gene encoding RNase E and the rnj gene encoding RNase J. The transcriptomes of the S. meliloti Rm2011 wild type, and rne and rnj mutants were compared.
Project description:Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 65:32:5, in 78 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation events potentially involved in rhizobial adaptation to diverse stresses were also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion). These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology.
Project description:In this experiment we have compared the transcriptome of the S. meliloti Sm2B3001 strain and that of a mutant derivative lacking the gene encoding the almost ubiquitous bacterial endoribonuclease YbeY. Both, the parent and mutant strains were grown to either exponential or stationary phases in complete TY medium. Total RNA of parent and mutant strains (four replicates in each growth condition) were probed by competitive hybridization on Sm14kOLI microarrays. SmYbeY loss-of-function compromised expression of core energy and RNA metabolism genes, whilst promoting accumulation of motility, late symbiotic and transport mRNAs. Up-regulated transcripts are putative SmYbeY substrates representing both bulk and sRNA-regulated mRNAs.
Project description:Hfq-dependent transcriptional alterations in the nitrogen-fixing endosymbiont S. meliloti. Comparison: S. meliloti 1021 strain Vs S. meliloti 1021Dhfq (containing a deletion of the hfq ORF).
Project description:The RNA-binding protein Hfq is a global regulator, which controls diverse cellular processes in bacteria. To begin understanding the role of Hfq in the Sinorhizobium meliloti-Medicago truncatula nitrogen-fixing symbiosis, we defined free-living and symbiotic phenotypes of an hfq mutant. Over 500 transcripts were differentially accumulated in the hfq mutant of S. meliloti Rm1021 when grown in a shaking culture. Gene expression profiling of Sinorhizobium meliloti Rm1021 or its isogenic hfq deletion mutant, grown to late exponential phase in rich medium, was performed using custom Affymetrix GeneChips.
Project description:Mechanistic studies have revealed that TLR8 senses single-stranded RNA (ssRNA) fragments, processed via synergistic cleavage by ribonucleases (RNase) T2 and RNase A family members. Processing by these RNases releases uridines and purine-terminated residues resulting in TLR8 activation. Monocytes show high expression of RNase 6, yet this RNase has not been analyzed for its physiological contribution to recognition of bacterial RNA by TLR8. Herein, we characterized molecular RNase 6 cleavage mechanisms. BLaER1 RNASE6-/- cells showed a dampened TLR8-dependent response upon stimulation with isolated bacterial RNA (bRNA) but also upon infection with live whole bacteria. Pretreatment of bacterial RNA with recombinant RNase 6 generated fragments that induced stimulation in RNASE6 knockout cells. 2’O-RNA methyl modification, when introduced at the first uridine in the UA dinucleotide, impaired upstream processing by RNase 6 and dampened TLR8 stimulation. In summary, this data shows that RNase 6 plays a critical role in the processing of bacterial RNA by generating uridine-terminated breakdown products that ultimately activate TLR8.
Project description:Sinorhizobium meliloti establishes symbiotic relationship with compatible leguminous plants by inducing root nodule formation, colonizing such nodules, and fixing molecular nitrogen for the host in exchange for carbon compounds. This mutualistic process requires complex communication and tight regulation to allow yet constrain infection to specific tissues. Production of succinoglycan, or exopolysaccharide-I (EPS-I), enables S. meliloti to invade the root cortex via infection threads. A previous genetic screen identified jspA (SMc03872), encoding an extracytoplasmic protease, as a regulator of EPS-I production. To elucidate its molecular role, we performed transcriptome analyses of strains overexpressing wild-type or mutant alleles of jspA. We observed changes in gene expression suggesting that JspA contributes to symbiosis efficiency by modulating the critical ExoR-ExoS-ChvI signaling pathway.
Project description:Sinorhizobium meliloti can live as a soil saprophyte, and can engage in a nitrogen fixing symbiosis with plant roots. To succeed in such diverse environments, the bacteria must continually adjust gene expression. Transcriptional plasticity in eubacteria is often mediated by alternative sigma factors interacting with core RNA polymerase. The S. meliloti genome encodes 14 of these alternative sigmas, including two putative RpoH (heat shock) sigmas. We used custom Affymetrix Symbiosis Chips to characterize the global transcriptional response of S. meliloti rpoH1, rpoH2 and rpoH1 rpoH2 mutants during heat shock and stationary phase growth. Under these conditions, expression of over 300 genes is dependent on rpoH1 and rpoH2.
Project description:Within this work we identified and characterized SMc03169 (hhrA) as a new Sinorhizobium meliloti gene product with relevance to biological nitrogen fixation symbiosis with leguminous plants. HhrA belongs to the TetR-family of repressors and its deletion from S. meliloti genome affected considerably gene expression as well as several phenotypic traits. For expression profiling, triplicates of the wild-type Sinorhizobium meliloti 1021 or the SMc03169 (delta hhrA) deletion mutant were grown in GMS liquid medium supplemented with 10 micromolar of luteolin for 4 hours at 30M-BM-:C and 180 rpm, followed by total RNA extraction.
Project description:Sinorhizobium meliloti lives as a soil saprophyte, and engages in a nitrogen fixing symbiosis with plant roots. To succeed in such diverse environments, the bacteria must continually adjust gene expression. Transcriptional plasticity in eubacteria is often mediated by alternative sigma factors interacting with core RNA polymerase. The S. meliloti genome encodes 14 of these alternative sigmas, including 11 extracytoplasmic function (ECF) sigmas. We used custom Affymetrix Symbiosis Chips to characterize the global transcriptional response of S. meliloti overexpressing the ECF sigma factor, RpoE2. Our work identifies over 200 genes whose expression is dependent on RpoE2.
Project description:The Alphaproteobacterium Sinorhizobium meliloti lives in soil and is capable of fixing molecular nitrogen in symbiosis with legume plants. In this work, the small proteome of S. meliloti strain 2011 was studied to uncover translation of both annotated and novel small open reading frame (sORF)-encoded proteins (SEPs).