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Diverse CRISPRs Evolving in Human Microbiomes
Rho, Mina
Wu, Yu-Wei
Tang, Haixu
Doak, Thomas G.
Ye, Yuzhen
PLoS Genetics
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with cas (CRISPR–associated) genes, form the CRISPR/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human Microbiome Project (HMP) enables us to explore the distribution and diversity of known CRISPRs in human-associated microbial communities and to discover new CRISPRs. We propose a targeted assembly strategy to reconstruct CRISPR arrays, which whole-metagenome assemblies fail to identify. For each known CRISPR type (identified from reference genomes), we use its direct repeat consensus sequence to recruit reads from each HMP dataset and then assemble the recruited reads into CRISPR loci; the unique spacer sequences can then be extracted for analysis. We also identified novel CRISPRs or new CRISPR variants in contigs from whole-metagenome assemblies and used targeted assembly to more comprehensively identify these CRISPRs across samples. We observed that the distributions of CRISPRs (including 64 known and 86 novel ones) are largely body-site specific. We provide detailed analysis of several CRISPR loci, including novel CRISPRs. For example, known streptococcal CRISPRs were identified in most oral microbiomes, totaling ∼8,000 unique spacers: samples resampled from the same individual and oral site shared the most spacers; different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences on the evolution of CRISPR defenses. We further demonstrate potential applications of CRISPRs to the tracing of rare species and the virus exposure of individuals. This work indicates the importance of effective identification and characterization of CRISPR loci to the study of the dynamic ecology of microbiomes.
Author Summary
Human bodies are complex ecological systems in which various microbial organisms and viruses interact with each other and with the human host. The Human Microbiome Project (HMP) has resulted in >700 datasets of shotgun metagenomic sequences, from which we can learn about the compositions and functions of human-associated microbial communities. CRISPR/Cas systems are a widespread class of adaptive immune systems in bacteria and archaea, providing acquired immunity against foreign nucleic acids: CRISPR/Cas defense pathways involve integration of viral- or plasmid-derived DNA segments into CRISPR arrays (forming spacers between repeated structural sequences), and expression of short crRNAs from these single repeat-spacer units, to generate interference to future invading foreign genomes. Powered by an effective computational approach (the targeted assembly approach for CRISPR), our analysis of CRISPR arrays in the HMP datasets provides the very first global view of bacterial immunity systems in human-associated microbial communities. The great diversity of CRISPR spacers we observed among different body sites, in different individuals, and in single individuals over time, indicates the impact of subtle niche differences on the evolution of CRISPR defenses and indicates the key role of bacteriophage (and plasmids) in shaping human microbial communities.
Related Articles
Nasty Viruses, Costly Plasmids, Population Dynamics, and the Conditions for Establishing and Maintaining CRISPR-Mediated Adaptive Immunity in Bacteria
Levin, Bruce R.
PLoS Genetics
Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) abound in the genomes of almost all archaebacteria and nearly half the eubacteria sequenced. Through a genetic interference mechanism, bacteria with CRISPR regions carrying copies of the DNA of previously encountered phage and plasmids abort the replication of phage and plasmids with these sequences. Thus it would seem that protection against infecting phage and plasmids is the selection pressure responsible for establishing and maintaining CRISPR in bacterial populations. But is it? To address this question and provide a framework and hypotheses for the experimental study of the ecology and evolution of CRISPR, I use mathematical models of the population dynamics of CRISPR-encoding bacteria with lytic phage and conjugative plasmids. The results of the numerical (computer simulation) analysis of the properties of these models with parameters in the ranges estimated for Escherichia coli and its phage and conjugative plasmids indicate: (1) In the presence of lytic phage there are broad conditions where bacteria with CRISPR-mediated immunity will have an advantage in competition with non-CRISPR bacteria with otherwise higher Malthusian fitness. (2) These conditions for the existence of CRISPR are narrower when there is envelope resistance to the phage. (3) While there are situations where CRISPR-mediated immunity can provide bacteria an advantage in competition with higher Malthusian fitness bacteria bearing deleterious conjugative plasmids, the conditions for this to obtain are relatively narrow and the intensity of selection favoring CRISPR weak. The parameters of these models can be independently estimated, the assumption behind their construction validated, and the hypotheses generated from the analysis of their properties tested in experimental populations of bacteria with lytic phage and conjugative plasmids. I suggest protocols for estimating these parameters and outline the design of experiments to evaluate the validity of these models and test these hypotheses.
Author Summary
CRISPR is the acronym for the adaptive immune system that has been found in almost all archaebacteria and nearly half the eubacteria examined. Unlike the other defenses bacteria have for protection from phage and other deleterious DNAs, CRISPR has the virtues of specificity, memory, and the capacity to abort infections with a virtually indefinite diversity of deleterious DNAs. In this report, mathematical models of the population dynamics of bacteria, phage, and plasmids are used to determine the conditions under which CRISPR can become established and will be maintained in bacterial populations and the contribution of this adaptive immune system to the ecology and (co)evolution of bacteria and bacteriophage. The models predict realistic and broad conditions under which bacteria bearing CRISPR regions can invade and be maintained in populations of higher fitness bacteria confronted with bacteriophage and narrower conditions when the confrontation is with competitors carrying conjugative plasmids. The models predict that CRISPR can facilitate long-term co-evolutionary arms races between phage and bacteria and between phage- rather than resource-limited bacterial communities. The parameters of these models can be independently estimated, the assumptions behind their construction validated, and the hypotheses generated from the analysis of their properties tested with experimental populations of bacteria.
Related Articles
A PNPase Dependent CRISPR System in Listeria
Sesto, Nina
Touchon, Marie
Andrade, José Marques
Kondo, Jiro
Rocha, Eduardo P. C.
Arraiano, Cecilia Maria
Archambaud, Cristel
Westhof, Éric
Romby, Pascale
Cossart, Pascale
PLoS Genetics
The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in "CRISPRology".
Author Summary
CRISPR-Cas systems confer to bacteria and archaea an adaptive immunity that protects them against invading bacteriophages and plasmids. In this study, we characterize a CRISPR (RliB-CRISPR) that is present in all L. monocytogenes strains at the same genomic locus but is never associated with a cas operon. It is an unusual CRISPR that, as we demonstrate, has a secondary structure consisting of basepair interactions between the repeat sequence and the adjacent spacer. We show that the RliB-CRISPR is processed by the endogenously encoded polynucleotide phosphorylase enzyme (PNPase). In addition, we show that the RliB-CRISPR system requires PNPase and presence of trans encoded cas genes of a second CRISPR-Cas system, to mediate DNA interference directed against a plasmid carrying a matching protospacer. Altogether, our data reveal a novel type of CRISPR system in bacteria that requires endogenously encoded PNPase enzyme for its processing and interference activity.
Related Articles
Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands
Vercoe, Reuben B.
Chang, James T.
Dy, Ron L.
Taylor, Corinda
Gristwood, Tamzin
Clulow, James S.
Richter, Corinna
Przybilski, Rita
Pitman, Andrew R.
Fineran, Peter C.
PLoS Genetics
In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.
Author Summary
Bacteria have evolved mechanisms that provide protection from continual invasion by viruses and other foreign elements. Resistance systems, known as CRISPR/Cas, were recently discovered and equip bacteria and archaea with an "adaptive immune system." This adaptive immunity provides a highly evolvable sequence-specific small RNA–based memory of past invasions by viruses and foreign genetic elements. There are many cases where these systems appear to target regions within the bacterial host's own genome (a possible autoimmunity), but the evolutionary rationale for this is unclear. Here, we demonstrate that CRISPR/Cas targeting of the host chromosome is highly toxic but that cells survive through mutations that alleviate the immune mechanism. We have used this phenotype to gain insight into how these systems function and show that large changes in the bacterial genome can occur. For example, targeting of a chromosomal pathogenicity island, important for virulence of the potato pathogen Pectobacterium atrosepticum, resulted in deletion of the island, which constituted ∼2% of the bacterial genome. These results have broad significance for the role of CRISPR/Cas systems and their impact on the evolution of bacterial genomes and virulence. In addition, this study demonstrates their potential as a tool for the targeted deletion of specific regions of bacterial chromosomes.
Related Articles
Crass: identification and reconstruction of CRISPR from unassembled metagenomic data
Skennerton, Connor T.
Imelfort, Michael
Tyson, Gene W.
Nucleic Acids Research
Clustered regularly interspaced short palindromic repeats (CRISPR) constitute a bacterial and archaeal adaptive immune system that protect against bacteriophage (phage). Analysis of CRISPR loci reveals the history of phage infections and provides a direct link between phage and their hosts. All current tools for CRISPR identification have been developed to analyse completed genomes and are not well suited to the analysis of metagenomic data sets, where CRISPR loci are difficult to assemble owing to their repetitive structure and population heterogeneity. Here, we introduce a new algorithm, Crass, which is designed to identify and reconstruct CRISPR loci from raw metagenomic data without the need for assembly or prior knowledge of CRISPR in the data set. CRISPR in assembled data are often fragmented across many contigs/scaffolds and do not fully represent the population heterogeneity of CRISPR loci. Crass identified substantially more CRISPR in metagenomes previously analysed using assembly-based approaches. Using Crass, we were able to detect CRISPR that contained spacers with sequence homology to phage in the system, which would not have been identified using other approaches. The increased sensitivity, specificity and speed of Crass will facilitate comprehensive analysis of CRISPRs in metagenomic data sets, increasing our understanding of phage-host interactions and co-evolution within microbial communities.
Related Articles
Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in Lactococcus lactis
Millen, Anne M.
Horvath, Philippe
Boyaval, Patrick
Romero, Dennis A.
PLoS ONE
Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes.
Related Articles
The Population and Evolutionary Dynamics of Phage and Bacteria with CRISPR–Mediated Immunity
Levin, Bruce R.
Moineau, Sylvain
Bushman, Mary
Barrangou, Rodolphe
PLoS Genetics
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with associated genes (cas), form the CRISPR–cas adaptive immune system, which can provide resistance to viruses and plasmids in bacteria and archaea. Here, we use mathematical models, population dynamic experiments, and DNA sequence analyses to investigate the host–phage interactions in a model CRISPR–cas system, Streptococcus thermophilus DGCC7710 and its virulent phage 2972. At the molecular level, the bacteriophage-immune mutant bacteria (BIMs) and CRISPR–escape mutant phage (CEMs) obtained in this study are consistent with those anticipated from an iterative model of this adaptive immune system: resistance by the addition of novel spacers and phage evasion of resistance by mutation in matching sequences or flanking motifs. While CRISPR BIMs were readily isolated and CEMs generated at high rates (frequencies in excess of 10−6), our population studies indicate that there is more to the dynamics of phage–host interactions and the establishment of a BIM–CEM arms race than predicted from existing assumptions about phage infection and CRISPR–cas immunity. Among the unanticipated observations are: (i) the invasion of phage into populations of BIMs resistant by the acquisition of one (but not two) spacers, (ii) the survival of sensitive bacteria despite the presence of high densities of phage, and (iii) the maintenance of phage-limited communities due to the failure of even two-spacer BIMs to become established in populations with wild-type bacteria and phage. We attribute (i) to incomplete resistance of single-spacer BIMs. Based on the results of additional modeling and experiments, we postulate that (ii) and (iii) can be attributed to the phage infection-associated production of enzymes or other compounds that induce phenotypic phage resistance in sensitive bacteria and kill resistant BIMs. We present evidence in support of these hypotheses and discuss the implications of these results for the ecology and (co)evolution of bacteria and phage.
Author Summary
The evidence that the CRISPR regions of the genomes of archaea and bacteria play a role in the ecology and (co)evolution of these microbes and their viruses is overwhelming: (i) the spacers (variable sequences of 26–72 bp of DNA between the repeats of this region) of these prokaryotes are homologous to the DNA of viruses in their communities; (ii) experimentally, the acquisition and incorporation of spacers of viral DNA can protect these organisms from subsequent infection by these viruses; (iii) experimentally, viruses evade this immunity by mutation in homologous protospacers or protospacer-adjacent motifs (PAMs). Not so clear are the nature and magnitude of the role CRISPR plays in this ecology and evolution. Here, we use mathematical models, experiments with Streptococcus thermophilus and the phage 2972, and DNA sequence analyses to explore the contribution of CRISPR–cas immunity to the ecology and (co)evolution of bacteria and their viruses. The results of this study suggest that the contribution of CRISPR to the ecology of bacteria and phage is more modest and limited, and the conditions for a CRISPR–mediated coevolutionary arms race between these organisms more restrictive, than anticipated from models based on the canonical view of phage infection and CRISPR–cas immunity.
Related Articles
CRISPR Interference Directs Strand Specific Spacer Acquisition
Swarts, Daan C.
Mosterd, Cas
van Passel, Mark W. J.
Brouns, Stan J. J.
PLoS ONE
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.
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 co-occurrence 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.
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 – in our case the first acquired spacer - and spacers acquired thereafter, possibly through the use of specific DNA degradation products of the CRISPR interference machinery by the CRISPR adaptation machinery. This loop enables a rapid expansion of the spacer repertoire against an actively present DNA element that is already targeted, amplifying the CRISPR interference effect.
Related Articles
Self vs. non-self discrimination during CRISPR RNA-directed immunity
Marraffini, Luciano A.
Sontheimer, Erik J.
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.
Related Articles
Multidrug-Resistant Enterococci Lack CRISPR-cas
Palmer, Kelli L.
Gilmore, Michael S.
Clustered, regularly interspaced short palindromic repeats (CRISPR) provide bacteria and archaea with sequence-specific, acquired defense against plasmids and phage. Because mobile elements constitute up to 25% of the genome of multidrug-resistant (MDR) enterococci, it was of interest to examine the codistribution of CRISPR and acquired antibiotic resistance in enterococcal lineages. A database was built from 16 Enterococcus faecalis draft genome sequences to identify commonalities and polymorphisms in the location and content of CRISPR loci. With this data set, we were able to detect identities between CRISPR spacers and sequences from mobile elements, including pheromone-responsive plasmids and phage, suggesting that CRISPR regulates the flux of these elements through the E. faecalis species. Based on conserved locations of CRISPR and CRISPR-cas loci and the discovery of a new CRISPR locus with associated functional genes, CRISPR3-cas, we screened additional E. faecalis strains for CRISPR content, including isolates predating the use of antibiotics. We found a highly significant inverse correlation between the presence of a CRISPR-cas locus and acquired antibiotic resistance in E. faecalis, and examination of an additional eight E. faecium genomes yielded similar results for that species. A mechanism for CRISPR-cas loss in E. faecalis was identified. The inverse relationship between CRISPR-cas and antibiotic resistance suggests that antibiotic use inadvertently selects for enterococcal strains with compromised genome defense.
For many bacteria, including the opportunistically pathogenic enterococci, antibiotic resistance is mediated by acquisition of new DNA and is frequently encoded on mobile DNA elements such as plasmids and transposons. Certain enterococcal lineages have recently emerged that are characterized by abundant mobile DNA, including numerous viruses (phage), and plasmids and transposons encoding multiple antibiotic resistances. These lineages cause hospital infection outbreaks around the world. The striking influx of mobile DNA into these lineages is in contrast to what would be expected if a self (genome)-defense system was present. Clustered, regularly interspaced short palindromic repeat (CRISPR) defense is a recently discovered mechanism of prokaryotic self-defense that provides a type of acquired immunity. Here, we find that antibiotic resistance and possession of complete CRISPR loci are inversely related and that members of recently emerged high-risk enterococcal lineages lack complete CRISPR loci. Our results suggest that antibiotic therapy inadvertently selects for enterococci with compromised genome defense.
Related Articles
Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer
Richter, Corinna
Dy, Ron L.
McKenzie, Rebecca E.
Watson, Bridget N.J.
Taylor, Corinda
Chang, James T.
McNeil, Matthew B.
Staals, Raymond H.J.
Fineran, Peter C.
Nucleic Acids Research
Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.
Related Articles
Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli
Pougach, Ksenia
Semenova, Ekaterina
Bogdanova, Ekaterina
Datsenko, Kirill A.
Djordjevic, Marko
Wanner, Barry L.
Severinov, Konstantin
Molecular microbiology
CRISPR/Cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of DNA loci called CRISPR cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. When a CRISPR spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. The CRISPR/Cas systems function through small RNAs originating from longer CRISPR cassette transcripts. While laboratory strains of Escherichia coli contain a functional CRISPR/Cas system (as judged by appearance of phage resistance at conditions of artificial co-overexpression of Cas genes and a CRISPR cassette engineered to target a λ phage), no natural phage resistance due to CRISPR system function was observed in this best-studied organism and no E. coli CRISPR spacer matches sequences of well-studied E. coli phages. To better understand the apparently "silent" E. coli CRISPR/Cas system, we systematically characterized processed transcripts from CRISPR cassettes. Using an engineered strain with genomically located spacer matching phage λ we show that endogenous levels of CRISPR cassette and cas genes expression allow only weak protection against infection with the phage. However, derepression of the CRISPR/Cas system by disruption of the hns gene leads to high level of protection.
Related Articles
CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli
Díez-Villaseñor, César
Guzmán, Noemí M.
Almendros, Cristóbal
García-Martínez, Jesús
Mojica, Francisco J.M.
RNA Biology
Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.
CRISPR-spacer acquisition; Cascade; Escherichia coli K12; O157:H7; RNA-guided immunity; cas genes; protospacer adjacent motif; reporter plasmids; ruler mechanism; spacer orientation
Related Articles
A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes
Haft, Daniel H
Selengut, Jeremy
Mongodin, Emmanuel F
Nelson, Karen E
PLoS Computational Biology
Clustered regularly interspaced short palindromic repeats (CRISPRs) are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21–37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas) protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer "immunity" against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.
The family of clustered regularly interspaced short palindromic repeats (CRISPRs) describes a class of DNA repeats found in nearly half of all bacterial and archaeal genomes. These DNA repeat regions have a remarkably regular structure: unique sequences of constant size, called spacers, sit between each pair of repeats. The DNA repeats do not encode proteins, but appear to be transcribed and processed into small RNAs that may have any number of functions, including resistance to any phage (i.e., virus of bacteria) whose sequence matches a spacer; spacers change rapidly as microbial strains evolve. This work describes 41 new CRISPR-associated (cas) gene families, which are always found near these repeats, in addition to the four previously known. It shows that CRISPR systems belong to different classes, with different repeat patterns, sets of genes, and species ranges. Most of these seem to come and go rather rapidly from their host genomes. These possibly beneficial mobile genetic elements may play an important role in driving prokaryotic evolution.
Related Articles
Viral Diversity Threshold for Adaptive Immunity in Prokaryotes
Weinberger, Ariel D.
Wolf, Yuri I.
Lobkovsky, Alexander E.
Gilmore, Michael S.
Koonin, Eugene V.
Bacteria and archaea face continual onslaughts of rapidly diversifying viruses and plasmids. Many prokaryotes maintain adaptive immune systems known as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). CRISPR-Cas systems are genomic sensors that serially acquire viral and plasmid DNA fragments (spacers) that are utilized to target and cleave matching viral and plasmid DNA in subsequent genomic invasions, offering critical immunological memory. Only 50% of sequenced bacteria possess CRISPR-Cas immunity, in contrast to over 90% of sequenced archaea. To probe why half of bacteria lack CRISPR-Cas immunity, we combined comparative genomics and mathematical modeling. Analysis of hundreds of diverse prokaryotic genomes shows that CRISPR-Cas systems are substantially more prevalent in thermophiles than in mesophiles. With sequenced bacteria disproportionately mesophilic and sequenced archaea mostly thermophilic, the presence of CRISPR-Cas appears to depend more on environmental temperature than on bacterial-archaeal taxonomy. Mutation rates are typically severalfold higher in mesophilic prokaryotes than in thermophilic prokaryotes. To quantitatively test whether accelerated viral mutation leads microbes to lose CRISPR-Cas systems, we developed a stochastic model of virus-CRISPR coevolution. The model competes CRISPR-Cas-positive (CRISPR-Cas+) prokaryotes against CRISPR-Cas-negative (CRISPR-Cas−) prokaryotes, continually weighing the antiviral benefits conferred by CRISPR-Cas immunity against its fitness costs. Tracking this cost-benefit analysis across parameter space reveals viral mutation rate thresholds beyond which CRISPR-Cas cannot provide sufficient immunity and is purged from host populations. These results offer a simple, testable viral diversity hypothesis to explain why mesophilic bacteria disproportionately lack CRISPR-Cas immunity. More generally, fundamental limits on the adaptability of biological sensors (Lamarckian evolution) are predicted.
A remarkable recent discovery in microbiology is that bacteria and archaea possess systems conferring immunological memory and adaptive immunity. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (CRISPR-Cas) are genomic sensors that allow prokaryotes to acquire DNA fragments from invading viruses and plasmids. Providing immunological memory, these stored fragments destroy matching DNA in future viral and plasmid invasions. CRISPR-Cas systems also provide adaptive immunity, keeping up with mutating viruses and plasmids by continually acquiring new DNA fragments. Surprisingly, less than 50% of mesophilic bacteria, in contrast to almost 90% of thermophilic bacteria and Archaea, maintain CRISPR-Cas immunity. Using mathematical modeling, we probe this dichotomy, showing how increased viral mutation rates can explain the reduced prevalence of CRISPR-Cas systems in mesophiles. Rapidly mutating viruses outrun CRISPR-Cas immune systems, likely decreasing their prevalence in bacterial populations. Thus, viral adaptability may select against, rather than for, immune adaptability in prokaryotes.
Related Articles
The Small, Slow and Specialized CRISPR and Anti-CRISPR of Escherichia and Salmonella
Touchon, Marie
Rocha, Eduardo P. C.
PLoS ONE
Prokaryotes thrive in spite of the vast number and diversity of their viruses. This partly results from the evolution of mechanisms to inactivate or silence the action of exogenous DNA. Among these, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are unique in providing adaptive immunity against elements with high local resemblance to genomes of previously infecting agents. Here, we analyze the CRISPR loci of 51 complete genomes of Escherichia and Salmonella. CRISPR are in two pairs of loci in Escherichia, one single pair in Salmonella, each pair showing a similar turnover rate, repeat sequence and putative linkage to a common set of cas genes. Yet, phylogeny shows that CRISPR and associated cas genes have different evolutionary histories, the latter being frequently exchanged or lost. In our set, one CRISPR pair seems specialized in plasmids often matching genes coding for the replication, conjugation and antirestriction machinery. Strikingly, this pair also matches the cognate cas genes in which case these genes are absent. The unexpectedly high conservation of this anti-CRISPR suggests selection to counteract the invasion of mobile elements containing functional CRISPR/cas systems. There are few spacers in most CRISPR, which rarely match genomes of known phages. Furthermore, we found that strains divergent less than 250 thousand years ago show virtually identical CRISPR. The lack of congruence between cas, CRISPR and the species phylogeny and the slow pace of CRISPR change make CRISPR poor epidemiological markers in enterobacteria. All these observations are at odds with the expectedly abundant and dynamic repertoire of spacers in an immune system aiming at protecting bacteria from phages. Since we observe purifying selection for the maintenance of CRISPR these results suggest that alternative evolutionary roles for CRISPR remain to be uncovered.
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Dealing with the Evolutionary Downside of CRISPR Immunity: Bacteria and Beneficial Plasmids
Jiang, Wenyan
Maniv, Inbal
Arain, Fawaz
Wang, Yaying
Levin, Bruce R.
Marraffini, Luciano A.
PLoS Genetics
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.
Author Summary
In addition to the virtue of protecting archaea and bacteria from the ravages of lethal viruses (phage), the immunity generated by the CRISPR-Cas systems have an evolutionary downside; they can prevent the acquisition of genes and genetic elements required for the adaptation and even the survival of these microbes. Using mathematical models and experiments with Staphylococcus epidermidis and the staphylococcal conjugative plasmid pG0400, we explore how bacteria deal with this evolutionary downside of CRISPR-Cas immunity. Although there are mechanisms by which immune populations of bacteria can acquire essential plasmids without the loss of CRISPR-Cas immunity, the results of our conjugation and fitness cost experiments suggest the most likely mechanism is the deactivation and deletion of this region. These results provide an explanation for the considerable variation in the existence, number and function of CRISPR-Cas within and between species of microbes. Along with other observations our work also suggests that the CRISPR-Cas loci are in a continuous state of flux: acquired by horizontal gene transfer, ascend when populations are confronted with phage and are rapidly lost when infectiously transmitted genes and genetic elements are required for the adaptation and survival of the population.
Related Articles
Non-Identity-Mediated CRISPR-Bacteriophage Interaction Mediated via the Csy and Cas3 Proteins ▿#
Cady, Kyle C.
O'Toole, George A.
Journal of Bacteriology
Studies of the Escherichia, Neisseria, Thermotoga, and Mycobacteria clustered regularly interspaced short palindromic repeat (CRISPR) subtypes have resulted in a model whereby CRISPRs function as a defense system against bacteriophage infection and conjugative plasmid transfer. In contrast, we previously showed that the Yersinia-subtype CRISPR region of Pseudomonas aeruginosa strain UCBPP-PA14 plays no detectable role in viral immunity but instead is required for bacteriophage DMS3-dependent inhibition of biofilm formation by P. aeruginosa. The goal of this study is to define the components of the Yersinia-subtype CRISPR region required to mediate this bacteriophage-host interaction. We show that the Yersinia-subtype-specific CRISPR-associated (Cas) proteins Csy4 and Csy2 are essential for small CRISPR RNA (crRNA) production in vivo, while the Csy1 and Csy3 proteins are not absolutely required for production of these small RNAs. Further, we present evidence that the core Cas protein Cas3 functions downstream of small crRNA production and that this protein requires functional HD (predicted phosphohydrolase) and DEXD/H (predicted helicase) domains to suppress biofilm formation in DMS3 lysogens. We also determined that only spacer 1, which is not identical to any region of the DMS3 genome, mediates the CRISPR-dependent loss of biofilm formation. Our evidence suggests that gene 42 of phage DMS3 (DMS3-42) is targeted by CRISPR2 spacer 1 and that this targeting tolerates multiple point mutations between the spacer and DMS3-42 target sequence. This work demonstrates how the interaction between P. aeruginosa strain UCBPP-PA14 and bacteriophage DMS3 can be used to further our understanding of the diverse roles of CRISPR system function in bacteria.
Related Articles
A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action
Makarova, Kira S
Grishin, Nick V
Shabalina, Svetlana A
Wolf, Yuri I
Koonin, Eugene V
Biology Direct
All archaeal and many bacterial genomes contain Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR) and variable arrays of the CRISPR-associated (cas) genes that have been previously implicated in a novel form of DNA repair on the basis of comparative analysis of their protein product sequences. However, the proximity of CRISPR and cas genes strongly suggests that they have related functions which is hard to reconcile with the repair hypothesis.
The protein sequences of the numerous cas gene products were classified into ~25 distinct protein families; several new functional and structural predictions are described. Comparative-genomic analysis of CRISPR and cas genes leads to the hypothesis that the CRISPR-Cas system (CASS) is a mechanism of defense against invading phages and plasmids that functions analogously to the eukaryotic RNA interference (RNAi) systems. Specific functional analogies are drawn between several components of CASS and proteins involved in eukaryotic RNAi, including the double-stranded RNA-specific helicase-nuclease (dicer), the endonuclease cleaving target mRNAs (slicer), and the RNA-dependent RNA polymerase. However, none of the CASS components is orthologous to its apparent eukaryotic functional counterpart. It is proposed that unique inserts of CRISPR, some of which are homologous to fragments of bacteriophage and plasmid genes, function as prokaryotic siRNAs (psiRNA), by base-pairing with the target mRNAs and promoting their degradation or translation shutdown. Specific hypothetical schemes are developed for the functioning of the predicted prokaryotic siRNA system and for the formation of new CRISPR units with unique inserts encoding psiRNA conferring immunity to the respective newly encountered phages or plasmids. The unique inserts in CRISPR show virtually no similarity even between closely related bacterial strains which suggests their rapid turnover, on evolutionary scale. Corollaries of this finding are that, even among closely related prokaryotes, the most commonly encountered phages and plasmids are different and/or that the dominant phages and plasmids turn over rapidly.
We proposed previously that Cas proteins comprise a novel DNA repair system. The association of the cas genes with CRISPR and, especially, the presence, in CRISPR units, of unique inserts homologous to phage and plasmid genes make us abandon this hypothesis. It appears most likely that CASS is a prokaryotic system of defense against phages and plasmids that functions via the RNAi mechanism. The functioning of this system seems to involve integration of fragments of foreign genes into archaeal and bacterial chromosomes yielding heritable immunity to the respective agents. However, it appears that this inheritance is extremely unstable on the evolutionary scale such that the repertoires of unique psiRNAs are completely replaced even in closely related prokaryotes, presumably, in response to rapidly changing repertoires of dominant phages and plasmids.
This article was reviewed by: Eric Bapteste, Patrick Forterre, and Martijn Huynen.
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Reviewed by Eric Bapteste, Patrick Forterre, and Martijn Huynen.
For the full reviews, please go to the Reviewers' comments section.
Related Articles
Function and Regulation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR Associated (Cas) Systems
Richter, Corinna
Chang, James T.
Fineran, Peter C.
Phages are the most abundant biological entities on earth and pose a constant challenge to their bacterial hosts. Thus, bacteria have evolved numerous 'innate' mechanisms of defense against phage, such as abortive infection or restriction/modification systems. In contrast, the clustered regularly interspaced short palindromic repeats (CRISPR) systems provide acquired, yet heritable, sequence-specific 'adaptive' immunity against phage and other horizontally-acquired elements, such as plasmids. Resistance is acquired following viral infection or plasmid uptake when a short sequence of the foreign genome is added to the CRISPR array. CRISPRs are then transcribed and processed, generally by CRISPR associated (Cas) proteins, into short interfering RNAs (crRNAs), which form part of a ribonucleoprotein complex. This complex guides the crRNA to the complementary invading nucleic acid and targets this for degradation. Recently, there have been rapid advances in our understanding of CRISPR/Cas systems. In this review, we will present the current model(s) of the molecular events involved in both the acquisition of immunity and interference stages and will also address recent progress in our knowledge of the regulation of CRISPR/Cas systems.
phages; plasmids; horizontal gene transfer; CRISPR; Cas; cascade; PAM; crRNA; resistance
Related Articles
Abundant and Diverse Clustered Regularly Interspaced Short Palindromic Repeat Spacers in Clostridium difficile Strains and Prophages Target Multiple Phage Types within This Pathogen
Hargreaves, Katherine R.
Flores, Cesar O.
Lawley, Trevor D.
Clokie, Martha R. J.
Clostridium difficile is an important human-pathogenic bacterium causing antibiotic-associated nosocomial infections worldwide. Mobile genetic elements and bacteriophages have helped shape C. difficile genome evolution. In many bacteria, phage infection may be controlled by a form of bacterial immunity called the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system. This uses acquired short nucleotide sequences (spacers) to target homologous sequences (protospacers) in phage genomes. C. difficile carries multiple CRISPR arrays, and in this paper we examine the relationships between the host- and phage-carried elements of the system. We detected multiple matches between spacers and regions in 31 C. difficile phage and prophage genomes. A subset of the spacers was located in prophage-carried CRISPR arrays. The CRISPR spacer profiles generated suggest that related phages would have similar host ranges. Furthermore, we show that C. difficile strains of the same ribotype could either have similar or divergent CRISPR contents. Both synonymous and nonsynonymous mutations in the protospacer sequences were identified, as well as differences in the protospacer adjacent motif (PAM), which could explain how phages escape this system. This paper illustrates how the distribution and diversity of CRISPR spacers in C. difficile, and its prophages, could modulate phage predation for this pathogen and impact upon its evolution and pathogenicity.
Clostridium difficile is a significant bacterial human pathogen which undergoes continual genome evolution, resulting in the emergence of new virulent strains. Phages are major facilitators of genome evolution in other bacterial species, and we use sequence analysis-based approaches in order to examine whether the CRISPR/Cas system could control these interactions across divergent C. difficile strains. The presence of spacer sequences in prophages that are homologous to phage genomes raises an extra level of complexity in this predator-prey microbial system. Our results demonstrate that the impact of phage infection in this system is widespread and that the CRISPR/Cas system is likely to be an important aspect of the evolutionary dynamics in C. difficile.
Related Articles
Structure and Genetic Content of the Megaplasmids of Neurotoxigenic Clostridium butyricum Type E Strains from Italy
Iacobino, Angelo
Scalfaro, Concetta
Franciosa, Giovanna
PLoS ONE
We determined the genetic maps of the megaplasmids of six neutoroxigenic Clostridium butyricum type E strains from Italy using molecular and bioinformatics techniques. The megaplasmids are circular, not linear as we had previously proposed. The differently-sized megaplasmids share a genetic region that includes structural, metabolic and regulatory genes. In addition, we found that a 168 kb genetic region is present only in the larger megaplasmids of two tested strains, whereas it is absent from the smaller megaplasmids of the four remaining strains. The genetic region unique to the larger megaplasmids contains, among other features, a locus for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (cas) genes, i.e. a bacterial adaptive immune system providing sequence-specific protection from invading genetic elements. Some CRISPR spacer sequences of the neurotoxigenic C. butyricum type E strains showed homology to prophage, phage and plasmid sequences from closely related clostridia species or from distant species, all sharing the intestinal habitat, suggesting that the CRISPR locus might be involved in the microorganism adaptation to the human or animal intestinal environment. Besides, we report here that each of four distinct CRISPR spacers partially matched DNA sequences of different prophages and phages, at identical nucleotide locations. This suggests that, at least in neurotoxigenic C. butyricum type E, the CRISPR locus is potentially able to recognize the same conserved DNA sequence of different invading genetic elements, besides targeting sequences unique to previously encountered invading DNA, as currently predicted for a CRISPR locus. Thus, the results of this study introduce the possibility that CRISPR loci can provide resistance to a wider range of invading DNA elements than previously appreciated. Whether it is more advantageous for the peculiar neurotoxigenic C. butyricum type E strains to maintain or to lose the CRISPR-cas system remains an open question.
Related Articles
A New Group of Phage Anti-CRISPR Genes Inhibits the Type I-E CRISPR-Cas System of Pseudomonas aeruginosa
Pawluk, April
Bondy-Denomy, Joseph
Cheung, Vivian H. W.
Maxwell, Karen L.
Davidson, Alan R.
CRISPR-Cas systems are one of the most widespread phage resistance mechanisms in prokaryotes. Our lab recently identified the first examples of phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. A key question arising from this work was whether there are other types of anti-CRISPR genes. In the current work, we address this question by demonstrating that some of the same phages carrying type I-F anti-CRISPR genes also possess genes that mediate inhibition of the type I-E CRISPR-Cas system of P. aeruginosa. We have discovered four distinct families of these type I-E anti-CRISPR genes. These genes do not inhibit the type I-F CRISPR-Cas system of P. aeruginosa or the type I-E system of Escherichia coli. Type I-E and I-F anti-CRISPR genes are located at the same position in the genomes of a large group of related P. aeruginosa phages, yet they are found in a variety of combinations and arrangements. We have also identified functional anti-CRISPR genes within nonprophage Pseudomonas genomic regions that are likely mobile genetic elements. This work emphasizes the potential importance of anti-CRISPR genes in phage evolution and lateral gene transfer and supports the hypothesis that more undiscovered families of anti-CRISPR genes exist. Finally, we provide the first demonstration that the type I-E CRISPR-Cas system of P. aeruginosa is naturally active without genetic manipulation, which contrasts with E. coli and other previously characterized I-E systems.
The CRISPR-Cas system is an adaptive immune system possessed by the majority of prokaryotic organisms to combat potentially harmful foreign genetic elements. This study reports the discovery of bacteriophage-encoded anti-CRISPR genes that mediate inhibition of a well-studied subtype of CRISPR-Cas system. The four families of anti-CRISPR genes described here, which comprise only the second group of anti-CRISPR genes to be identified, encode small proteins that bear no sequence similarity to previously studied phage or bacterial proteins. Anti-CRISPR genes represent a newly discovered and intriguing facet of the ongoing evolutionary competition between phages and their bacterial hosts.
Related Articles
Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus▿
Deveau, Hélène
Barrangou, Rodolphe
Garneau, Josiane E.
Labonté, Jessica
Fremaux, Christophe
Boyaval, Patrick
Romero, Dennis A.
Horvath, Philippe
Moineau, Sylvain
Journal of Bacteriology
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.
Related Articles
The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli
Sapranauskas, Rimantas
Gasiunas, Giedrius
Fremaux, Christophe
Barrangou, Rodolphe
Horvath, Philippe
Siksnys, Virginijus
Nucleic Acids Research
The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.
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High-density UPS
Active Power's CleanSource High Density (CSHD) 750 kVA UPS product line is designed for data center environments and other mission critical operations.
Active Power's CleanSource High Density (CSHD) 750 kVA UPS product line is designed for data center environments and other mission critical operations. CSHD is a three-phase, line interactive UPS system that has been optimized for cost, efficiency, and overall system performance. The new and innovative engineering approach to the design and development of CSHD dramatically improves upon its successful predecessor, the CleanSource UPS 300 kVA product line. The CSHD has a 750 kVA power rating and delivers up to 97% efficiency at full load.
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Condola Rashad
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Today, while running late to my sister's wedding and rushing to get ready, I accidentally grabbed my travel size shaving cream can in place of my body spray, and quickly drew a blue foaming line across my rental tux. FML
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by Anonymous / 05/27/2010 at 12:10am / United States (Nebraska) / Intimacy
Today, I picked up my 3 friends from a party. One of them drank too much and claimed she needed to throw up. I pulled over multiple times so she could. It didn't happen until we were right in front of her house and still inside my car. FML
by Anonymous / 05/26/2010 at 10:30pm / United States (California) / Transportation
Today, I found out I'm allergic to the pills my doctor prescribed for coughing, which I really need because I ripped a muscle in my stomach. Now my whole upper body is covered in a terrible itchy rash. I also found out it will last for at least another week. FML
by Anonymous / 05/26/2010 at 4:14pm / Switzerland (Geneve) / Health
Today, I found out my ex-boyfriend is moving out to California to be with a girl he met while on Chatroulette. We broke up because he thought we were moving too fast. FML
by Sarahfizzeller / 05/26/2010 at 4:13pm / United States (Ohio) / Love
Today, a few friends and I ran through sprinklers at our school until we were all soaked. Then we were told that they put fertilizer in the water. I'm soaked in manure. Now I know why it tasted weird. FML
by noraidk / 05/26/2010 at 2:35pm / United States (Texas) / Miscellaneous
- 1Today, my parents let me babysit my baby sister for the first time. About an hour after they left,… 2Today, I saw an elderly lady fall over in the street. Nobody bothered to do anything, so I went… 3Today, I was fired for being late to work, even though the only reason I was late was because I had…


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- Miss Florida 2017 stripped of crown days after win - US Weekly
- Lindsay Lohan steps out amid relationship drama - TMZ
- Was Taylor Swift snubbed by the VMAs? - Gossip Cop
- Rachel Platten sings "Fight Song" with lots of stars - Just Jared Jr
- Farrah Abraham sizzles in a revealing bikini - Radar
- New pics from the Clooneys' Italian summer - Lainey Gossip
- Fox News snubs major DNC moment - The Hollywood Reporter
15 July 2013 at 6:56 PM
Katie Holmes Picks Suri Up from Gym Class
About this photo set: Katie Holmes holds her daughter Suri's hand while they leave a gym class on Monday afternoon (July 15) in New York City. The day before, the 34-year-old actress… Read More Here
Photo #: 2909915
Posted to Celebrity Babies, Katie Holmes, Suri Cruise, Tom Cruise
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text:
Stripe Ruby Bindings
The Stripe Ruby bindings provide a small SDK for convenient access to the Stripe API from applications written in the Ruby language. It provides a pre-defined set of classes for API resources that initialize themselves dynamically from API responses which allows the bindings to tolerate a number of different versions of the API.
The bindings also provide other features. For example:
- Easy configuration path for fast setup and use.
- Helpers for pagination.
- Tracking of "fresh" values in API resources so that partial updates can be executed.
- Built-in mechanisms for the serialization of parameters according to the expectations of Stripe's API.
See the Ruby API docs.
You don't need this source code unless you want to modify the gem. If you just want to use the Stripe Ruby bindings, you should run:
gem install stripe
If you want to build the gem from source:
gem build stripe.gemspec
- Ruby 1.9.3 or above.
- rest-client
If you are installing via bundler, you should be sure to use the https rubygems source in your Gemfile, as any gems fetched over http could potentially be compromised in transit and alter the code of gems fetched securely over https:
source 'https://rubygems.org'
gem 'rails'
gem 'stripe'
Run all tests:
bundle exec rake
Run a single test suite:
bundle exec ruby -Ilib/ test/stripe/util_test.rb
Run a single test:
bundle exec ruby -Ilib/ test/stripe/util_test.rb -n /should.convert.names.to.symbols/
Update bundled CA certificates from the Mozilla cURL release:
bundle exec rake update_certs
The location of a file containing a bundle of CA certificates. By default the library will use an included bundle that can successfully validate Stripe certificates.
max_network_retries is set to a positive integer, stripe will retry
requests that fail on a network error. Idempotency keys will be added to post
and get requests to ensure the safety of retrying. There will be a short delay
between each retry, with an exponential backoff algorithm used to determine the
length of the delay. Default value is 0.
Stripe.max_network_retries = 2


text:
Top Definition
A shield that a man keeps up, refusing to talk about his feelings in fear of being called a gay.
I wish Adam Thomas would drop his man shield so we could talk about how we really feel <3
de Drewziph 10 décembre 2010
Trending Now
Les e-mails sont envoyés par email@example.com. Nous ne vous enverrons jamais de spam.×


text:
Caraïbes Bonheur, Guadeloupe (islands)
- Free WiFi and free parking
Main amenities
- 20 guestrooms
- Near the beach
- Outdoor pool
- Breakfast available
- Bar/lounge
- Airport shuttle
- Terrace
- Air conditioning
- Garden
- Library
- Self-serve laundry
- Multilingual staff
What's around
- Fort Royal Beach (0.5 mi / 0.8 km)
- Pearl Beach (1 mi / 1.7 km)
- Deshaies Botanical Garden (3.7 mi / 5.9 km)
- Sofaia Waterfall (7.4 mi / 11.9 km)
For families
- Free cribs/infant beds
- Children's pool
- Sofa bed
- Kitchen
- Cookware, dishware, and utensils
- Stovetop
Caraïbes Bonheur, Guadeloupe (islands)
Hotel Info: 866-539-8117from $107
- Bungalow (6)
- Bungalow (4)
- Bungalow (2)
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 20 rooms
- Check-in time 2 PM-Midnight
- Check-out time is 10 AM
If you are planning to arrive after 6 PM please contact the property in advance using the information on the booking confirmation.
24-hour airport shuttle service is available. Contact the property in advance to get details.
Required at check in
- Credit card deposit required
- Government-issued photo ID required
- Minimum check-in age is 18
- Minimum Spring Break check-in age is 18
Travelling with others
- Children (5 years old and younger) stay free when occupying the parent or guardian's room, using existing bedding
- Pets not allowed
- Restrictions apply *
- Free WiFi in public areas
- Free WiFi in rooms
- Airport shuttle (available 24 hours) *
- Free self parking
- Free nearby parking
- Area shuttle *
Nearby Four Star Guadeloupe (islands) hotels
In the hotel
- Continental breakfast daily (surcharge)
- Bar/lounge
- Barbeque grills
- Outdoor pool
- Children's pool
- Front desk (limited hours)
- Tours/ticket assistance
- Laundry facilities
- Luggage storage
- Multilingual staff
- Year Built 2002
- Garden
- Terrace
- Library
- Accessible bathroom
- In-room accessibility
- Roll-in shower
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Coffee/tea maker
- Iron/ironing board (on request)
- Blackout drapes/curtains
- Sofa bed
- Sleep Number by Select Comfort mattress
- Balcony or patio
- Private bathroom
- Shower only
- Hair dryer (on request)
- Satellite TV channels
- Free WiFi
- Full-sized refrigerator/freezer
- Microwave
- Kitchen
- Stovetop
- Cookware, dishware, and utensils
- Free bottled water
- In-room safe
Top Guadeloupe (islands) hotels
Recently reviewed hotels in Guadeloupe (islands)
- E. Gwada Hostel - Auberge de jeunesseFrom
"Just stayed here for one night before flying out from the airport the next morning. Hostel is conveniently located nearby everything in Point-a-Pitre and is very well maintained.…"Feb 17, 2016
- Hotel Village SoleilFrom
"The hotel was pretty good. Our room was nicer than we expected. The hotel staff in the office isn't too friendly though. And they refuse to give you more towels when asked. They…"Feb 4, 2016
- Mahogany Hôtel Résidence & SpaFrom
"I've travelled to Guadeloupe 5 times for business and this is by far the best hotel I've found. I shall use it again."Oct 10, 2015
- Hôtel La Maison CréoleFrom
"Had a last minute layover in Guadalupe due to hurricane. I speak zero french, but the hotel staff was very accommodating and willing to help us find some food and a taxi. The…"Aug 28, 2015
Special features
On Site
- Fishing on site
- Horse riding/rental on site
- Personal motorized watercraft on site
- Bicycle rentals nearby
- Ecotours nearby
- Golfing nearby
- Hiking/biking trails nearby
- Kayaking nearby
- Parasailing nearby
- Sailing nearby
- Scuba diving nearby
- Segway rental/tour nearby
- Skydiving nearby
- Snorkeling nearby
- Surfing/boogie boarding nearby
- Windsurfing nearby
Caraïbes Bonheur, Guadeloupe (islands)'s small print
Also known as
- Caraïbes Bonheur
- Caraibes Bonheur Deshaies
- Caraïbes Bonheur Deshaies
- Caraibes Bonheur Guadeloupe/Deshaies
- Caraïbes Bonheur Hotel
- Caraibes Bonheur Hotel Deshaies
- Caraïbes Bonheur Hotel Deshaies
- Caraïbes Bonheur House Deshaies
Pet-friendly rooms can be requested by contacting the property at the number on the booking confirmation.
Mandatory fees
You'll be asked to pay the following charges at check in or check out:
- A tax is imposed by the city: EUR 1.00 per person, per night. This tax does not apply to children under 12 years of age.
Optional extras
Rollaway beds are available for an additional charge
Continental breakfast is offered for an extra charge of EUR 9 for adults and EUR 9 for children (approximately)
Airport shuttle service is offered for an extra charge of EUR 80 per vehicle (roundtrip)
Area shuttle service is offered for an extra charge
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Is the description of this hotel not correct? Tell us


text:
W.Va.-based center offers virtualization and teleconferencing
The Veterans Affairs Department has expanded its use of virtual technologies and distance learning in its new National Information Technology Training Academy that began operating a month ago in West Virginia.
The goal of the center is to train up to 200 VA employees at a time to strengthen their IT support skills and to improve transparency and information sharing in the department, VA Secretary Eric Shinseki said March 12.
The National IT Training Academy, which opened Feb. 12, is linked with four existing regional VA training classrooms in Colorado, the District of Columbia, Florida and Texas. It will be linked with two more training classrooms to open later this year in Utah and Washington State.
The national center has remote virtualization and teleconferencing capabilities, permitting more frequent training at reduced cost, the VA said. In addition to interactive video, the training will be presented in Web-based courses, on-demand recordings and live meetings.
About the Author
Alice Lipowicz is a staff writer covering government 2.0, homeland security and other IT policies for Federal Computer Week.
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The latest version of the Defense Department-wide Enterprise Service Management Framework places more emphasis on managing IT risk.
Despite GSA's efforts to accelerate the FedRAMP approval process, the lack of agency reciprocity puts the program's central goals at risk.
Got some early-career colleagues who are doing great things in federal IT? Nominate them for FCW's 2016 Rising Star awards.
The agency software inventories required under the draft category management policy will be treasure maps that lead to efficient, effective, streamlined buying and unprecedented savings.
As experts warned of the "dire" threats posed by outdated federal technology, lawmakers grilled top feds, debated workforce issues and inched closer to backing a $3.1 billion fix-it fund.
The agency is making strides in developing and deploying border technologies but still has some weaknesses.
Agencies have spent almost $23 billion on legacy IT over the past three years, according to reports to Congress. Is a revolving fund the answer?
The United Kingdom will go live with its governmentwide digital identity platform, GOV.UK Verify, in the coming days. The U.S. government will need a little more time.
What contracting officer representatives really think is needed to fix contract management.
Would like information about VA IT Training Centers.
"train up to 200 VA employees at a time to strengthen their IT support skills and to improve transparency and information sharing in the department" Improve Transparency?? Maybe they could TQM it while there at it (how 90's). Transparency, the lastest buzzword in Federal Govt.
8609 Westwood Center Drive, Suite 500Vienna, VA 22182-2215 703-876-5100
© 1996-2013 1105 Media, Inc. All Rights Reserved. This copy is for your personal, non-commercial use only.To order presentation-ready copies for distribution to colleagues, clients or customers, visit: www.1105Reprints.com


text:
- Architecture
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- Executive Order 9066 and the Residents of Santa Cruz County
- Films
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- In the 19th Century
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Santa Cruz County History - Articles
Browsing Local History Articles tagged "bridges"
Santa Cruz Owes Site to Spud Rush, by Ross Eric GibsonThe clock tower marks the heart of Santa Cruz. But this is hardly an ideal place for a downtown, and ... [Read more] Read more]
San Lorenzo Once was Full of Fish: The River was Santa Cruz's No. 2 Tourist Draw, by Ross Eric GibsonBefore flood-control measures were taken by the Army Corps of Engineers in 1959, the San Lorenzo River was Santa Cruz's ... [Read more]
Bridges Span Santa Cruz's Past: From Felton's Covered Redwood Span to West Cliff Drive's Iron Landmark, by Ross Eric GibsonThe early evolution of bridge construction in Santa Cruz shows a progression of problem-solving and building techniques. [Read more]
Covered Bridges, by John V. YoungSanta Cruz County has the most covered bridges still standing of any county in the state, all of them deep ... [Read more]
The Soquel Avenue Bridge, by City of Santa Cruz Public Works DepartmentThere have been several bridges previously located at this site. In 1874 a covered bridge was constructed. [Read more]
Water Street Bridge Fact Sheet, by City of Santa Cruz Public Works DepartmentThe Water Street Bridge Rehabilitation Project involved the removal and replacement of the northern half of the bridge and the ... [Read more]
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Explore Santa Cruz County History!
Doctor Knight and his family were among the most prominent residents of Santa Cruz County during its first century. Not only did they occupy numerous important positions in the community, but they were held in the highest esteem by the citizenry.
It has been said that the measure of a ..." [More]
Excerpted from A Walk Through Time: Doctor Benjamin K. Knight and Family by Janet Krassow and Randy Krassow


text:
Huge list I've been saving for OH's retirement but haven't actually written down for fear of frightening him so I plan to spring them one at a time after preparing the proverbial and metaphorical ground. He's had January off to get used to not having to get up and catch a train to work and we've just had the build up to the dance club's 40th anniversary ball this weekend and are off to the Hague tomorrow so nothing doing till next week really.
He knows about the compost heaps needing turning and emptying and fixing. He knows about the trellis panels swaps and repairs and replacements that need doing. Just waiting for decent weather.
Then there's finishing the edging of the holly hedge bed with granite pavers and laying chipped bark. Ditto one edge of the back lawn without the bark, pruning dead branches from our prunus cerastifera, cutting back other shrubs, wading into the pond to clear flag iris and so on and so forth.
Grass likes sunlight. You have shade. Go with the flow and redesign. Loads of fabulous plants like shady woodland situations. The RHS Plant Selector lets you search for plants by aspect, available light, soil etc. I typed in perennials and partial shade and it returned 1358 plants - https://www.rhs.org.uk/Plants/Search-Results?form-mode=true&context=l%3Den%26q%3D%2523all%26sl%3DplantForm%26r%3Df%252Fplant_sunlight%252Fpartial%2Bshade%26r%3Df%252Fplant_plant_type%252Fherbaceous%2Bperennial
You could do the same search entering your soil type, drainage, more shade etc.
It may be worth looking at raising the canopy of your oaks to let in more light. This just means removing the lower branches to a a certain height without changing the shape or size or look of the rest of the tree. You can consult a tree surgeon if you like and they should chip any small offcuts and leave bigger ones so you can use them as mulch or woodpiles for habitat according to preference.
Have fun.
I loathe the look of cane supports in gardens plus tehy rot and have to be re-done and they can poke your eye out. I use the bent metal rods with great success - discreet, can be made to order for the plant and cheap and everlasting.
I buy 5m lengths at the local building supplies shop and then cut them in half or 3 depending on the height of plant to be supported which can be 2m high helianthemums, slightly shorter echinops, then phlomis (the purple one), occasional floppy roses and so on. I bend them round an upright railway sleeper which supports a retaining wall then stand on a plank to bend the legs up. They push into the soil easily, go deep without snapping, no need for strings and become invisible very quickly as the plants grow.
I have a Betty Corning. Once she gets going she's a good strong grower and very hardy. Needs cutting back to just above ground level every year in late winter/early spring and then a good dollop of slow release feed.
Gentle perfume and lovely flowers. I grow mine on a trellis with rosa Constance Spry so the flowers succeed each other but you could combine it with a repeat flowering rambler or climber.
Three main reason - drumming to make a noise to mark out territory for the breeding season, drilling to make a hole to nest and tapping to forage for insects for food.
I saw a biodynamic gardener on TV once who soaked her peas in paraffin before sowing them. The smell puts off the rodents.
I do my peas and beans in toilet rolls up on a shelf where the peskies can't get to them since I lost the lot in a cold frame some years ago.
I wouldn't at this stage but maybe in a couple of years when they're really established you can. In the mean time, give them a good feed and wait and see what you've got.
You can use this website to help identify them. It's a research website so has no commercial axe to grind. You can search by colour of tepals, stamens, flowering period and so on.
Glad you enjoyed it Hogweed. I tend to use fewer bowls than many recipes think are needed.
Think I might make us one next week.
I treat all my group 2s as group 3s as they usually get their tops frozen to bits in a normal winter.
Gave up growing group 1s as they too get frozen and then die as they don't respond well to being cut back hard unless they've been going for years and years and have good strong roots to drive recovery.
At this size I would leave it and see what grows this season. It is a group 1 and can be trimmed, if needs be, after any flowers have formed in April/May/June.
This clematis website gives pruning info and can be searched for info on hundreds of clematis and their growing habits - http://www.clematis.hull.ac.uk/new-pruning.cfm


text:
Top Definition
A reference to Santa Claus, often mispelled as Chris Cringle
Kris Kringle a.k.a. Santa Claus
by BigWang117 December 04, 2008
Trending Now
The act of farting on your own ball sack.
Yukon Cornelius was the first man to ever successfully give himself a kris kringle.
by Beck C Men November 29, 2010


text:
Solution Environment:
MS Visual Studio 2010 Ultimate, MS Office 2010 Professional, Microsoft Interop classes
I've created a .Net application which uses the Interop classes to interact with MS Word. Consider this: An application which mimics a key-value pair table. When the user clicks on the "value" field, the .net application opens up a winword window where the user can enter some notes. Upon clicking close (winword), .net app will do a winword.wordopenxml and save the data in the field. For this to happen, i've added event handles to winword. The application runs perfectly fine. The problem arises when i have multiple instances of winword opened (separately by the user but not thru the .net application) . The event handlers from the .net application is applied to all instances of winword and hence, upon closing just one instance word, all the other instances gets closed too. I tried different ways to initialize the word object in the application. I also tried the work around "Initializing two instances of winword and quit the first instance" -> This approach also did not work well.
Gurus: Could you please help me find a suitable solution for this problem ?


text:
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text: Yanzhou Coal Mining A similar setup is forming in shares of Yanzhou Coal Mining ( YZC), the sixth-largest coal miner in the People's Republic. Like LFC, Yanzhou is forming a long-term ascending triangle setup that's been forming since the latter half of the third quarter. The resistance level to watch in YZC right now is $27. Looking at the technicals in YZC from a more economics-driven perspective, the setup in this stock all comes down to a case of supply and demand. Essentially, there's an excess of supply of shares above the $27 level -- that is, it's a price above which sellers become more eager to sell and take gains than buyers are to buy. The result is a price level that acts as a sort of ceiling for shares of Yanzhou. The uptrending support level below shares gives us an important clue about investor sentiment in this stock. Because Yanzhou is posting higher lows, we have a hint that buyers have some control over price action; a breakdown below that trend line indicates that that's no longer the case. A move above $27 is significant because it shows us that the upside barriers to movement have been squashed; keep a close eye on that price. Follow @stockpickr Prev 2 of 6 Next


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