title: sRNA154a newly identified regulator of nitrogen fixation in Methanosarcina mazei strain Gö1.
abstract: Trans-encoded sRNA154is exclusively expressed under nitrogen (N)-deficiency in Methanosarcina mazei strain Gö1. The sRNA154deletion strain showed a significant decrease in growth under N-limitation, pointing toward a regulatory role of sRNA154in N-metabolism. Aiming to elucidate its regulatory function we characterized sRNA154by means of biochemical and genetic approaches. 24 homologs of sRNA154were identified in recently reported draft genomes of Methanosarcina strains, demonstrating high conservation in sequence and predicted secondary structure with two highly conserved single stranded loops. Transcriptome studies of sRNA154deletion mutants by an RNA-seq approach uncovered nifH- and nrpA-mRNA, encoding the α-subunit of nitrogenase and the transcriptional activator of the nitrogen fixation (nif)-operon, as potential targets besides other components of the N-metabolism. Furthermore, results obtained from stability, complementation and western blot analysis, as well as in silico target predictions combined with electrophoretic mobility shift-assays,argue for a stabilizing effect of sRNA154on the polycistronic nif-mRNA and nrpA-mRNA by binding with both loops. Further identified N-related targets were studied, which demonstrates that translation initiation of glnA2-mRNA, encoding glutamine synthetase2, appears to be affected by sRNA154masking the ribosome binding site, whereas glnA1-mRNA appears to be stabilized by sRNA154. Overall, we propose that sRNA154has a crucial regulatory role in N-metabolism in M. mazei by stabilizing the polycistronic mRNA encoding nitrogenase and glnA1-mRNA, as well as allowing a feed forward regulation of nif-gene expression by stabilizing nrpA-mRNA. Consequently, sRNA154represents the first archaeal sRNA, for which a positive posttranscriptional regulation is demonstrated as well as inhibition of translation initiation.
authors.affiliation: a Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität zu Kiel , Am Botanischen Garten 1-9, Kiel , Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Prasse
authors.affiliation: b Zentrum für Infektionsforschung , Universität Würzburg , Josef Schneider-Str. 2/ Bau D15, Würzburg.
authors.forename: Konrad U
authors.identifier: 0000-0002-1481-2996
authors.initials: KU
authors.lastname: Förstner
authors.affiliation: a Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität zu Kiel , Am Botanischen Garten 1-9, Kiel , Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Jäger
authors.affiliation: c Institut für Informatik, Albert-Ludwigs-Universität zu Freiburg , Georges-Koehler-Allee, Freiburg , Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Backofen
authors.affiliation: a Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität zu Kiel , Am Botanischen Garten 1-9, Kiel , Germany.
authors.forename: Ruth A
authors.identifier: 0000-0002-6788-0829
authors.initials: RA
authors.lastname: Schmitz
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D017124:Conserved Sequence; D019848:Gene Expression Regulation, Archaeal; D020745:Genome, Archaeal; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism; D017020:Methanosarcina / Q000235:genetics* / Q000378:metabolism; D009584:Nitrogen / Q000172:deficiency; D009586:Nitrogen Fixation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D009876:Operon; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D019642:RNA, Archaeal / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA
chemical_list: D019843:Archaeal Proteins; D019642:RNA, Archaeal; D012333:RNA, Messenger; D010088:Oxidoreductases; C029130:nitrogenase reductase; C083818:glutamine synthetase 2; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen
keywords: Methanosarcina mazei; RNA stability; nitrogen fixation; nitrogenase; regulatory RNAs