An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA - PubMed (original) (raw)

An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA

Susan Fischer et al. J Biol Chem. 2012.

Abstract

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.

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Figures

FIGURE 1.

FIGURE 1.

The CRISPR/Cas system of H. volcanii. Two CRISPR genes (P1 and P2) are encoded on minichromosome pHV4 flanking the cas gene cluster. The third CRISPR gene is encoded on the chromosome (C). The cas gene cluster codes for the Cas proteins Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, and Cas8b. Sequencing of the CRISPR P1 locus of the Haloferax strain used in this study (H119) showed that part of this locus was deleted. 23 repeats and 23 spacers are missing in the P1 locus in strain H119. In Haloferax strain DS2, the P1 locus contains 40 repeats and 39 spacers. The repeat sequences of the three CRISPR RNAs are identical except for one nucleotide (bottom right).

FIGURE 2.

FIGURE 2.

Expression of CRISPR genes. RNA from Haloferax cultures grown under different conditions was isolated, separated on 8% denaturing polyacrylamide gels, transferred to membranes, and then hybridized to either a P1 repeat probe (A) or the first spacer of each CRISPR locus (B–D). The P1 repeat probe binds to the repeats of all three CRISPR loci (only one mismatch to the repeat sequences of P2 and C; see Fig. 1). The other probes (spacer1 from P1 (B), spacer1 from locus P2 (C), and spacer1 from locus C (D)) are specific to their respective loci. Lanes no, standard conditions; lanes lowT, lower temperature (30 °C); lanes ex, exponential phase; lanes st, stationary phase. The sizes of a DNA marker are shown on the left in nucleotides. The crRNA is shown schematically on the right.

FIGURE 3.

FIGURE 3.

A plasmid-based invader assay for the identification of PAM sequences. A, the spacer sequence P1-1 was cloned adjacent to potential PAM sequences into the vector pTA409. For initial screening, the potential PAM sequence was cloned upstream as well as downstream of the spacer sequence. The second screening was done with constructs containing the PAM sequences only upstream of the spacer sequence. Selection for transformants was achieved by growth without uracil, which is only possible with the pyrE2 selection marker encoded on the vector. Cells can grow if the invader plasmid is retained, such as when it does not trigger a CRISPR/Cas interference response. If a nucleotide combination is active as a PAM, the plasmid will be recognized by the CRISPR/Cas system and degraded, so precluding growth of transformants on selective medium. B, transformation of Haloferax cells with invader plasmids carrying the correct PAM sequence results in failure to grow for almost all cells (right plate, pTA409-PAM9 (ACT)), whereas transformation with the vector (without PAM and P1-1) (left plate, pTA409; only a dilution of the transformed cells has been plated) results in a lawn of cells.

FIGURE 4.

FIGURE 4.

Analysis of CRISPR/Cas escape mutants. Thirty individual colonies of escape mutants that grew on selective medium after challenge with invader plasmid pTA409-PAM9 (ACT) were analyzed using PCR and Southern blot analyses and grouped into five categories according to the lesions identified. Most surviving colonies (77%) had deletions or mutations of the cas gene cluster. Additional ways of escape were the deletion of the spacer P1-1 from the CRISPR locus (10%) or the plasmid (7%). For one escape mutant (3%), the mutations leading to survival could not be identified. One mutant (3%) had a mutation in the PAM sequence, rendering it non-functional. One mutant had a deletion that covered the cas gene cluster and CRISPR locus P1 (including spacer1); this mutant is only shown under cas gene deletion.

FIGURE 5.

FIGURE 5.

Analyses of escape mutants for cas gene cluster. A, PCR analyses. Twenty-six escape mutants were analyzed by PCR on chromosomal DNA for the presence of the cas genes cas3–cas1. The cas gene locus is shown schematically at the top. Primers hybridized as indicated with arrows in the cas3 gene and cas1 gene. For 10 escape mutants, a full-length PCR product is detected (lanes 1, 3, 4, 8, 11, 14, 17, and 24–26); for the other escape mutants, a shorter PCR product of approximately 1 kb is visible. Lanes 1–26, PCR products with chromosomal DNA from escape mutants; lane M, DNA size marker (sizes are given at both sides in kilobase pairs); lane wt, DNA from H119; lane −, control reaction without DNA. B, Southern blot of escape mutants. The cas gene locus is shown schematically at the top. Chromosomal DNA of escape mutants was digested with SmaI, separated on an agarose gel, and blotted. A DIG-labeled probe was used for hybridization as indicated with the red line below the cas1 gene in the scheme. If no deletion in the locus occurred, a 9.2-kb fragment should be detected. The wild-type cas gene locus fragment is present in escape mutants 1, 3, 4, 8, 11, 14, 17, 24–27, and 29, confirming the PCR results (see A and Table 3). The probe used does not pick up any signal for escape mutants 2, 5–7, 9, 10, 12, 13, 15, 16, 18–20, 21–23, 28, and 30. Lanes 1–30, DNA from wild-type and escape mutants; lane wt, DNA from H119; lanes M1 and M2, DNA size marker (sizes are given at the left in kilobase pairs).

FIGURE 6.

FIGURE 6.

A, PCR analysis of escape mutants for the presence of spacer P1-1. Sixteen escape mutants were analyzed by PCR on chromosomal DNA for the presence of spacer P1-1. The CRISPR locus P1 is shown schematically at the top. Repeats are shown as diamonds, and spacers are shown as rectangles. PCR primers bind as indicated by arrows in the leader region and spacer4 of the CRISPR locus P1. If P1-1 is present, the expected PCR product would have a size of 395 bp. Three escape mutants generated a full-length PCR product (lanes 3, 4, and 11), whereas one escape mutant showed a shorter PCR product of approximately 250 bp (lane 8). Sequence analysis showed that this mutant had a deletion of two repeats and two spacers (spacer1 and spacer2). For all other mutants, no product was generated, suggesting that there is a longer deletion in this region that prevents binding of one or both PCR primers. Lanes 1–16, PCR products with chromosomal DNA from escape mutants; lane M, DNA size marker (sizes are given at the left of the gel in base pairs); lane −, control PCR without addition of template DNA. B, Southern blot of escape mutants. The CRISPR locus P1 and flanking regions as well as the invader plasmid carrying the P1-1 and PAM sequences are shown schematically at the top. Repeats are shown as diamonds, and spacers are shown as rectangles. Total DNA (genomic DNA and plasmids (pTA409-PAM9)) was isolated from escape mutants, digested with SmaI, separated on an agarose gel, blotted onto a membrane, and hybridized to a DIG-labeled oligonucleotide complementary to spacer P1-1 (indicated by the gray line below spacer1 in the diagram). Using this probe, the P1-1 sequence can be detected on the chromosome as well as on the plasmid pTA409-PAM9. If no deletion in the CRISPR locus occurred, a 2.7-kb fragment should be detected. If the P1-1 spacer was not removed from the plasmid, a 720-bp fragment should be visible. The wild-type CRISPR locus fragment is present in escape mutants 3, 4, and 11, confirming the PCR results (see A). The probe did not pick up any signal for escape mutants 1, 8, and 14. In escape mutant 8, at least spacer1 is deleted, but the leader and spacer4 are, according to the PCR product, still present (see A). Mutants 2, 5–7, 9, 10, 12, 13, and 15 show a fragment of about 3.5 kb, which is larger than the wild-type band, suggesting a major change in this region. This was confirmed later as all these mutants had the complete cas gene cluster and parts of the adjoining CRISPR loci deleted (Fig. 5). All escape mutants analyzed here still contain the P1-1 spacer on the plasmid because in all lanes the 700-bp fragment is visible. Lanes 1–15, plasmid and chromosomal DNA from escape mutants; lane wt, DNA from H119; lanes M1 and M2, DNA size marker (sizes are given at the left in kilobase pairs).

FIGURE 7.

FIGURE 7.

Analyses of escape mutants 16–30 for the presence of spacer P1-1. A, PCR analysis. Fourteen escape mutants were analyzed by PCR on chromosomal DNA for the presence of spacer P1-1. The CRISPR locus P1 is shown schematically at the top. Repeats are shown as diamonds, and spacers are shown as rectangles. Primers used are indicated with arrows in the leader region and spacer4 of the CRISPR locus P1. If P1-1 is not deleted, the expected PCR product would have a size of 395 bp (arrow wt). For nine escape mutants, a full-length PCR product was detected (lanes 17, 20, 21, and 24–29). For all other mutants, no product was generated, suggesting that there is a longer deletion in this region that prevents binding of one or both PCR primers. Lanes 16–30, PCR products with chromosomal DNA from escape mutants; lane M, DNA size marker (sizes are given at the left of the gel in base pairs); lane wt, control PCR with wild-type DNA. B, Southern blot of escape mutants. The CRISPR locus P1 and flanking regions as well as the invader plasmid carrying the P1-1 and PAM sequences are shown schematically at the top. Repeats are shown as diamonds, and spacers are shown as rectangles. Chromosomal DNA and plasmids of escape mutants were digested with SmaI, separated on an agarose gel, and blotted. A DIG-labeled oligonucleotide complementary to spacer P1-1 was used for hybridization as indicated with the red line below spacer1 in the scheme. Using this probe, the P1-1 sequence could be detected on the chromosome as well as on the plasmid (if present). If no deletion in the CRISPR locus occurred, a 2.7-kb fragment should be detected (arrow wt), and if the P1-1 was not removed from the plasmid, a 720-bp fragment should be visible. The wild-type CRISPR locus fragment of 2.7 kb is present in escape mutants 17, 24–27, and 29, confirming the PCR results (see A). A fragment longer than the wild-type fragment is detected in mutants 18–23, 28, and 30. In these mutants, a rearrangement of this locus has occurred as observed later upon analysis of the neighboring cas gene cluster (Figs. 5 and 6). Some mutants show the presence of spacer1 in the Southern blot (escape mutants 18, 19, 22, 23, and 30) (data not shown) with fragments larger than the expected wild-type fragment of 2.7 kb, but they do not give a signal in the PCR (see A and Table 3). This is due to the rearrangement in this locus that deletes the complete cas gene cluster along with parts of the adjoining CRISPR loci. In these cases, spacer4 from CRISPR locus P1 was also deleted; therefore, the PCR primer cannot bind, and the PCR could not work, but spacer1 is still present. Except for mutants 25 and 26, all escape mutants analyzed here still contain the P1-1 spacer on the plasmid because in these lanes the 700-bp fragment is visible. Lanes 16–30, plasmid and chromosomal DNA from escape mutants; lane wt, DNA from H119; lanes M1 and M2, DNA size marker (sizes are given at the left in kilobase pairs).

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