Many bacteria and archaea protect themselves from viruses and other invasive genomes through a genetic interference pathway. The small RNAs that guide this defence specify the direct cleavage of foreign DNA.

Sequence-directed genetic interference pathways control gene expression and preserve genome integrity in all kingdoms of life. The importance of such pathways is highlighted by the extensive study of RNA interference (RNAi) and related processes in eukaryotes. In many bacteria and most archaea, clustered, regularly interspaced short palindromic repeats (CRISPRs) are involved in a more recently discovered interference pathway that protects cells from bacteriophages and conjugative plasmids. CRISPR sequences provide an adaptive, heritable record of past infections and express CRISPR RNAs — small RNAs that target invasive nucleic acids. Here, we review the mechanisms of CRISPR interference and its roles in microbial physiology and evolution. We also discuss potential applications of this novel interference pathway.

All immune systems must distinguish self from non-self to repel invaders without inducing autoimmunity. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci protect bacteria and archaea from invasion by phage and plasmid DNA through a genetic interference pathway1–9. CRISPR loci are present in ~ 40% and ~90% of sequenced bacterial and archaeal genomes respectively10 and evolve rapidly, acquiring new spacer sequences to adapt to highly dynamic viral populations1, 11–13. Immunity requires a sequence match between the invasive DNA and the spacers that lie between CRISPR repeats1–9. Each cluster is genetically linked to a subset of the cas (CRISPR-associated) genes14–16 that collectively encode >40 families of proteins involved in adaptation and interference. CRISPR loci encode small CRISPR RNAs (crRNAs) that contain a full spacer flanked by partial repeat sequences2, 17–19. CrRNA spacers are thought to identify targets by direct Watson-Crick pairing with invasive “protospacer” DNA2, 3, but how they avoid targeting the spacer DNA within the encoding CRISPR locus itself is unknown. Here we have defined the mechanism of CRISPR self/non-self discrimination. In Staphylococcus epidermidis, target/crRNA mismatches at specific positions outside of the spacer sequence license foreign DNA for interference, whereas extended pairing between crRNA and CRISPR DNA repeats prevents autoimmunity. Hence, this CRISPR system uses the base-pairing potential of crRNAs not only to specify a target but also to spare the bacterial chromosome from interference. Differential complementarity outside of the spacer sequence is a built-in feature of all CRISPR systems, suggesting that this mechanism is a broadly applicable solution to the self/non-self dilemma that confronts all immune pathways.

In this issue of Structure, Wiedenheft et al. describe the structure and activity of Cas1, the only protein associated with all CRISPR loci. Cas1 is a metal-dependent deoxyribonuclease, consistent with a role in the adaptation phase of CRISPR immunity against invading nucleic acids.

Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced short palindromic repeats (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, indicating that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.

Vegetative forms of Bacillus anthracis replicate in tissues of an infected host and precipitate lethal anthrax disease. Upon host death, bacilli form dormant spores that contaminate the environment, thereby gaining entry into new hosts where spores germinate and once again replicate as vegetative forms. We show here that sortase C, an enzyme that is required for the formation of infectious spores, anchors BasI polypeptide to the envelope of predivisional sporulating bacilli. BasI anchoring to the cell wall requires the active site cysteine of sortase C and an LPNTA motif sorting signal at the C-terminal end of the BasI precursor. The LPNTA motif of BasI is cleaved between the threonine (T) and the alanine (A) residue; the C-terminal carboxyl group of threonine is subsequently amide linked to the side chain amino group of diaminopimelic acid within the wall peptides of B. anthracis peptidoglycan.

The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.

Cell wall-anchored surface proteins of gram-positive pathogens play important roles during the establishment of many infectious diseases, but the contributions of surface proteins to the pathogenesis of anthrax have not yet been revealed. Cell wall anchoring in Staphylococcus aureus occurs by a transpeptidation mechanism requiring surface proteins with C-terminal sorting signals as well as sortase enzymes. The genome sequence of Bacillus anthracis encodes three sortase genes and eleven surface proteins with different types of cell wall sorting signals. Purified B. anthracis sortase A cleaved peptides encompassing LPXTG motif-type sorting signals between the threonine (T) and the glycine (G) residues in vitro. Sortase A activity could be inhibited by thiol-reactive reagents, similar to staphylococcal sortases. B. anthracis parent strain Sterne 34F2, but not variants lacking the srtA gene, anchored the collagen-binding MSCRAMM (microbial surface components recognizing adhesive matrix molecules) BasC (BA5258/BAS4884) to the bacterial cell wall. These results suggest that B. anthracis SrtA anchors surface proteins bearing LPXTG motif sorting signals to the cell wall envelope of vegetative bacilli.