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Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli.

ABSTRACT: The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.

SUBMITTER: Yosef I

PROVIDER: S-EPMC3384332 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli.

Yosef Ido I Goren Moran G MG Qimron Udi U

Nucleic acids research 20120308 12

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-media ...[more]

PMID: 22402487

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Project description:Antimicrobial resistance threatens our current standards of care for the treatment and prevention of infectious disease. Antibiotics that have multiple targets have a lower propensity for the development of antibiotic resistance than those that have single targets and therefore represent an important tool in the fight against antimicrobial resistance. In this work, groups of essential paralogous proteins were identified in the important Gram-negative pathogen Escherichia coli that could represent novel targets for multitargeting antibiotics. These groups include targets from a broad range of essential macromolecular and biosynthetic pathways, including cell wall synthesis, membrane biogenesis, transcription, translation, DNA replication, fatty acid biosynthesis, and riboflavin and isoprenoid biosynthesis. Importantly, three groups of clinically validated antibiotic multitargets were identified using this method: the two subunits of the essential topoisomerases, DNA gyrase and topoisomerase IV, and one pair of penicillin-binding proteins. An additional eighteen protein groups represent potentially novel multitargets that could be explored in drug discovery efforts aimed at developing compounds having multiple targets in E. coli and other bacterial pathogens. IMPORTANCE Many types of bacteria have gained resistance to existing antibiotics used in medicine today. Therefore, new antibiotics with novel mechanisms must continue to be developed. One tool to prevent the development of antibiotic resistance is for a single drug to target multiple processes in a bacterium so that more than one change must arise for resistance to develop. The work described here provides a comprehensive search for proteins in the bacterium Escherichia coli that could be targets for such multitargeting antibiotics. Several groups of proteins that are already targets of clinically used antibiotics were identified, indicating that this approach can uncover clinically relevant antibiotic targets. In addition, eighteen currently unexploited groups of proteins were identified, representing new multitargets that could be explored in antibiotic research and development.

Dataset Information

Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli.

Publications

Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli.

Similar Datasets

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