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Methanosarcina mazei Go1

ABSTRACT: sRNA154 a newly identified regulator of nitrogen fixation in Methanosarcina mazei strain Gö1

PROVIDER: PRJNA338498 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR4018514.fastq.gz	Fastqsanger.gz
	SRR4018515.fastq.gz	Fastqsanger.gz
	SRR4018516.fastq.gz	Fastqsanger.gz
	SRR4018517.fastq.gz	Fastqsanger.gz

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sRNA154 a newly identified regulator of nitrogen fixation in Methanosarcina mazei strain Gö1

Project description:Trans-encoded sRNA154 is exclusively expressed under nitrogen (N)-deficiency in Methanosarcina mazei strain Gö1. The respective deletion strain showed a significant growth defect under N-limitation, pointing towards a regulatory role of sRNA154 in the N-metabolism. Aiming to elucidate this regulatory function we characterized sRNA154 by biochemical and genetic approaches. 24 homologs of sRNA154 were identified in recently reported draft genomes of Methanosarcina strains, demonstrating high conservation in sequence and predicted secondary structure with two highly conserved single stranded loop regions. In silico target prediction uncovered multiple potential interactions of both conserved loops with mRNA targets 5´untranslated region and coding sequence) encoding key components of the N-metabolism. In line with the computational prediction transcriptome studies of the sRNA154 deletion mutant by an RNA-seq approach uncovered nrpA-mRNA as a potential target, encoding the transcriptional activator of the nitrogen fixation (nif)-operon. Further evidence obtained by electromobility shift-, stability- and complementation assays, strongly argues for a stabilizing effect of sRNA154 on nrpA-mRNA by binding with both loops. Studying the further predicted N-related targets showing lower transcript levels in the absence of sRNA154, demonstrated that nifH transcript levels are most likely indirectly affected by sRNA154 due to enhanced stability of the nrpA transcripts. Besides, translation of glnA2-mRNA, encoding glutamine synthetase, appears to be affected by sRNA154 masking the ribosome binding site (RBS), whereas glnA1-mRNA appears to be stabilized by sRNA154. Overall, we propose that sRNA154 has a crucial role in N-metabolism in M. mazei and allows a feed forward regulation of nif-gene expression by stabilizing nrpA mRNA.

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Exploring the hidden world of Methanosarcina mazei: Unravelling the secrets of its small proteome and its nitrogen-dependent regulation

Project description:The mesophilic methanogenic archaeal model organism Methanosarcina mazei strain Gö1, is crucial for climate and environmental research due to its ability to produce methane from H2 plus CO2, acetate, and methylamines. Here, we established the first Ribo-seq protocol for M. mazei strain Gö1 under two growth conditions (nitrogen sufficiency (+N) versus nitrogen limitation (-N)). Translation of 93 previously annotated and 311 novel small open reading frames (sORFs), coding for proteins ≤ 70 amino acids in length was predicted with high confidence based on Ribo-seq data. Epitope tagging followed by immunoblotting analysis confirmed the translation of 12 out of 15 selected novel sORFs, the remaining three were validated by LC-MS-MS analysis. In total, LC-MS-MS analysis validated translation for 60 annotated sORFs and 26 novel sORFs. A comprehensive differential expression analysis revealed that 29 of 311 novel sORFs were differentially regulated in response to nitrogen availability at the transcriptional and 49 at the translational level. Several reported sRNAs are now emerging as dual-functional sRNAs, including sRNA154, the central regulatory sRNA in regulation of nitrogen metabolism. Numerous of the novel sORFs identified in this study are conserved in Methanosarcina species, some of which showed a phenotype when overproduced, pinpointing important physiological functions. Overall, the comprehensive analysis opens a new avenue to start elucidating the function(s) of small proteins in M. mazei.

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Project description:We used ribosome profiling (Ribo-seq) to generate a translatome map for the archaeon M. mazei under two growth conditions. The analysis of the datasets allowed to generate a high confidence inventory of small proteins in the model archaeon M. mazei and their regulation under nitrogen limiting conditions.

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