Characterization of CRISPR RNA biogenesis and Cas6 cleavage-mediated inhibition of a provirus in the haloarchaeon Haloferax mediterranei

Abstract

The adaptive immune system comprising CRISPR (clustered regularly interspaced short palindromic repeats) arrays and cas (CRISPR-associated) genes has been discovered in a wide range of bacteria and archaea and has recently attracted comprehensive investigations. However, the subtype I-B CRISPR-Cas system in haloarchaea has been less characterized. Here, we investigated Cas6-mediated RNA processing in Haloferax mediterranei. The Cas6 cleavage site, as well as the CRISPR transcription start site, was experimentally determined, and processing of CRISPR transcripts was detected with a progressively increasing pattern from early log to stationary phase. With genetic approaches, we discovered that the lack of Cas1, Cas3, or Cas4 unexpectedly resulted in a decrease of CRISPR transcripts, while Cas5, Cas6, and Cas7 were found to be essential in stabilizing mature CRISPR RNA (crRNA). Intriguingly, we observed a CRISPR- and Cas3-independent inhibition of a defective provirus, in which the putative Cascade (CRISPR-associated complex for antiviral defense) proteins (Cas5, Cas6, Cas7, and Cas8b) were indispensably required. A sequence carried by a proviral transcript was found to be homologous to the CRISPR repeat RNA and vulnerable to Cas6-mediated cleavage, implying a distinct interference mechanism that may account for this unusual inhibition. These results provide fundamental information for the subtype I-B CRISPR-Cas system in halophilic archaea and suggest diversified mechanisms and multiple physiological functions for the CRISPR-Cas system.

Fig 1

Depiction of the genomic contexts of CRISPR loci interspersed within the H. mediterranei genome. Each CRISPR locus is indicated by an array of white bars (each bar represents a repeat-spacer unit); the black bars represent the leader sequence. The gray rectangles represent two proviruses, and the small black and gray arrows around provirus 1, respectively, indicate the test or control primers used for detecting its excision from the chromosome. The proviral genes (int) encoding integrases are shown.

Fig 2

Determination of the transcription start site (TSS; solid triangles) and pre-crRNA cleavage site (PCS; empty triangles). (A) Multiple alignments of partial leader sequences (consensus sequence on the top) and the first repeats (consensus sequence on the bottom) of the H. mediterranei arrays. Black or grey shading indicates the same nucleotide at this position in all or most sequences, respectively. The putative promoter motifs (BRE motif, TATA box, and −10 element) and the AT(C/T)GG(G/C)CATG leader motif (immediately preceding the first repeat), are boxed in black. The TRTR element containing the TSS is underlined. The black arrows at −37 and −31 indicate the 5′ ends of two truncated C2 arrays used in the experiment shown in panel C. (B) The primer extension result with a primer specific to the first spacer of P12. (C) Northern blot analysis with a repeat probe validating the prediction of promoter motifs. pC2-37 and pC2-31 are pWL502 vectors carrying C2 arrays with differently truncated 5′ leaders (shown in panel A). These plasmids were transformed into a CRISPR-free (CRF) strain, and total RNA was sampled at stationary phase. Values to the left indicate RNA size markers, in nucleotides. (D) Predicted secondary structure of the repeat RNA from P12 using the RNAfold server.

Fig 3

Analysis of crRNA biogenesis pattern. (A) Northern analysis against H. mediterranei DF50 total RNA sampled at different time points with a repeat probe. Smaller processed products are labeled. (B) Nonspecific degradation was detected when the endonuclease Cas6 was missing. The RNA samples were separated by an 8% PAGE gel (left) or a 1.2% agarose gel (right). (C) Northern analysis of a stationary-phase RNA sample with a mixture of probes against the transcribed leader regions. (D) Schematic diagram showing the generation of smaller products and their sizes; the distribution of various spacer (SP) sizes is also shown. (E) Transcriptional analysis of each CRISPR locus with spacer-specific probes. For P12, probes against the first (S1) and third (S3) spacers were used for two separate experiments. The product pattern of the one-spacer array C2 was different, maybe due to the degeneration of the second repeat. Values to the left indicate RNA size markers, in nucleotides.

Fig 4

Dissection of the roles of Cas proteins in crRNA biogenesis. (A) Northern blot of total RNA from the cas mutants and their parental strain (DF50) with the repeat probe. After exposure and stripping, a second hybridization was performed with the 7S RNA-specific probe serving as the internal control. (B) Northern blot with a repeat probe analyzing the products from the modified pWL502 vectors carrying a wild-type C2 array (pC2) or a variant array (pVC2) lacking the AT(C/T)GG(G/C)CATG leader motif (Fig. 2A). These plasmids were transformed into a CRISPR-free strain. Values to the left indicate RNA size markers, in nucleotides.

Fig 5

Detection of attA site restoration of provirus 1 in strains with various CRISPR-Cas backgrounds. The PCR product indicating attA site restoration and a control product were labeled in panels A and D, and the corresponding primers are diagramed in Fig. 1. (A) Detection of attA site restoration in strains with different cas gene environments. The Δcas6::cas6 and Δcas6::ctrl strains are Δcas6 strains complemented with a pWL502 vector carrying the gene cas6 or not, respectively. (B) Northern blot analysis with a repeat probe confirming the extinction of crRNA in a CRF strain. (C) Validation of the genotype of the CRISPR-free (CRF) strain. Forward and backward primers were ∼600 bp upstream or downstream from each deleted CRISPR array; thus, smaller products (∼1.2 kb) for the CRF strain indicate the absence of these arrays. (D) The effect of CRISPR deficiency on attA site restoration. Lanes M, molecular size markers.

Fig 6

Analysis of a noncognate repeat-like sequence on the proviral phiH transcript. (A) The repeat-like sequence (boxed) on the phiH transcript. The transcription start (TSS) and termination (TTS) sites determined in a cas6 mutant strain are labeled. The putative BRE and TATA elements are underlined, and the start and stop codons are in bold. The Cas6 cleavage site within the repeat-like sequence determined in the DF50 strain is indicated with a small black triangle. (B) Northern analysis of crRNA production from a modified C2 array (pNCG) whose first repeat has been replaced by the proviral noncognate sequence, with a probe against the spacer sequence. Values to the left indicate RNA size markers, in nucleotides. (C) Alignment of the noncognate repeat of the phiH transcript to the consensus sequence of the haloarchaeal CRISPR repeats (Hbor6C to Npha5C: Hbor, Halogeometricum borinquense; Hhis, Haloarcula hispanica; Hmar, Haloarcula marismortui; Hmed, H. mediterranei; Hmuk, Halomicrobium mukohataei; Hwal, Haloquadratum walsbyi; Hvol, Haloferax volcanii; Huta, Halorhabdus utahensis; Nmag, Natrialba magadii; Npha, Natronomonas pharaonis). Mismatches are indicated by asterisks. P or C represents the location on a plasmid or a chromosome, respectively, and the number denotes the repeat number. Black or grey shading indicates the same nucleotide at this position in all or most sequences, respectively. (D) Secondary structure of the repeat-like RNA sequence predicted by the RNAfold server.