title: Two CRISPR-Cas systems in Methanosarcina mazei strain Gö1 display common processing features despite belonging to different types I and III.
abstract: The clustered regularly interspaced short palindromic repeats (CRISPR) system represents a highly adaptive and heritable defense system against foreign nucleic acids in bacteria and archaea. We analyzed the two CRISPR-Cas systems in Methanosarcina mazei strain Gö1. Although belonging to different subtypes (I-B and III-B), the leaders and repeats of both loci are nearly identical. Also, despite many point mutations in each array, a common hairpin motif was identified in the repeats by a bioinformatics analysis and in vitro structural probing. The expression and maturation of CRISPR-derived RNAs (crRNAs) were studied in vitro and in vivo. Both respective potential Cas6b-type endonucleases were purified and their activity tested in vitro. Each protein showed significant activity and could cleave both repeats at the same processing site. Cas6b of subtype III-B, however, was significantly more efficient in its cleavage activity compared with Cas6b of subtype I-B. Northern blot and differential RNAseq analyses were performed to investigate in vivo transcription and maturation of crRNAs, revealing generally very low expression of both systems, whereas significant induction at high NaCl concentrations was observed. crRNAs derived proximal to the leader were generally more abundant than distal ones and in vivo processing sites were clarified for both loci, confirming the previously well-established 8 nt 5' repeat tags. The 3'-ends were more diverse, but generally ended in a prefix of the following repeat sequence (3'-tag). The analysis further revealed a 5'-hydroxy and 3'-phosphate termini architecture of small crRNAs specific for cleavage products of Cas6 endonucleases from type I-E and I-F and type III-B.
authors.affiliation: Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität zu Kiel, Am Botanischen Garten 1-9, Kiel, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Nickel
authors.forename: Katrin
authors.initials: K
authors.lastname: Weidenbach
authors.forename: Dominik
authors.initials: D
authors.lastname: Jäger
authors.forename: Rolf
authors.initials: R
authors.lastname: Backofen
authors.forename: Sita J
authors.initials: SJ
authors.lastname: Lange
authors.forename: Nadja
authors.initials: N
authors.lastname: Heidrich
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Schmitz
mesh_terms: D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems; D019295:Computational Biology; D017020:Methanosarcina / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012323:RNA Processing, Post-Transcriptional; D019642:RNA, Archaeal / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA; D012965:Sodium Chloride
chemical_list: D064130:CRISPR-Associated Proteins; D019642:RNA, Archaeal; D012965:Sodium Chloride
keywords: CRISPR-Cas system; Methanosarcina mazei; immunity of prokaryotes; methanoarchaea; phages; regulatory RNA