The CARF Protein MM_0565 Affects Transcription of the Casposon-Encoded cas1-solo Gene in Methanosarcina mazei Gö1.

Andrea Ulbricht,Claudia Kießling,Herman Vargas Gebauer,Ruth A Schmitz,Lisa Nickel,Konrad U Förstner,Katrin Weidenbach

doi:10.3390/biom10081161

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.

Highlights

In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas system has gained much interest due to its versatile usability for editing of target genomes
The CRISPR-Cas system has been identified as a prokaryotic adaptive defense system against foreign nucleic acids like viruses, phages and plasmids and can be found in about one third of bacterial and most archaeal genomes sequenced today [4,5,6,7,8]
It is known that some systems are induced, e.g., upon phage challenge, resulting in increased transcription of the CRISPR array and the associated cas genes, as it is reported for Streptococcus thermophilus [20], or general stress conditions

Summary

Introduction

The CRISPR-Cas (clustered regulatory interspaced short palindromic repeat and CRISPR-associated) system has gained much interest due to its versatile usability for editing of target genomes (reviewed in [1,2,3]). The CRISPR-Cas system has been identified as a prokaryotic adaptive defense system against foreign nucleic acids like viruses, phages and plasmids and can be found in about one third of bacterial and most archaeal genomes sequenced today [4,5,6,7,8]. A CRISPR locus is composed of CRISPR-associated (cas) genes, often organized in an operon, the CRISPR array, with variable numbers of direct repeats interrupted by invasive nucleic acid-derived spacer sequences and a leader sequence in front of it. New spacers get incorporated into the CRISPR array after the attack of foreign nucleic acids. Since CRISPR-Cas systems function as defense systems against foreign nucleic acids, they are assumed to be highly active in the presence of invading elements. The transcriptional regulator Csa3b as well as the type I-A interference complex were required for inhibition of transcription of the interference cassette [24]

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Conclusion