Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition

This is the final version of the article. Available from the publisher via the DOI in this record.Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.This work was financially supported by an NWO Vidi grant to SJJB (864.11.005). RNJ and BW are supported by the National Institutes of Health (GM 103500) and the Montana State University Agricultural Experimental Station. ES, KAD and KS are supported by an NIH grant RO1 GM10407, a program grant in Molecular and Cell Biology from Presidium of Russian Academy of Sciences and a Russian Foundation for Basic Research grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A capture approach for supercoiled plasmid DNA using a triplex-forming oligonucleotide

This is the final version of the article. Available from the publisher via the DOI in this record.Proteins that recognize and bind specific sites in DNA are essential for regulation of numerous biological functions. Such proteins often require a negative supercoiled DNA topology to function correctly. In current research, short linear DNA is often used to study DNA-protein interactions. Although linear DNA can easily be modified, for capture on a surface, its relaxed topology does not accurately resemble the natural situation in which DNA is generally negatively supercoiled. Moreover, specific binding sequences are flanked by large stretches of non-target sequence in vivo. Here, we present a straightforward method for capturing negatively supercoiled plasmid DNA on a streptavidin surface. It relies on the formation of a temporary parallel triplex, using a triple helix forming oligonucleotide containing locked nucleic acid nucleotides. All materials required for this method are commercially available. Lac repressor binding to its operator was used as model system. Although the dissociation constants for both the linear and plasmid-based operator are in the range of 4 nM, the association and dissociation rates of Lac repressor binding to the plasmid-based operator are ~18 times slower than on a linear fragment. This difference underscores the importance of using a physiologically relevant DNA topology for studying DNA-protein interactions.Netherlands Organisation for Scientific Research and the Netherlands Institute for Space Research [ALW-GO-PL/ 08-08]; NWO Vidi grant [864.11.005 to S.J.J.B.]. Funding for open access charge: Microbiology department/ Wageningen UR library

Addiction systems antagonize bacterial adaptive immunity.

This is the author accepted manuscript. The final version is available from Oxford University Press (OUP) via the DOI in this record. CRISPR-Cas systems provide adaptive immunity against mobile genetic elements, but employment of this resistance mechanism is often reported with a fitness cost for the host. Whether or not CRISPR-Cas systems are important barriers for the horizontal spread of conjugative plasmids, which play a crucial role in the spread of antibiotic resistance, will depend on the fitness costs of employing CRISPR-based defences and the benefits of resisting conjugative plasmids. To estimate these costs and benefits we measured bacterial fitness associated with plasmid immunity using Escherichia coli and the conjugative plasmid pOX38-Cm. We find that CRISPR-mediated immunity fails to confer a fitness benefit in the absence of antibiotics, despite the large fitness cost associated with carrying the plasmid in this context. Similar to many other conjugative plasmids, pOX38-Cm carries a CcdAB toxin-antitoxin (TA) addiction system. These addiction systems encode long-lived toxins and short-lived anti-toxins, resulting in toxic effects following the loss of the TA genes from the bacterial host. Our data suggest that the lack of a fitness benefit associated with CRISPR-mediated defence is due to expression of the TA system before plasmid detection and degradation. As most antibiotic resistance plasmids encode TA systems this could have important consequences for the role of CRISPR-Cas systems in limiting the spread of antibiotic resistance.European CommissionBiotechnology & Biological Sciences Research Council (BBSRC)Natural Environment Research CouncilRoyal Society of Biological SciencesNetherlands Organization of Scientific Research (NWO)Wellcome Trus

CRISPR Interference Directs Strand Specific Spacer Acquisition

Background: CRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference. Results: We describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A cooccurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner. Conclusions: The E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster – i

Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS

Background: CRISPR-Cas systems provide adaptive immunity to mobile genetic elements in prokaryotes. In many bacteria, including E. coli, a specialized ribonucleoprotein complex called Cascade enacts immunity by “an interference reaction" between CRISPR encoded RNA (crRNA) and invader DNA sequences called “protospacers”. Cascade recognizes invader DNA via short “protospacer adjacent motif” (PAM) sequences and crRNA-DNA complementarity. This triggers degradation of invader DNA by Cas3 protein and in some circumstances stimulates capture of new invader DNA protospacers for incorporation into CRISPR as “spacers” by Cas1 and Cas2 proteins, thus enhancing immunity. Co-expression of Cascade, Cas3 and crRNA is effective at giving E. coli cells resistance to phage lysis, if a transcriptional repressor of Cascade and CRISPR, H-NS, is inactivated (Δhns). We present further genetic analyses of the regulation of CRISPR-Cas mediated phage resistance in Δhns E. coli cells. Results: We observed that E. coli Type I-E CRISPR-Cas mediated resistance to phage λ was strongly temperature dependent, when repeating previously published experimental procedures. Further genetic analyses highlighted the importance of culture conditions for controlling the extent of CRISPR immunity in E. coli. These data identified that expression levels of cas3 is an important limiting factor for successful resistance to phage. Significantly, we describe the new identification that cas3 is also under transcriptional control by H-NS but that this is exerted only in stationary phase cells. Conclusions: Regulation of cas3 is responsive to phase of growth, and to growth temperature in E. coli, impacting on the efficacy of CRISPR-Cas immunity in these experimental systems

CRISPR Inhibition of Prophage Acquisition in Streptococcus pyogenes

Streptococcus pyogenes, one of the major human pathogens, is a unique species since it has acquired diverse strain-specific virulence properties mainly through the acquisition of streptococcal prophages. In addition, S. pyogenes possesses clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems that can restrict horizontal gene transfer (HGT) including phage insertion. Therefore, it was of interest to examine the relationship between CRISPR and acquisition of prophages in S. pyogenes. Although two distinct CRISPR loci were found in S. pyogenes, some strains lacked CRISPR and these strains possess significantly more prophages than CRISPR harboring strains. We also found that the number of spacers of S. pyogenes CRISPR was less than for other streptococci. The demonstrated spacer contents, however, suggested that the CRISPR appear to limit phage insertions. In addition, we found a significant inverse correlation between the number of spacers and prophages in S. pyogenes. It was therefore suggested that S. pyogenes CRISPR have permitted phage insertion by lacking its own spacers. Interestingly, in two closely related S. pyogenes strains (SSI-1 and MGAS315), CRISPR activity appeared to be impaired following the insertion of phage genomes into the repeat sequences. Detailed analysis of this prophage insertion site suggested that MGAS315 is the ancestral strain of SSI-1. As a result of analysis of 35 additional streptococcal genomes, it was suggested that the influences of the CRISPR on the phage insertion vary among species even within the same genus. Our results suggested that limitations in CRISPR content could explain the characteristic acquisition of prophages and might contribute to strain-specific pathogenesis in S. pyogenes

The Complete Genome Sequence of Thermoproteus tenax: A Physiologically Versatile Member of the Crenarchaeota

Here, we report on the complete genome sequence of the hyperthermophilic Crenarchaeum Thermoproteus tenax (strain Kra 1, DSM 2078(T)) a type strain of the crenarchaeotal order Thermoproteales. Its circular 1.84-megabase genome harbors no extrachromosomal elements and 2,051 open reading frames are identified, covering 90.6% of the complete sequence, which represents a high coding density. Derived from the gene content, T. tenax is a representative member of the Crenarchaeota. The organism is strictly anaerobic and sulfur-dependent with optimal growth at 86 degrees C and pH 5.6. One particular feature is the great metabolic versatility, which is not accompanied by a distinct increase of genome size or information density as compared to other Crenarchaeota. T. tenax is able to grow chemolithoautotrophically (CO2/H-2) as well as chemoorganoheterotrophically in presence of various organic substrates. All pathways for synthesizing the 20 proteinogenic amino acids are present. In addition, two presumably complete gene sets for NADH:quinone oxidoreductase (complex I) were identified in the genome and there is evidence that either NADH or reduced ferredoxin might serve as electron donor. Beside the typical archaeal A(0)A(1)-ATP synthase, a membrane-bound pyrophosphatase is found, which might contribute to energy conservation. Surprisingly, all genes required for dissimilatory sulfate reduction are present, which is confirmed by growth experiments. Mentionable is furthermore, the presence of two proteins (ParA family ATPase, actin-like protein) that might be involved in cell division in Thermoproteales, where the ESCRT system is absent, and of genes involved in genetic competence (DprA, ComF) that is so far unique within Archaea

CRISPR-based adaptive and heritable immunity in prokaryotes.

The recently discovered CRISPR (clustered regularly interspaced short palindromic repeat) defense system protects bacteria and archaea against mobile genetic elements. This immunity system has the potential to continuously adjust its reach at the genomic level, implying that both gain and loss of information is inheritable. The CRISPR system consists of typical stretches of interspaced repetitive DNA (CRISPRs) and associated cas genes. Three distinct stages are recognized in the CRISPR defense mechanism: (i) adaptation of the CRISPR via the integration of short sequences of the invaders as spacers; (ii) expression of CRISPRs and subsequent processing to small guide RNAs; and (iii) interference of target DNA by the crRNA guides. Recent analyses of key Cas proteins indicate that, despite some functional analogies, this fascinating prokaryotic system shares no phylogenetic relation with the eukaryotic RNA interference system