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A stress-induced small RNA modulates alpha-rhizobial cell cycle progression.

Robledo M, Frage B, Wright PR, Becker A - PLoS Genet. (2015)

Bottom Line: Induced EcpR1 overproduction led to cell elongation and increased DNA content, while deletion of ecpR1 resulted in reduced competitiveness.Evidence is presented for EcpR1 promoting RNase E-dependent degradation of the dnaA mRNA.We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions.

View Article: PubMed Central - PubMed

Affiliation: LOEWE Center for Synthetic Microbiology and Faculty of Biology, Philipps-University Marburg, Marburg, Germany.

ABSTRACT
Mechanisms adjusting replication initiation and cell cycle progression in response to environmental conditions are crucial for microbial survival. Functional characterization of the trans-encoded small non-coding RNA (trans-sRNA) EcpR1 in the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti 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 S. meliloti, this trans-sRNA is encoded downstream of the divK-pleD operon. ecpR1 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 ecpR1 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 gcrA and dnaA 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 dnaA mRNA. We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions.

No MeSH data available.


Related in: MedlinePlus

ecpR1 genomic locus and transcriptional regulation.(A) 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 gcrA mRNA is boxed. Below, chromosomal region including the ecpR1 gene and RNAseq coverage profile of the EcpR1 sRNA in S. meliloti Rm1021. Genome coordinates of the full length ecpR1 variant are denoted. Black and grey areas represent coverages from samples enriched for processed and primary transcripts, respectively [21]. 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) Schematic representation of the fragments included in the ecpR1 transcriptional fusions and fluorescence values of stationary phase Rm2011 wild type and derivative cells harbouring the indicated constructs: 5’1, pPecpR1_5’1; 5’2, pPecpR1_5’2; 5’2-Pσ70, pPecpR1_5’2-Pσ70; 5’1–204, pPecpR1_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. (C) qRT-PCR analysis and Northern blot detection of EcpR1 transcript abundance in Rm2011 and the relA 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.
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pgen.1005153.g001: ecpR1 genomic locus and transcriptional regulation.(A) 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 gcrA mRNA is boxed. Below, chromosomal region including the ecpR1 gene and RNAseq coverage profile of the EcpR1 sRNA in S. meliloti Rm1021. Genome coordinates of the full length ecpR1 variant are denoted. Black and grey areas represent coverages from samples enriched for processed and primary transcripts, respectively [21]. 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) Schematic representation of the fragments included in the ecpR1 transcriptional fusions and fluorescence values of stationary phase Rm2011 wild type and derivative cells harbouring the indicated constructs: 5’1, pPecpR1_5’1; 5’2, pPecpR1_5’2; 5’2-Pσ70, pPecpR1_5’2-Pσ70; 5’1–204, pPecpR1_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. (C) qRT-PCR analysis and Northern blot detection of EcpR1 transcript abundance in Rm2011 and the relA 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.

Mentions: Targets predicted for the sRNA family established by the S. meliloti trans-sRNA SmelC291 show a significant enrichment (P-value = 2.5*10-5) of cell cycle-related mRNAs (n = 7) among the top-ranked candidates (P≤0.01, n = 89; S1 Table) [27]. The 23 family members are broadly distributed among the Rhizobiales including members in the Rhizobiaceae, Phyllobacteriaceae, Xanthobacteriaceae, Beijerinckaceae, and Hyphomicrobiaceae. SmelC291, previously named SmrC10 or Sra33, was first identified by comparative genomic predictions of sRNAs [31] and confirmed by RNAseq [21]. In this study we renamed it EcpR1 (elongated cell phenotype RNA1) according to the phenotype induced by its overproduction (see below). In S. meliloti, ecpR1 is located in the intergenic region between the divK-pleD operon coding for an essential cell cycle response regulator and a diguanylate cyclase, respectively [32] and rpmG encoding the 50S ribosomal protein L33 (Fig 1A). In the Rhizobiacea, this genomic locus is highly microsyntenic [27]. Northern blot hybridizations confirmed ecpR1 expression from an independent transcription unit [33] and RNAseq coverage data suggested variants of different length with a dominant 101 nt sRNA [21] which is predicted to form a stable structure with two defined stem-loop domains, SL1 and SL2 (Fig 1A, S1A Fig). SL1 is strongly conserved and positions C16 to G36 (according to the numbering of EcpR1 nucleotides in Fig 1A) including the loop sequence are identical in all species with EcpR1 homologs analyzed by Reinkensmeier et al. [27]. The 3’-region harbors a putative Rho-independent terminator and 4 terminal U residues (S1A Fig).


A stress-induced small RNA modulates alpha-rhizobial cell cycle progression.

Robledo M, Frage B, Wright PR, Becker A - PLoS Genet. (2015)

ecpR1 genomic locus and transcriptional regulation.(A) 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 gcrA mRNA is boxed. Below, chromosomal region including the ecpR1 gene and RNAseq coverage profile of the EcpR1 sRNA in S. meliloti Rm1021. Genome coordinates of the full length ecpR1 variant are denoted. Black and grey areas represent coverages from samples enriched for processed and primary transcripts, respectively [21]. 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) Schematic representation of the fragments included in the ecpR1 transcriptional fusions and fluorescence values of stationary phase Rm2011 wild type and derivative cells harbouring the indicated constructs: 5’1, pPecpR1_5’1; 5’2, pPecpR1_5’2; 5’2-Pσ70, pPecpR1_5’2-Pσ70; 5’1–204, pPecpR1_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. (C) qRT-PCR analysis and Northern blot detection of EcpR1 transcript abundance in Rm2011 and the relA 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.
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Related In: Results  -  Collection

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pgen.1005153.g001: ecpR1 genomic locus and transcriptional regulation.(A) 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 gcrA mRNA is boxed. Below, chromosomal region including the ecpR1 gene and RNAseq coverage profile of the EcpR1 sRNA in S. meliloti Rm1021. Genome coordinates of the full length ecpR1 variant are denoted. Black and grey areas represent coverages from samples enriched for processed and primary transcripts, respectively [21]. 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) Schematic representation of the fragments included in the ecpR1 transcriptional fusions and fluorescence values of stationary phase Rm2011 wild type and derivative cells harbouring the indicated constructs: 5’1, pPecpR1_5’1; 5’2, pPecpR1_5’2; 5’2-Pσ70, pPecpR1_5’2-Pσ70; 5’1–204, pPecpR1_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. (C) qRT-PCR analysis and Northern blot detection of EcpR1 transcript abundance in Rm2011 and the relA 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.
Mentions: Targets predicted for the sRNA family established by the S. meliloti trans-sRNA SmelC291 show a significant enrichment (P-value = 2.5*10-5) of cell cycle-related mRNAs (n = 7) among the top-ranked candidates (P≤0.01, n = 89; S1 Table) [27]. The 23 family members are broadly distributed among the Rhizobiales including members in the Rhizobiaceae, Phyllobacteriaceae, Xanthobacteriaceae, Beijerinckaceae, and Hyphomicrobiaceae. SmelC291, previously named SmrC10 or Sra33, was first identified by comparative genomic predictions of sRNAs [31] and confirmed by RNAseq [21]. In this study we renamed it EcpR1 (elongated cell phenotype RNA1) according to the phenotype induced by its overproduction (see below). In S. meliloti, ecpR1 is located in the intergenic region between the divK-pleD operon coding for an essential cell cycle response regulator and a diguanylate cyclase, respectively [32] and rpmG encoding the 50S ribosomal protein L33 (Fig 1A). In the Rhizobiacea, this genomic locus is highly microsyntenic [27]. Northern blot hybridizations confirmed ecpR1 expression from an independent transcription unit [33] and RNAseq coverage data suggested variants of different length with a dominant 101 nt sRNA [21] which is predicted to form a stable structure with two defined stem-loop domains, SL1 and SL2 (Fig 1A, S1A Fig). SL1 is strongly conserved and positions C16 to G36 (according to the numbering of EcpR1 nucleotides in Fig 1A) including the loop sequence are identical in all species with EcpR1 homologs analyzed by Reinkensmeier et al. [27]. The 3’-region harbors a putative Rho-independent terminator and 4 terminal U residues (S1A Fig).

Bottom Line: Induced EcpR1 overproduction led to cell elongation and increased DNA content, while deletion of ecpR1 resulted in reduced competitiveness.Evidence is presented for EcpR1 promoting RNase E-dependent degradation of the dnaA mRNA.We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions.

View Article: PubMed Central - PubMed

Affiliation: LOEWE Center for Synthetic Microbiology and Faculty of Biology, Philipps-University Marburg, Marburg, Germany.

ABSTRACT
Mechanisms adjusting replication initiation and cell cycle progression in response to environmental conditions are crucial for microbial survival. Functional characterization of the trans-encoded small non-coding RNA (trans-sRNA) EcpR1 in the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti 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 S. meliloti, this trans-sRNA is encoded downstream of the divK-pleD operon. ecpR1 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 ecpR1 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 gcrA and dnaA 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 dnaA mRNA. We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions.

No MeSH data available.


Related in: MedlinePlus