engleski

Phenotypic effects of inactivating Cas-CRISPR genes in E. coli

Prokaryotes use a diversity of gene systems to defend against infection by viruses and promiscuous DNA elements. These systems are sometimes collectively called the “ bacteriophage resistome” . Knowledge of some parts of the resistome is well-established, for example general principles of bacterial restriction-modification systems to nullify invading DNA. Other components of the resistome are emerging, including Cas-CRISPR, which comprises clusters of repetitive DNA (CRISPR) that is associated with up to six core cas (crispr-associated) genes. Very recent evidence1 implicates cas-CRISPR in providing a mechanism for integration of bacteriophage DNA fragments into chromosomal sites to promote resistance to future infection: a form of acquired immunity. The same work also implicated one gene in particular (cas-5) as important for resistance to bacteriophage infection in Streptococcus thermophilus. The core cas gene cas-3 is absent from this organism. We report the construction and phenotypic effects of deleting cas-3 (ygcB) and cas-5 (ygcJ) genes in E. coli. The strain deleted for cas-5 ( ygcJ) had no obvious phenotypic difference from the MG1655 mother strain when challenged with plasmids, phage or mutagenic agents. Inactivation of ygcB ( ygcB) had multiple effects on DNA processing, including phenotypes typical of elevated mutagenesis and DNA repair defects, and a propensity for cells to integrate plasmid DNA molecules into the chromosome. We were surprised that  ygcB severely reduces the ability of various types of bacteriophage to infect this E. coli strain. It seems that functional ygcB may be required for phage DNA replication. We also used RT-PCR to detect expression of ygcB and ygcJ in E. coli, and found that functional ygcB is absolutely required for expression of ygcJ. Cas-3 has been predicted to encode a HD-nuclease/superfamily II helicase. We have begun biochemical analyses of DNA processing by the archaeal orthologue of E. coli (mth1086, COG1203), building upon our earlier work2. Our preliminary results describing zinc binding, ATPase and nuclease activities of mth1086 protein will be presented. 1.Barrangou et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709-1712. 2.Guy et al (2004) A novel nuclease-ATPase (Nar71) from archaea is part of a proposed thermophilic DNA repair system. Nucl. Acids Res. 32, 6176-6186.

phage immunity; DNA repair; E. coli

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