Small CRISPR RNAs guide antiviral defense in prokaryotes

Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an antiviral response that counteracts infection. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation. Our results demonstrate that the formation of mature guide RNAs by the CRISPR RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense

Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR-Cas effector complexes

Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction-modification (R-M) and CRISPR-Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR-Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR-Cas systems by lowering the affinity of the Cascade and Cas9-crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR-Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems

Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escheria coli

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80°C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80°C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80°C with a half-life of 4 h at 90°C and 1.5 h at 100°

High-resolution reconstruction of a Jumbo-bacteriophage infecting capsulated bacteria using hyperbranched tail fibers

NWO184.034.014Computer Systems, Imagery and Medi

Integrated molecular analysis of sugar metabolism of Sulfolobus solfataricus

cum laude graduation (with distinction

Molecular biology. A Swiss army knife of immunity

Selfish genetic elements are more than a daily nuisance in the life of prokaryotes. Whereas viruses can multiply by reprogramming host cells, or integrate in the host genome as “stowaways,” conjugative plasmids (transferrable extrachromosomal DNA) make cells addicted to plasmid-encoded antitoxin factors, thus preventing their disposal. Bacteria and archaea defend themselves against these invasive elements using an adaptive immune system based on clustered regularly interspaced short palindromic repeats (CRISPRs). On page 816 in this issue, Jinek et al. (1) show how the CRISPR effector enzyme Cas9 from bacteria is directed not by one, but two small RNAs to cleave invader DNA

The rise and fall of CRISPRs - dynamics of spacer acquisition and loss

Bacteria and Archaea are continuously exposed to mobile genetic elements (MGE), such as viruses and plasmids. MGEs may provide a selective advantage, may be neutral or may cause cell damage. To protect against invading DNA, prokaryotes utilize a number of defence systems, including the CRISPR/Cas system. CRISPR/Cas systems rely on integration of invader sequences (spacers) into CRISPR loci that act as a genetic memory of past invasions. Processed CRISPR transcripts are utilized as guides by Cas proteins to cleave complementary invader nucleic acids. In this issue, two groups report on spacer acquisition and turnover dynamics of CRISPR loci in a thermoacidophilic archeon and a pathogenic bacterium. Erdmann and Garrett demonstrate that three of the six CRISPR loci of Sulfolobus solfataricus rapidly acquire new spacer sequences from a conjugative plasmid present in a virus mixture. Intriguingly, two distinct mechanisms of spacer integration are utilized: leader adjacent and internal CRISPR spacer acquisition. Lopez-Sanchez and co-workers studied the type II system of Streptococcus agalactiae and observe heterogeneity in the bacterial population. A fraction of the population lost one or more anti-mobilome spacer sequences during its cultivation, allowing the transfer of a MGE in this subpopulation and a rapid response to altering selection pressure

DNA modification

The invention relates to the field of genetic engineering, and to the modification of nucleic acids and organismal genomes in particular. Specifically, the invention concerns the chemical modification of nucleic acids for the improved transformation of cells. Accordingly, the invention provides for modified nucleic acids, methods of modifying nucleic acids, methods of transforming cells and methods for the sequence-directed site-specific genetic modification of cells. The invention additionally encompasses cells transformed with modified nucleic acids and expression constructs for delivery and expression of modified nucleic acids. The nucleic acids, expression constructs, cells and methods of the invention find application in many areas of biotechnology, including, for example, genome editing

Distribution and mechanism of Type I-E CRISPR-Cas systems

Although the CRISPR type I system encompasses six different subtypes (I-A to I-F), only three subtypes have been studied in detail to date. This review includes an analysis of the distribution of CRISPR-Cas systems among the different bacterial and archaeal lineages, and will focus on our mechanistic understanding of the type I-E system of Escherichia coli. We will cover the overall organization of this system, starting with a detailed description of a typical type I-E gene cluster and its associated CRISPR array. In addition, we will describe recent insights on the three different stages in CRISPR-Cas type I-mediated defense: adaptation, expression and crRNA maturation, and interference. A comparison will be presented of the physical and functional characteristics of CRISPR effector complexes from the various subtype

RNAi: prokaryotes get in on the act

The small CRISPR-derived RNAs of bacteria and archaea provide adaptive immunity by targeting the DNA of invading viruses and plasmids. Hale et al. (2009) now report on a new variant CRISPR/Cas complex in the archaeon Pyrococcus furiosus that uses guide RNAs to specifically target and cleave RNA not DN