engleski

StpA represses CRISPR-Cas immunity in H-NS deficient Escherichia coli

Many bacteria and most archaea have CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-associated) systems which provide adaptive immunity against invading DNA elements. Fragments of DNA from previous infections are stored in CRISPR arrays, while products of cas genes provide immunity by integrating new DNA fragments into CRISPR arrays and using this information to recognize and destroy invading DNA. The CRISPR-Cas system in Escherichia coli belongs to class 1, type I-E systems in which foreign DNA targets are recognized by a crRNA-guided complex consisting of five proteins (CasA, CasB, CasC, CasD, CasE) called Cascade, and are degraded by the nuclease/helicase Cas3. In E. coli the CRISPR- Cas system is repressed by the histone-like nucleoid-structuring protein H-NS. H-NS repression can be alleviated either by inactivating the hns gene or by elevated levels of the H-NS antagonist LeuO, which induces higher transcript levels of cas genes than was observed for Δhns cells. This suggests that derepression in Δhns cells is incomplete and that an additional repressor could be involved in the silencing of the CRISPR-Cas system. The H-NS paralog protein StpA is one such candidate, as it has similar DNA binding preferences to those of H-NS. Here we show that overexpression of StpA in Δhns cells containing anti-lambda spacers abolishes resistance to λvir infection and reduces transcription of the casA gene. In cells lacking hns and stpA genes, the transcript levels of the casA gene are higher than in Δhns cells and similar to wt cells overexpressing LeuO. These results suggest that Cascade genes in E. coli are repressed by the StpA protein when H-NS is absent.

Cas3 ; StpA ; CRISPR ; genome editing

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