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Westra E.R.,Wageningen University | Semenova E.,Waksman Institute | Datsenko K.A.,Waksman Institute | Datsenko K.A.,Purdue University | And 6 more authors.
PLoS Genetics | Year: 2013

Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism. © 2013 westra et al. Source


Soutourina O.A.,Institute Pasteur Paris | Soutourina O.A.,University Paris Diderot | Monot M.,Institute Pasteur Paris | Boudry P.,Institute Pasteur Paris | And 13 more authors.
PLoS Genetics | Year: 2013

Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA-seq and differential 5′-end RNA-seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen. © 2013 Soutourina et al. Source


Westra E.,Wageningen University | van Erp P.,Wageningen University | Kunne T.,Wageningen University | Wong S.,Wageningen University | And 14 more authors.
Molecular Cell | Year: 2012

The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg 2+-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization. © 2012 Elsevier Inc. Source


Pougach K.,Russian Academy of Sciences | Semenova E.,Waksman Institute | Bogdanova E.,Waksman Institute | Datsenko K.A.,Purdue University | And 4 more authors.
Molecular Microbiology | Year: 2010

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. © 2010 Blackwell Publishing Ltd. Source


Novikova M.,Russian Academy of Sciences | Kazakov T.,Russian Academy of Sciences | Vondenhoff G.H.,Waksman Institute | Vondenhoff G.H.,Rega Institute for Medical Research | And 8 more authors.
Journal of Biological Chemistry | Year: 2010

The heptapeptide-nucleotide microcin C (McC) is a potent inhibitor of enteric bacteria growth. McC is excreted from producing cells by the MccC transporter. The residual McC that remainsin the producing cell can be processed by cellular amino-peptidases with the release of a non-hydrolyzable aspartyl-adenylate, a strong inhibitor of aspartyl-tRNA synthetase. Accumulation of processed McC inside producing cells should therefore lead to translation inhibition and cessation of growth. Here, we show that a product of another gene of the McC biosynthetic cluster, mccE, acetylates processed McC and converts it into a non-toxic compound. MccE also makes Escherichia coli resistant to albomycin, a Trojan horse inhibitor unrelated to McC that, upon processing, gives rise to a serine coupled to a thioxylofuranosyl pyrimidine, an inhibitor of seryl-tRNA synthetase. We speculate that MccE and related cellular acetyl-transferases of the Rim family may detoxify various aminoacyl- nucleotides, either exogenous or those generated inside the cell. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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