A putative viral defence mechanism in archaeal cells

Abstract

Clusters of regularly spaced direct repeats, separated by unconserved spacer sequences, are ubiquitous in archaeal chromosomes and occur in some plasmids. Some clusters constitute around 1% of chromosomal DNA. Similarly structured clusters, generally smaller, also occur in some bacterial chromosomes. Although early studies implicated these clusters in segregation/partition functions, recent evidence suggests that the spacer sequences derive from extrachromosomal elements, and, primarily, viruses. This has led to the proposal that the clusters provide a defence against viral propagation in cells, and that both the mode of inhibition of viral propagation and the mechanism of adding spacer-repeat units to clusters, are dependent on RNAs transcribed from the clusters. Moreover, the putative inhibitory apparatus (piRNA-based) may be evolutionarily related to the interference RNA systems (siRNA and miRNA), which are common in eukarya. Here, we analyze all the current data on archaeal repeat clusters and provide some new insights into their diverse structures, transcriptional properties and mode of structural development. The results are consistent with larger cluster transcripts being processed at the centers of the repeat sequences and being further trimmed by exonucleases to yield a dominant, intracellular RNA species, which corresponds approximately to the size of a spacer. Furthermore, analysis of the extensive clusters of Sulfolobus solfataricus strains P1 and P2B provides support for the presence of a flanking sequence adjoining a cluster being a prerequisite for the incorporation of new spacer-repeat units, which occurs between the flanking sequence and the cluster. An archaeal database summarizing the data will be maintained at http://dac.molbio.ku.dk/dbs/SRSR/.

Figure 1.

(A) A map of mthe-124 from M. thermoautotrophicus showing the locations of duplicated spacer-repeat units, or groups of units, labeled 1–5. Each triangle represents a spacer-repeat unit. Duplicated spacer-repeat units are shaded in gray. (B) Schematic representation of the six clusters which occur in the genome of S. solfataricus P2B (She et al. 2001). Each triangle represents a spacer-repeat unit. The colored triangles are coded to indicate the archaeal viruses, plasmids or chromosomes (S. solfataricus or A. fulgidus) that yield good matches with the spacer sequence. (C) A corresponding scheme is presented for three of the six clusters (ssol-95, ssol-32 and ssol-7) that are present in the chromosome of the closely related strain S. solfataricus P1 (Accession numbers: DQ831675, DQ831676 and DQ831677). Open triangles denote those spacer-repeat units that differ in sequence between the strains P1 and P2A. Abbreviation: del. indicates the two sites where deletion of several spacer-repeat units has occurred in strain P1 (or where insertions have occurred in strain P2B).

Figure 2.

(A) Alignment of the five conserved flanking sequences adjoining the first repeat (in bold type) of five clusters of the three Methanosarcina strains. A putative TATA-like box is outlined. (B) Alignment of two flanking regions of the P. abyssi genome where paby-1 appears to exhibit a defective start site.

Figure 3.

Typical composition and orientation of the major gene components of superoperons which are closely linked to repeat clusters in crenarchaea and euryarchaea. The cas genes and their COG identities are given in Tables 4 and 5.

Figure 4.

Northern blotting analyses of RNA transcripts obtained from the cluster saci-4 of S. acidocaldarius. (A) Transcripts from one strand were detected in total RNA extracted from cells grown to exponential (expon.) or stationary (stat.) phase. (B) Transcripts were observed from the complementary DNA strand at stationary phase. The approximate estimated nucleotide lengths of the RNA products are given. The minimal detection limit, using 26-nt. probes, was estimated at 15–16 nucleotides. (C) Denotes the location of the primers “a” and “b,” and “L” indicates the location of the putative transcriptional leader within the flanking sequence.