Involvement of E. coli 6S RNA in Oxidative Stress Response.

6S RNA, a small non-coding RNA present in almost all bacteria, inhibits transcription via direct binding to RNA polymerase holoenzymes. The mechanism of 6S RNA action was investigated to a large extent in E. coli, however, lack of 6S RNA (ΔssrS) was demonstrated to be unfavorable but not essential for cell survival under various growth conditions. In the present study, we revealed, for the first time, a lethal phenotype of the ΔssrS strain in the presence of high concentrations of H2O2. This phenotype was rescued by complementation of the ssrS gene on a plasmid. We performed comparative qRT-PCR analyses on an enlarged set of mRNAs of genes associated with the oxidative stress response, allowing us to identify four genes known to be involved in this pathway (soxS, ahpC, sodA and tpx) that had decreased mRNA levels in the ΔssrS strain. Finally, we performed comparative proteomic analyses of the wild-type and ΔssrS strains, confirming that ΔssrS bacteria have reduced levels of the proteins AhpC and Tpx involved in H2O2 reduction. Our findings substantiate the crucial role of the riboregulator 6S RNA for bacterial coping with extreme stresses.

6S RNA in Rhodobacter sphaeroides: 6S RNA and pRNA transcript levels peak in late exponential phase and gene deletion causes a high salt stress phenotype.

The function of 6S RNA, a global regulator of transcription, was studied in the photosynthetic α-proteobacterium Rhodobacter sphaeroides. The cellular levels of R. sphaeroides 6S RNA peak toward the transition to stationary phase and strongly decrease during extended stationary phase. The synthesis of so-called product RNA transcripts (mainly 12-16-mers) on 6S RNA as template by RNA polymerase was found to be highest in late exponential phase. Product RNA ≥ 13-mers are expected to trigger the dissociation of 6S RNA:RNA polymerase complexes. A 6S RNA deletion in R. sphaeroides had no impact on growth under various metabolic and oxidative stress conditions (with the possible exception of tert-butyl hydroperoxide stress). However, the 6S RNA knockout resulted in a robust growth defect under high salt stress (0.25 M NaCl). Remarkably, the sspA gene encoding the putative salt stress-induced membrane protein SspA and located immediately downstream of the 6S RNA (ssrS) gene on the antisense strand was expressed at elevated levels in the ΔssrS strain when grown in the presence of 250 mM NaCl.

Key interactions of RNA polymerase with 6S RNA and secondary channel factors during pRNA synthesis.

Small non-coding 6S RNA mimics DNA promoters and binds to the σ70 holoenzyme of bacterial RNA polymerase (RNAP) to suppress transcription of various genes mainly during the stationary phase of cell growth or starvation. This inhibition can be relieved upon synthesis of short product RNA (pRNA) performed by RNAP from the 6S RNA template. Here, we have shown that pRNA synthesis depends on specific contacts of 6S RNA with RNAP and interactions of the σ finger with the RNA template in the active site of RNAP, and is also modulated by the secondary channel factors. We have adapted a molecular beacon assay with fluorescently labeled σ70 to analyze 6S RNA release during pRNA synthesis. We found the kinetics of 6S RNA release to be oppositely affected by mutations in the σ finger and in the CRE pocket of core RNAP, similarly to the reported role of these regions in promoter-dependent transcription. Secondary channel factors, DksA and GreB, inhibit pRNA synthesis and 6S RNA release from RNAP, suggesting that they may contribute to the 6S RNA-mediated switch in transcription during stringent response. Our results demonstrate that pRNA synthesis depends on a similar set of contacts between RNAP and 6S RNA as in the case of promoter-dependent transcription initiation and reveal that both processes can be regulated by universal transcription factors acting on RNAP.

Similarities and differences between 6S RNAs from Bradyrhizobium japonicum and Sinorhizobium meliloti.

6S RNA, a conserved and abundant small non-coding RNA found in most bacteria, regulates gene expression by inhibiting RNA polymerase (RNAP) holoenzyme. 6S RNAs from α-proteobacteria have been studied poorly so far. Here, we present a first in-depth analysis of 6S RNAs from two α-proteobacteria species, Bradyrhizobium japonicum and Sinorhizobium meliloti. Although both belong to the order Rhizobiales and are typical nitrogen-fixing symbionts of legumes, their 6S RNA expression profiles were found to differ: B. japonicum 6S RNA accumulated in the stationary phase, thus being reminiscent of Escherichia coli 6S RNA, whereas S. meliloti 6S RNA level peaked at the transition to the stationary phase, similarly to Rhodobacter sphaeroides 6S RNA. We demonstrated in vitro that both RNAs have hallmarks of 6S RNAs: they bind to the σ70-type RNAP holoenzyme and serve as templates for de novo transcription of so-called product RNAs (pRNAs) ranging in length from ∼13 to 24 nucleotides, with further evidence of the synthesis of even longer pRNAs. Likewise, stably bound pRNAs were found to rearrange the 6S RNA structure to induce its dissociation from RNAP. Compared with B. japonicum 6S RNA, considerable conformational heterogeneity was observed for S. meliloti 6S RNA and its complexes with pRNAs, even though the two 6S RNAs share ∼75% sequence identity. Overall, our findings suggest that the two rhizobial 6S RNAs have diverged with respect to their regulatory impact on gene expression throughout the bacterial life cycle.

Giant caldera in the Arctic Ocean: Evidence of the catastrophic eruptive event.

A giant caldera located in the eastern segment of the Gakkel Ridge could be firstly seen on the bathymetric map of the Arctic Ocean published in 1999. In 2014, seismic and multibeam echosounding data were acquired at the location. The caldera is 80 km long, 40 km wide and 1.2 km deep. The total volume of ejected volcanic material is estimated as no less than 3000 km3 placing it into the same category with the largest Quaternary calderas (Yellowstone and Toba). Time of the eruption is estimated as ~1.1 Ma. Thin layers of the volcanic material related to the eruption had been identified in sedimentary cores located about 1000 km away from the Gakkel Ridge. The Gakkel Ridge Caldera is the single example of a supervolcano in the rift zone of the Mid-Oceanic Ridge System.

Phenotypic characterization and complementation analysis of Bacillus subtilis 6S RNA single and double deletion mutants.

6S RNA, a global regulator of transcription in bacteria, binds to housekeeping RNA polymerase (RNAP) holoenzymes to competitively inhibit transcription from DNA promoters. Bacillus subtilis encodes two 6S RNA homologs whose differential functions are as yet unclear. We constructed derivative strains of B. subtilis PY79 lacking 6S-1 RNA (ΔbsrA), 6S-2 RNA (ΔbsrB) or both (ΔbsrAB) to study the physiological role of the two 6S RNAs. We observed two growth phenotypes of mutant strains: (i) accelerated decrease of optical density toward extended stationary phase and (ii) faster outgrowth from stationary phase under alkaline stress conditions (pH 9.8). The first phenotype was observed for bacteria lacking bsrA, and even more pronounced for ΔbsrAB bacteria, but not for those lacking bsrB. The magnitude of the second phenotype was relatively weak for ΔbsrB, moderate for ΔbsrA and again strongest for ΔbsrAB bacteria. Whereas ΔbsrAB bacteria complemented with bsrB or bsrA (strains ΔbsrAB + B and ΔbsrAB + A) mimicked the phenotypes of the ΔbsrA and ΔbsrB strains, respectively, complementation with the gene ssrS encoding Escherichia coli 6S RNA failed to cure the "low stationary optical density" phenotype of the double mutant, despite ssrS expression, in line with previous findings. Finally, proteomics (two-dimensional differential gel electrophoresis, 2D-DIGE) of B. subtilis 6S RNA deletion strains unveiled a set of proteins that were expressed at higher levels particularly during exponential growth and preferentially in mutant strains lacking 6S-2 RNA. Several of these proteins are involved in metabolism and stress responses.