Dataset Information

The small 6C RNA of Corynebacterium glutamicum is involved in the SOS response.

ABSTRACT: The 6C RNA family is a class of small RNAs highly conserved in Actinobacteria, including the genera Mycobacterium, Streptomyces and Corynebacterium whose physiological function has not yet been elucidated. We found that strong transcription of the cgb_03605 gene, which encodes 6C RNA in C. glutamicum, was driven by the SigA- and SigB-dependent promoter Pcgb_03605. 6C RNA was detected at high level during exponential growth phase (180 to 240 molcules per cell) which even increased at the entry of the stationary phase. 6C RNA level did not decrease within 240 min after transcription had been stopped with rifampicin, which suggests high 6C RNA stability. The expression of cgb_03605 further increased approximately twofold in the presence of DNA-damaging mitomycin C (MMC) and nearly threefold in the absence of LexA. Deletion of the 6C RNA gene cgb_03605 resulted in a higher sensitivity of C. glutamicum toward MMC and UV radiation. These results indicate that 6C RNA is involved in the DNA damage response. Both 6C RNA level-dependent pausing of cell growth and branched cell morphology in response to MMC suggest that 6C RNA may also be involved in a control of cell division.

SUBMITTER: Pahlke J

PROVIDER: S-EPMC5014011 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The small 6C RNA of Corynebacterium glutamicum is involved in the SOS response.

Pahlke Jennifer J Dostálová Hana H Holátko Jiří J Degner Ursula U Bott Michael M Pátek Miroslav M Polen Tino T

RNA biology 20160630 9

The 6C RNA family is a class of small RNAs highly conserved in Actinobacteria, including the genera Mycobacterium, Streptomyces and Corynebacterium whose physiological function has not yet been elucidated. We found that strong transcription of the cgb_03605 gene, which encodes 6C RNA in C. glutamicum, was driven by the SigA- and SigB-dependent promoter Pcgb_03605. 6C RNA was detected at high level during exponential growth phase (180 to 240 molcules per cell) which even increased at the entry of ...[more]

PMID: 27362471

Similar Datasets

Project description:The (pp)pGpp metabolism is an important component of bacterial physiology as it is involved in various stress responses and mechanisms of cell homeostasis, e.g., the regulation of growth. However, in order to better understand the (pp)pGpp associated regulation, it is crucial to study the molecular mechanisms of (pp)pGpp metabolism. In recent years, bioinformatic analyses of the RelA/SpoT homolog (RSH) superfamily have led to the discovery of small monofunctional RSH derivatives in addition to the well-known bifunctional Rel proteins. These are also referred to as small alarmone synthetases (SASs) or small alarmone hydrolases (SAHs). In this study, the ORF cg1485 from C. glutamicum was identified as a putative SAH encoding gene, based on a high similarity of the corresponding amino acid sequence with the (pp)pGpp hydrolysis domain. The characterization of its gene product, designated as RelHCg, represents the first functional investigation of a bacterial representative of the SAH subfamily. The predicted pyrophosphohydrolase activity was demonstrated in vivo by expression in two E. coli strains, characterized by different alarmone basal levels, as well as by in vitro analysis of the purified protein. During the assay-based analysis of hydrolysis activity in relation to the three known alarmone species, both RelHCg and the bifunctional RSH enzyme RelCg were found to exhibit a pronounced substrate inhibition for alarmone concentrations of more than 0.75 mM. This characteristic of (pp)pGpp hydrolases could be an important mechanism for realizing the bistable character of the (pp)pGpp metabolism between a (pp)pGpp basal level and stress-associated alarmone production. The deletion of relHCg caused only a minor effect on growth behavior in both wild-type background and deletion mutants with deletion of (pp)pGpp synthetases. Based on this observation, the protein is probably only present or active under specific environmental conditions. The independent loss of the corresponding gene in numerous representatives of the genus Corynebacterium, which was found by bioinformatic analyses, also supports this hypothesis. Furthermore, growth analysis of all possible deletion combinations of the three active C. glutamicum RSH genes revealed interesting functional relationships which will have to be investigated in more detail in the future.

Project description:Corynebacterium glutamicum has been applied for the industrial production of various metabolites, such as amino acids. To understand the biosynthesis of the membrane protein in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP is found in all three domains of life and plays an important role in the membrane insertion of proteins. SRP RNA is initially transcribed as precursor molecules; however, relatively little is known about its maturation. In C. glutamicum, SRP consists of the Ffh protein and 4.5S RNA lacking an Alu domain. In this study, we found that 3'-to-5' exoribonuclease, polynucleotide phosphorylase (PNPase), and two endo-type RNases, RNase E/G and YbeY, are involved in the 3' maturation of 4.5S RNA in C. glutamicum The mature form of 4.5S RNA was inefficiently formed in ΔrneG Δpnp mutant cells, suggesting the existence of an alternative pathway for the 3' maturation of 4.5S RNA. Primer extension analysis also revealed that the 5' mature end of 4.5S RNA corresponds to that of the transcriptional start site. Immunoprecipitated Ffh protein contained immature 4.5S RNA in Δpnp, ΔrneG, and ΔybeY mutants, suggesting that 4.5S RNA precursors can interact with Ffh. These results imply that the maturation of 4.5S RNA can be performed in the 4.5S RNA-Ffh complex.IMPORTANCE Overproduction of a membrane protein, such as a transporter, is useful for engineering of strains of Corynebacterium glutamicum, which is a workhorse of amino acid production. To understand membrane protein biogenesis in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP contains the Ffh protein and SRP RNA and plays an important role in the membrane insertion of proteins. Although SRP RNA is highly conserved among the three domains of life, relatively little is known about its maturation. We show that PNPase, RNase E/G, and YbeY are involved in the 3' maturation of the SRP RNA (4.5S RNA) in this bacterium. This indicates that 3' end processing in this organism is different from that in other bacteria, such as Escherichia coli.

Project description:BACKGROUND: Recent discoveries on bacterial transcriptomes gave evidence that small RNAs (sRNAs) have important regulatory roles in prokaryotic cells. Modern high-throughput sequencing approaches (RNA-Seq) enable the most detailed view on transcriptomes offering an unmatched comprehensiveness and single-base resolution. Whole transcriptome data obtained by RNA-Seq can be used to detect and characterize all transcript species, including small RNAs. Here, we describe an RNA-Seq approach for comprehensive detection and characterization of small RNAs from Corynebacterium glutamicum, an actinobacterium of high industrial relevance and model organism for medically important Corynebacterianeae, such as C. diphtheriae and Mycobacterium tuberculosis. RESULTS: In our RNA-Seq approach, total RNA from C. glutamicum ATCC 13032 was prepared from cultures grown in minimal medium at exponential growth or challenged by physical (heat shock, cold shock) or by chemical stresses (diamide, H2O2, NaCl) at this time point. Total RNA samples were pooled and sequencing libraries were prepared from the isolated small RNA fraction. High throughput short read sequencing and mapping yielded over 800 sRNA genes. By determining their 5'- and 3'-ends and inspection of their locations, these potential sRNA genes were classified into UTRs of mRNAs (316), cis-antisense sRNAs (543), and trans-encoded sRNAs (262). For 77 of trans-encoded sRNAs significant sequence and secondary structure conservation was found by a computational approach using a whole genome alignment with the closely related species C. efficiens YS-314 and C. diphtheriae NCTC 13129. Three selected trans-encoded sRNAs were characterized by Northern blot analysis and stress-specific transcript patterns were found. CONCLUSIONS: The study showed comparable numbers of sRNAs known from genome-wide surveys in other bacteria. In detail, our results give deep insight into the comprehensive equipment of sRNAs in C. glutamicum and provide a sound basis for further studies concerning the functions of these sRNAs.

Dataset Information

The small 6C RNA of Corynebacterium glutamicum is involved in the SOS response.

Publications

The small 6C RNA of Corynebacterium glutamicum is involved in the SOS response.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets