engleski

The effect of the transcriptional repressor StpA on CRISPR-Cas activity in E. coli cells lacking H-NS

Clustered regularly interspaced short palindromic repeats – CRISPR associated genes (CRISPR-Cas) is a prokaryotic defence system that protects against phage infection and foreign DNA, such as plasmids. It is comprised of cas protein genes and the CRISPR locus which consists of repeat arrays interspaced with sequences originating from invading DNA, that are transcribed and processed into CRISPR RNA (crRNA). E. coli has a type I-E system, in which foreign DNA targets are recognized by Cascade, a crRNA-guided complex comprised of five proteins (CasA, CasB, CasC, CasD, CasE) and degraded by Cas3. In E. coli the CRISPR-Cas type I-E system is repressed by the histone-like nucleoid-structuring protein H-NS, a global transcriptional repressor. In wt cells H-NS repression can be relieved by elevated levels of LeuO transcription factor which induce higher transcript levels of cas genes than was observed for hns cells. This suggested that derepression in hns cells is incomplete and that an additional repressor could be involved in silencing. We wanted to test if StpA, a paralogue of H-NS with similar DNA binding preferences as H-NS, is another repressor of cas genes. By overexpressing stpA from the plasmid we have managed to abolish resistance to phage in naturally resistant Δhns cells. To confirm the exact mechanism of this phenomenon, we determined cas3 and casA transcript levels in different mutant cells (wt, ΔhnsΔstpA, Δhns, and Δhns cells overexpressing stpA). Our results show that in the absence of H-NS, the StpA protein is another repressor of cas genes.

Cas3 ; StpA ; CRISPR ; CasA

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