1. The CARF Protein MM_0565 Affects Transcription of the Casposon-Encoded cas1-solo Gene in Methanosarcina mazei Gö1.
- Author
-
Ulbricht A, Nickel L, Weidenbach K, Vargas Gebauer H, Kießling C, Förstner KU, and Schmitz RA
- Subjects
- Amino Acid Motifs genetics, CRISPR-Associated Proteins genetics, CRISPR-Cas Systems, Gene Expression Regulation, Archaeal genetics, Methanosarcina chemistry, Methanosarcina metabolism, Open Reading Frames genetics, Promoter Regions, Genetic, Protein Binding, Protein Folding, Protein Multimerization genetics, RNA-Seq, CRISPR-Associated Proteins chemistry, Clustered Regularly Interspaced Short Palindromic Repeats genetics, Methanosarcina genetics
- Abstract
Clustered Regularly Interspaced Short Palindromic Repeat (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 associated to the type I-B CRISPR-locus of Methanosarcina mazei Gö1. We show that purified MM_0565 composed of a CRISPR-Cas Associated Rossmann Fold (CARF) and a winged helix-turn-helix domain forms a dimer in solution; in vivo, the dimeric MM_0565 is strongly stabilized under high salt stress. While direct effects on CRISPR-Cas transcription were not detected by genetic approaches, specific binding of MM_0565 to the leader region of both CRISPR-Cas systems was observed by microscale thermophoresis and electromobility shift assays. Moreover, overexpression of MM_0565 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, and taking the absence of the expressed CRISPR locus-encoded Cas1 protein into account, we hypothesize that MM_0565 might modulate the activity of the CRISPR systems on different levels.
- Published
- 2020
- Full Text
- View/download PDF