In this work, we systematically studied the abundance of processed cse-subtype CRISPR cassette transcripts in E. coli. Our results indicate that there is no significant decrease in abundance of processed CRISPR transcripts as the distance from the leader located between the cas genes cluster and the CRISPR cassette is increased. Thus, whatever the biological function of CRISPR locus of E. coli is, “older” spacers should be as functional as new ones, at least in E. coli. It is possible however that a gradient in abundance of processed CRISPR transcripts exists in organisms with long cassettes.
Since CRISPR transcripts are not translated, it is likely that special antitermination mechanisms exist to ensure efficient transcription of CRISPR RNA. In the case of rDNA transcription, a poorly characterized antitermination system that relies on an upstream rut
element ensures efficient production of full-sized transcripts (Arnvig et al., 2008
). No sequences matching the rut
sequence are present in CRISPR transcripts. However, conserved elements present in CRISPR I and CRISPR II leader sequences between the transcription start point and beginning of the first repeat may act in cis
to ensure efficient transcription of the CRISPR cassette. Further experimentation in this direction seems to be warranted.
The apparent absence of processed CRISPR I spacer 13 transcript could be due to the fact that spacer 13 is followed by the most degenerate and probably the “oldest” repeat of CRISPR I cassette. The sequence of this repeat differs from other CRISPR repeat sequences. It is possible that the distinct sequence of the oldest repeat that is shared by all E. coli strains carrying CRISPR I cassette i) ensures transcription termination to prevent production of antisense transcripts of the iap gene located immediately downstream and ii) destabilizes the processed spacer 13 transcript that must arise upon the upstream processing event that generates processed spacer 12 transcript. Again, further experimentation will be needed to understand the events at this side of the cassette.
We identified promoters responsible for E. coli
CRISPR transcription. Both CRISPR I and CRISPR II promoters are quite weak. Absence of sequence conservation upstream of extended −10 elements of CRISPR I and CRISPR II promoters argues against the presence of additional DNA binding factors that ensure co-regulation of transcription of the two cassettes. Both CRISPR promoters contain a TG motif characteristic of promoters of the extended −10 class. The TG motif clearly contributes to CRISPR promoter strength, as fortuitous down mutation in the TG motif of CRISPR II promoter demonstrates. Yet, neither CRISPR promoter can function in the absence of σ70
conserved region 4.2 that is responsible for the recognition of the −35 promoter consensus element. Neither promoter contains sequences similar to the −35 consensus element, indicating that σ70
conserved region 4.2 makes favorable but non-specific interactions with CRISPR promoter DNA. While our work was in progress, Pul et al., (2010)
reported the mapping of E. coli
CRISPR promoters that matches our results. These authors went on to show that the activity of CRISPR promoters is negatively affected by H-NS, a histone-like architectural protein that appears to be employed by the cell to keep CRISPR transcription low.
The previous observation of Brouns et al., (2008)
on the very strong increase in the abundance of processed CRISPR transcripts upon disruption of casA, casB
, and, to a lesser extent, casE
, was difficult to rationalize. Here, we show this effect to be an artifact caused by read though transcription from the kanamycin resistance cassette that was used to disrupt cas
genes, which leads to the increased synthesis of CasE. The result shows that caution should be exercised when interpreting phenotypes of Keio collection strains harboring disruptions of genes, which are part of operons. CasE is a nuclease that is responsible for CRISPR RNA processing (Brouns et al., 2008
). Once processed, CRISPR transcripts are very stable, whether increased amounts of CasE are present or not. Disruption of casC
with a kanamycin resistance cassette has a similar stimulatory effect on casE
transcript abundance as disruption of casA
. Yet, disruption of casC
leads to much smaller increase in processed CRISPR transcript abundance. This effect suggests that CasC needs to be present for efficient CasE-dependent processing of the full-sized CRISPR transcript to occur. The strong increase of processed CRISPR transcripts abundance mentioned by Pul et al., (2010)
and confirmed by us is likely a consequence of increased casE
expression, since transcription of cas
operon is negatively regulated by H-NS (Pul et al., 2010
Since overexpression of CasE alone is sufficient for complete processing of the CRISPR transcript (and, conversely, virtually no processing occurs at basal levels of casE expression), it follows that other cas gene products are not limiting the processing rate at their physiological concentrations. A very strong increase in abundance of processed CRISPR transcripts upon CasE overexpression occurs despite very low steady-state levels of full-sized CRISPR transcripts observed in wild-type cells. We provide experimental data that explain this dramatic accumulation by differential stabilities of processed and unprocessed CRISPR transcripts. The short half-life of unprocessed CRISPR transcripts is determined by an as yet unidentified RNase, which may be an important negative regulator of CRISPR system function.
The principal result of our work is the demonstration that E. coli
CRISPR system can function in phage defense. Using an engineered strain with expanded CRISPR cassette containing a λ-specific spacer, we show that development of phage λ is inhibited in the context of wild-type cells and prevented in the context of an hns
mutant. The result provides an answer to a perplexing observation that CRISPR system was never recovered as a functional phage-resistance mechanism despite years of research by some of the giants of early molecular biology (Young, 2008
). It now appears that E. coli
CRISPR system, while clearly active based on the enormous variability of the locus (Díez-Villaseñor et al., 2010
and our unpublished observations), is kept inactive at laboratory conditions by (at least) H-NS and an unidentified RNase that degrades full-sized CRISPR transcripts. Determination of physiological conditions that activate the E. coli
CRISPR system remain the subject of ongoing experiments in our laboratory.
Proto-spacer adjacent motifs (PAMs) within target sequences constitute a cornerstone component of at least some of CRISPR/Cas immune systems (Deveau et al., 2008
), allowing a self vs. non-self discrimination of target DNA molecules. Mutations in PAM have been shown to prevent CRISPR mediated immunity even at conditions of a perfect match between the spacer and protospacer sequences (Deveau et al., 2008
). The absence of PAM within the CRISPR locus (and the presence of repeats) explains why the CRISPR locus itself is not recognized by the cognate small CRISPR RNAs that are generated inside the cell (Marraffini and Sontheimer, 2010
). Analysis of matches between known E. coli
spacers and E. coli
phage and, most notably, plasmid sequences allowed Mojica et al. (2009)
to identify a likely PAM consensus AWG. On the other hand, Brouns et al. (2008)
had shown, that at least at conditions of artificial CRISPR cassette and cas
genes co-overexpression, engineered CRISPR spacers provide efficient protection from phage λ infection even in the absence of PAM-like sequences adjacent to protospacer. In particular, the protospacer matching their T3 spacer that was also used in our work has a TGG sequence instead of PAM, with only one position (a G immediately adjacent to the protospacer) matching the proposed PAM consensus. While the relaxed requirement for PAM in the Brouns et al. experimental system could have been caused by co-overexpression of CRISPR/Cas components, in our case efficient interference with phage λ infection is achieved even with genomically located T3 spacer. The result thus may suggest that the stringency of requirements for PAM vary in bacteria (less stringent in E. coli
, more stringent in S. thermophilus
) or, alternatively, that elements within the spacer itself can override the requirement for PAM. These questions are currently being investigated in our laboratory.