The immune systems that protect organisms from infectious agents invariably have a cost for the host. In bacteria and archaea CRISPR-Cas loci can serve as adaptive immune systems that protect these microbes from infectiously transmitted DNAs. When those DNAs are borne by lytic viruses (phages), this protection can provide a considerable advantage. CRISPR-Cas immunity can also prevent cells from acquiring plasmids and free DNA bearing genes that increase their fitness. Here, we use a combination of experiments and mathematical-computer simulation models to explore this downside of CRISPR-Cas immunity and its implications for the maintenance of CRISPR-Cas loci in microbial populations. We analyzed the conjugational transfer of the staphylococcal plasmid pG0400 into Staphylococcus epidermidis RP62a recipients that bear a CRISPR-Cas locus targeting this plasmid. Contrary to what is anticipated for lytic phages, which evade CRISPR by mutations in the target region, the evasion of CRISPR immunity by plasmids occurs at the level of the host through loss of functional CRISPR-Cas immunity. The results of our experiments and models indicate that more than 10−4 of the cells in CRISPR-Cas positive populations are defective or deleted for the CRISPR-Cas region and thereby able to receive and carry the plasmid. Most intriguingly, the loss of CRISPR function even by large deletions can have little or no fitness cost in vitro. These theoretical and experimental results can account for the considerable variation in the existence, number and function of CRISPR-Cas loci within and between bacterial species. We postulate that as a consequence of the opposing positive and negative selection for immunity, CRISPR-Cas systems are in a continuous state of flux. They are lost when they bear immunity to laterally transferred beneficial genes, re-acquired by horizontal gene transfer, and ascend in environments where phage are a major source of mortality.

Precise RNA processing is fundamental to all small RNA-mediated interference pathways. In prokaryotes, clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode small CRISPR RNAs (crRNAs) that protect against invasive genetic elements by antisense targeting. CRISPR loci are transcribed as a long precursor that is cleaved within repeat sequences by CRISPR-associated (Cas) proteins. In many organisms, this primary processing generates crRNA intermediates that are subject to additional nucleolytic trimming to render mature crRNAs of specific lengths. The molecular mechanisms underlying this maturation event remain poorly understood. Here, we defined the genetic requirements for crRNA primary processing and maturation in Staphylococcus epidermidis. We show that changes in the position of the primary processing site result in extended or diminished maturation to generate mature crRNAs of constant length. These results indicate that crRNA maturation occurs by a ruler mechanism anchored at the primary processing site. We also show that maturation is mediated by specific cas genes distinct from those genes involved in primary processing, showing that this event is directed by CRISPR/Cas loci.conjugation | genetic interference | antisense RNA C lustered, regularly interspaced, short palindromic repeat (CRISPR) sequences are present in ∼40% of eubacterial genomes and nearly all archaeal genomes sequenced to date. CRISPR loci consist of short (∼24-48 nt) repeats separated by similarly sized unique spacers (1-4). CRISPR systems protect against bacteriophage and plasmid infection by a genetic interference mechanism that relies on the identity between CRISPR spacers and the invading targets (5-7). CRISPR arrays are transcribed into a long precursor containing spacers and repeats that are processed into small CRISPR RNAs (crRNAs) by dedicated CRISPR-associated (Cas) endoribonucleases (6,8,9). crRNAs act as guides for a targeting complex (10-15) that cleaves the genetic material of the invading bacteriophage or plasmid (16).In many prokaryotes, the biogenesis of mature crRNAs can be divided into two stages: (i) a primary cleavage of the crRNA precursor within repeat sequences that generates intermediate crRNAs containing a full spacer flanked by partial repeats, and (ii) a final maturation event where intermediate crRNAs are subject to additional nucleolytic digestion at one end. Repeat spacer arrays and their adjacent cas genes are classified into three CRISPR/Cas types (I-III) (17) that undergo different mechanisms of crRNA processing. In Types I and III CRISPR/Cas systems, primary processing is achieved by Cas6 endoribonucleases and results in crRNA intermediates with 5′-hydroxyl and 3′-phosphate or 2′-3′-cyclic phosphate ends (9,10,12,13). Cleavage occurs 8 nt upstream of the beginning of the spacer sequence, leaving a 5′ handle (6) or crRNA tag (18) on the 5′ end. In Type III systems, crRNA intermediates are subject to additional nucleolytic attack at the 3′ end, generating mature crRNAs with reduced...

Background: CRISPR immune systems protect prokaryotes from their viruses using small interfering RNAs (crRNAs), which require maturation events during their biogenesis. Results: In Staphylococcus epidermidis, crRNAs undergo maturation in a Cas10⅐Csm ribonucleoprotein complex; Csm3 modulates the extent of maturation. Conclusion: Csm3 acts as a ruler for crRNAs. Significance: Investigating CRISPR immunity is important to understand prokaryotic ecology and to develop biotechnological applications.

Many prokaryotes possess an adaptive immune system encoded by clustered regularly interspaced short palindromic repeats (CRISPRs). CRISPR loci produce small guide RNAs (crRNAs) that, in conjunction with flanking CRISPR-associated (cas) genes, combat viruses and block plasmid transfer by an antisense targeting mechanism. CRISPR-Cas systems have been classified into three types (I to III) that employ distinct mechanisms of crRNA biogenesis and targeting. The type III-A system in Staphylococcus epidermidis RP62a blocks the transfer of staphylococcal conjugative plasmids and harbors nine cas-csm genes. Previous biochemical analysis indicated that Cas10, Csm2, Csm3, Csm4, and Csm5 form a crRNA-containing ribonucleoprotein complex; however, the roles of these genes toward antiplasmid targeting remain unknown. Here, we determined the cas-csm genes that are required for antiplasmid immunity and used genetic and biochemical analyses to investigate the functions of predicted motifs and domains within these genes. We found that many mutations affected immunity by impacting the formation of the Cas10-Csm complex or crRNA biogenesis. Surprisingly, mutations in the predicted nuclease domains of the members of the Cas10-Csm complex had no detectable effect on antiplasmid immunity or crRNA biogenesis. In contrast, the deletion of csm6 and mutations in the cas10 Palm polymerase domain prevented CRISPR immunity without affecting either complex formation or crRNA production, suggesting their involvement in target destruction. By delineating the genetic requirements of this system, our findings further contribute to the mechanistic understanding of type III CRISPR-Cas systems.

ARTS (Sept4_i2) is a pro-apoptotic tumor suppressor protein that functions as an antagonist of X-linked IAP (XIAP) to promote apoptosis. It is generally thought that mitochondrial outer membrane permeabilization (MOMP) occurs before activation of caspases and is required for it. Here, we show that ARTS initiates caspase activation upstream of MOMP. In living cells, ARTS is localized to the mitochondrial outer membrane. In response to apoptotic signals, ARTS translocates rapidly to the cytosol in a caspase-independent manner, where it binds XIAP and promotes caspase activation. This translocation precedes the release of cytochrome C and SMAC/Diablo, and ARTS function is required for the normal timing of MOMP. We also show that ARTS-induced caspase activation leads to cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation that can promote MOMP. This leads to the subsequent release of additional mitochondrial factors, including cytochrome C and SMAC/Diablo, which then amplifies the caspase cascade and causes apoptosis. Apoptosis is important for regulating cell numbers and maintaining tissue homeostasis. The main executioners of apoptosis are caspases, a family of cysteine proteases that cleave substrates after aspartate. 1 In the mitochondrial pathway, release of pro-apoptotic factors, including cytochrome C (cytoC) and Smac/Diablo (SMAC), from the mitochondrial intermembrane space (IMS) to the cytosol promotes caspase activation. This release requires mitochondrial outer membrane permeabilization (MOMP). 2 A holoenzyme complex known as the 'apoptosome' is formed when cytoC is released from mitochondria and binds to apoptotic protease-related factor-1 (Apaf-1) to activate procaspase-9. 3 The best-studied family of caspase inhibitors is the inhibitor of apoptosis (IAP) proteins. 4 IAP proteins contain at least one baculoviral IAP repeat (BIR) domain which can directly interact with caspases and inhibit their apoptotic activity, a RING domain that bestows E3-ubiquitin ligase activity and an Ubiquitin-associated (UBA) domain, which enables the binding of polyubiquitin conjugates via lysine 63. 5-7 X-linked IAP (XIAP) directly inhibits caspases-3, -7 and -9. 8 XIAP is considered to be the most potent inhibitor of caspases in vitro, and elevated levels of this protein are found in human cancers. 9 Although XIAP-null mice are viable, it was recently shown that loss of XIAP function causes elevated caspase-3 activity and sensitizes certain primary cells toward apoptosis. 7 In dying cells, apoptosis can be overcome through the release of caspases from their binding to IAP proteins. [10][11][12][13] Several mammalian XIAP antagonists have been identified, including SMAC, 14,15 Omi/HtrA2 16 and ARTS. 17,18 SMAC and Omi/HtrA2 are located in the mitochondrial IMS, contain a conserved IAP-binding motif (IBM) and are released to the cytosol upon apoptotic induction. 14,15 Genetic inactivation of SMAC and Omi/HtrA2 has fail...