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.
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.
