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.

2019-07-30 | GSE85456 | GEO

Methanosarcina mazei Go1

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

| PRJNA338498 | ENA

MM_0565 orchestrates CRISPR-based immunity in Methanosarcina mazei by transcriptional regulation of components essential for the adaptation

Project description:CRISPR loci are found in bacterial and archaeal genomes where they provide the molecular machinery for acquisition of immunity against foreign DNA. In addition to the cas genes fundamentally required for CRISPR activity, a second class of genes is associated with the CRISPR loci, of which many have no reported function in CRISPR-mediated immunity. Here, we characterize MM_0565 of Methanosarcina mazei Gö1 associated to the type I-B CRISPR-locus providing evidence for its relevance in regulating this system. We show that MM_0565 is composed of a modified Rossmann-like fold and a winged helix-turn-helix domain and forms a dimer in solution. While direct effects on CRISPR-Cas transcription were not detected by genetic approaches, binding to the leader region of both CRISPR-Cas systems was observed by microscale thermophoresis and electromobility shift assays. Overexpression of MM_0565 however, strongly induced transcription of the cas1-solo gene located in the recently reported casposon, the gene product of which shows high similarity to classical Cas1 proteins. Based on our findings we hypothesize that Cas1-solo is involved in the adaptation of CRISPR-mediated immunity in M. mazei, and that MM_0565 modulates the activity of the CRISPR systems amongst potential other hypnotized actions by activating the transcription of the cas1-solo gene.

2020-09-08 | GSE151372 | GEO

Transcription profiling of Methanosarcina mazei Goe1 in response to bismuth nitrate.

Project description:Pattern of gene expression of M. mazei in response to 10 uM bismuth nitrate was analyzed relative to gene expression pattern of M. mazei grown in the presence of 30 uM potassium nitrate (control cultures). Therefore, five pre-cultures of M. mazei was grown to mid-exponential growth-phase. Then, the pre-cultures were divided into aliquotes, one half spiked with bismuth nitrate and the other half with potassium nitrate (control) to match nitrate concentration of the bismuth spike. Samples were propagated for two days. Then, total RNA was isolated and transformed into Cy3 or Cy5 labeled cDNA. Labeled cDNA derived from one culture treated with bismuth and from one control culture, both obtained from the same pre-culture, was hybridized with microarray chips. Hybridized chips were scanned, derived data processed and relative expression levels of orfs from M. mazei in response to bismuth calculated.

2011-10-01 | E-MTAB-787 | biostudies-arrayexpress

Methanosarcina barkeri JCM 10043

Project description:Genome sequencing of Methanosarcina barkeri JCM 10043

| PRJDB882 | ENA