Identification and characterization of E. coli CRISPR‐cas promoters and their silencing by H‐NS

Abstract

SummaryInheritable bacterial defence systems against phage infection and foreign DNA, termed CRISPR (clustered regularly interspaced short palindromic repeats), consist of cas protein genes and repeat arrays interspaced with sequences originating from invaders. The Cas proteins together with processed small spacer-repeat transcripts (crRNAs) cause degradation of penetrated foreign DNA by unknown mechanisms. Here, we have characterized previously unidentified promoters of the Escherichia coli CRISPR arrays and cas protein genes. Transcription of precursor crRNA is directed by a promoter located within the CRISPR leader. A second promoter, directing cas gene transcription, is located upstream of the genes encoding proteins of the Cascade complex. Furthermore, we demonstrate that the DNA-binding protein H-NS is involved in silencing the CRISPR-cas promoters, resulting in cryptic Cas protein expression. Our results demonstrate an active involvement of H-NS in the induction of the CRISPRcas system and suggest a potential link between two prokaryotic defence systems against foreign DNA.

Csy4 is responsible for CRISPR RNA processing inPectobacterium atrosepticum

“…46 In E. coli, crRNA abundance increased with elevated levels of CasE. 23,39,46 In agreement, we observe a similar increase in crRNA levels for the Δcas strain overexpressing Csy4 compared with the wt (compare Fig. 2C and B).…”

“…This 5'UTR is longer than average but it is interesting to note that the +1 for casA in E. coli was at position -180. 39 We speculate that the cas mRNA might be subject to post-transcriptional regulatory mechanisms involving the 5'UTR.…”

“…38,39 Interestingly, analysis of microarray data 40 indicated that all cas genes are expressed in planta during potato infection (data not shown). The transcriptional start site for the cas operon in P. atrosepticum was approximately 140 nt upstream of cas1.…”

“…It is yet to be determined what regulates CRISPR/Cas expression in P. atrosepticum. H-NS and LeuO differentially regulate CRISPR/Cas activity in E. coli, 39,46,47 and therefore may also have a role in P. atrosepticum.…”

Sensing danger

“…Little is known about the regulation of the CRISPR-Cas immune response. The CRISPR-Cas locus present in Escherichia coli has been shown to be tightly repressed by HNS, a negative regulator of widespread effects in bacteria (7). Repression can be relieved by the LeuO activator (8), but environmental or physiological conditions that lead to the expression of the CRISPR-Cas machinery have not been identified.…”