Prokaryotes have developed several strategies to defend themselves against foreign genetic elements. One of those defense mechanisms is the recently identified CRISPR/Cas system, which is used by approximately half of all bacterial and almost all archaeal organisms. The CRISPR/Cas system differs from the other defense strategies because it is adaptive, hereditary and it recognizes the invader by a sequence specific mechanism. To identify the invading foreign nucleic acid, a crRNA that matches the invader DNA is required, as well as a short sequence motif called protospacer adjacent motif (PAM). We recently identified the PAM sequences for the halophilic archaeon Haloferax volcanii, and found that several motifs were active in triggering the defense reaction. In contrast, selection of protospacers from the invader seems to be based on fewer PAM sequences, as evidenced by comparative sequence data. This suggests that the selection of protospacers has stricter requirements than the defense reaction. Comparison of CRISPR-repeat sequences carried by sequenced haloarchaea revealed that in more than half of the species, the repeat sequence is conserved and that they have the same CRISPR/Cas type.

Non-coding RNAs are key players in many cellular processes within organisms from all three domains of life. The range and diversity of small RNA functions beyond their involvement in translation and RNA processing was first recognized for eukaryotes and bacteria. Since then, small RNAs were also found to be abundant in archaea. Their functions include the regulation of gene expression and the establishment of immunity against invading mobile genetic elements. This review summarizes our current knowledge about small RNAs used for regulation and defence in archaea.

Background: CRISPR/Cas systems allow archaea and bacteria to resist invasion by foreign nucleic acids.

Results: The CRISPR/Cas system in Haloferax recognized six different PAM sequences that could trigger a defense response.

Conclusion: The PAM sequence specificity of the defense response in type I CRISPR systems is more relaxed than previously thought.

Significance: The PAM sequence requirements for interference and adaptation appear to differ markedly.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.