Abstract
Acquisition of spacers confers the CRISPR–Cas system with the memory to defend against invading mobile genetic elements. We previously reported that the CRISPR-associated factor Csa3a triggers CRISPR adaptation in Sulfolobus islandicus. However, a feedback regulation of CRISPR adaptation remains unclear. Here we show that another CRISPR-associated factor, Csa3b, binds a cyclic oligoadenylate (cOA) analog (5′-CAAAA-3′) and mutation at its CARF domain, which reduces the binding affinity. Csa3b also binds the promoter of adaptation cas genes, and the cOA analog enhances their binding probably by allosteric regulation. Deletion of the csa3b gene triggers spacer acquisition from both plasmid and viral DNAs, indicating that Csa3b acted as a repressor for CRISPR adaptation. Moreover, we also find that Csa3b activates the expression of subtype cmr-α and cmr-β genes according to transcriptome data and demonstrate that Csa3b binds the promoters of cmr genes. The deletion of the csa3b gene reduces Cmr-mediated RNA interference activity, indicating that Csa3b acts as a transcriptional activator for Cmr-mediated RNA interference. In summary, our findings reveal a novel pathway for the regulation of CRISPR adaptation and CRISPR–Cmr RNA interference in S. islandicus. Our results also suggest a feedback repression of CRIPSR adaptation by the Csa3b factor and the cOA signal produced by the Cmr complex at the CRISPR interference stage.
Highlights
CRISPR–Cas is a prokaryotic immune system that defends bacteria and archaea against invasive plasmids and viruses (Barrangou et al, 2007; Makarova et al, 2011)
The plasmid transformation efficiencies of both wild-type and csa3b cells were very low (Figure 5D), compared with the transformation efficiency of the empty vector pSeSD, suggesting that Csa3b has less effect on the regulation of Cmr-mediated DNA interference. These results suggested that the cmr genes were expressed in the absence of the csa3b gene, and their expression level was probably sufficient for efficient Cmr-mediated DNA interference in this study using a plasmid as the targeted mobile genetic element (MGE)
Csa3a carries a CARF domain and was previously suggested to bind a ligand for its regulatory effect (Lintner et al, 2011). This ligand was not identified until recently, where cyclic oligoadenylate (cOA) were found to be bound by the CARF domain of Csm6 (Kazlauskiene et al, 2017; Niewoehner et al, 2017) or Csx1 (Han et al, 2018) to regulate their ribonuclease activity
Summary
CRISPR–Cas is a prokaryotic immune system that defends bacteria and archaea against invasive plasmids and viruses (Barrangou et al, 2007; Makarova et al, 2011). Spacer acquisition into CRISPR arrays constitutes the first stage of the immune reaction (Brouns et al, 2008; Westra et al, 2012). Short DNA fragments, called protospacers, are taken from the invasive genetic elements and integrated into the CRISPR arrays at the leader proximal end facilitated by the conserved core proteins Cas and Cas (Makarova et al, 2011) and, in some cases, additional subunits such as Cas family proteins (Kieper et al, 2018; Lee et al, 2018; Shiimori et al, 2018; Zhang et al, 2019). The first successful demonstration of spacer acquisition under laboratory conditions was based on Streptococcus thermophilus subtype II-A system (Barrangou et al, 2007), after which more studies have focused mainly on type I systems (Sternberg et al, 2016), including Sulfolobus subtype I-A systems (Erdmann and Garrett, 2012; Erdmann et al, 2014b; Liu et al, 2015), Haloarcula hispanica subtype I-B system (Li et al, 2014a,b), Escherichia coli subtype I-E system (Swarts et al, 2012; Yosef et al, 2012, 2013), and Pseudomonas aeruginosa (Cady et al, 2012) and Pectobacterium atrosepticum (Richter et al, 2014) subtype I-F systems
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