Abstract
The CRISPR/Cas system protects bacteria against bacteriophage and plasmids through a sophisticated mechanism where cas operon plays a crucial role consisting of cse1 and cas3. However, comprehensive studies on the regulation of cas3 operon of the Type I-E CRISPR/Cas system are scarce. Herein, we investigated the regulation of cas3 in Escherichia coli. The mutation in gcvP or crp reduced the CRISPR/Cas system interference ability and increased bacterial susceptibility to phage, when the casA operon of the CRISPR/Cas system was activated. The silence of the glycine cleavage system (GCS) encoded by gcvTHP operon reduced cas3 expression. Adding N5, N10-methylene tetrahydrofolate (N5, N10-mTHF), which is the product of GCS-catalyzed glycine, was able to activate cas3 expression. In addition, a cAMP receptor protein (CRP) encoded by crp activated cas3 expression via binding to the cas3 promoter in response to cAMP concentration. Since N5, N10-mTHF provides one-carbon unit for purine, we assumed GCS regulates cas3 through associating with CRP. It was evident that the mutation of gcvP failed to further reduce the cas3 expression with the crp deletion. These results illustrated a novel regulatory pathway which GCS and CRP co-regulate cas3 of the CRISPR/Cas system and contribute to the defence against invasive genetic elements, where CRP is indispensable for GCS regulation of cas3 expression.
Highlights
Prokaryotic viruses occur ubiquitously and pose a serious threat to bacteria and archaea [1].To resist these extremely large numbers of predators, bacteria have developed multiple resistant mechanisms [2,3,4]
Our study suggested that glycine cleavage system (GCS) affected the bacterial susceptibility to phage by altering cas3 expression, and cAMP receptor protein (CRP) was dispensable for the GCS to regulate cas3 expression
The results showed that the cas3 expression was activated by the minimal of 1 mM glycine in wild type (WT) but it failed to be activated by glycine in ∆gcvP (Figure 3B), which indicated the knock-out of gcvP led to the silence of GCS that the CCS was unable to regulate cas3 expression through glycine usage
Summary
Prokaryotic viruses occur ubiquitously and pose a serious threat to bacteria and archaea [1]. To resist these extremely large numbers of predators, bacteria have developed multiple resistant mechanisms [2,3,4]. As one of the important defence mechanisms, arrays of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute the CRISPR/Cas system which has attracted much attention in terms of its function and application in recent years [5,6]. CRISPR arrays are composed of the spacer sequences acquired from foreign DNA between. A group of cas genes encoding Cas proteins is generally located near the CRISPR loci, and it displays immune function associated with CRISPR arrays to battle with foreign DNA invasion [9].
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