The effect of the temperature and StpA protein on the antiCas transcript in the CRISPR-Cas mediated protection of Escherichia coli

Mnoge bakterije i većina arheja koriste funkcionalne sustave CRISPR-Cas u obrani protiv strane invazivne DNA. Ekspresija komponenata sustava CRISPR-Cas u E. coli regulirana je s četiri promotora i moguća je samo ako je transkripcijski represor H-NS inaktiviran (Δhns). S promotora anti-Pcas se prepisuje transkript gena antiCas koji nepoznatim mehanizmom utječe na aktivnost sustava CRISPR-Cas. Paralog proteina H-NS, protein StpA u suvišku ukida otpornost na infekciju λvir fagom u Δhns stanicama na 30℃ tako što se ponaša kao dodatni represor. U ovom radu istražena je mogućnost da protein StpA svojom ulogom RNA šaperona utječe na tercijarnu strukturu transkripta antiCas u ovisnosti o temperaturi i time na nukleaznu aktivnost proteina Cas3. Praćenjem otpornosti stanica na infekciju fagom λvir utvrđeno je da suvišak transkripta antiCas pri 31°C inhibira aktivnost CRISPR-Cas sustava neovisno o proteinu StpA, dok je pri suvišku proteina Cas3 na 37°C potreban RNA šaperon StpA kako bi očuvao negativni učinak. Povećana ekspresija transkripta antiCas u mutantima s inducibilnim genima cas uzrokuje lizu stanica i na 31°C i 37°C.Many bacteria and most archaea use functional CRISPR-Cas systems in defense against invading DNA. The expression of CRISPR-Cas system components in E. coli is regulated by four promoters and is only possible if the transcriptional repressor H-NS is inactivated (Δhns). The antiCas gene transcript is transcribed from the anti-Pcas promoter, which affects the activity of the CRISPR-Cas system by an unknown mechanism. A paralog of H-NS protein, StpA protein in excess abolish resistance to λvir phage infection in Δhns cells at 30℃ by acting as an additional repressor. In this paper it was investigated whether the protein StpA, with its role as an RNA chaperone, affects the tertiary structure of the antiCas transcript in temperature dependent manner and consequently affects the nuclease activity of the Cas3 protein. By monitoring the resistance to phage λvir infection, it was determined that the excess of antiCas transcript at 31°C inhibits the activity of the CRISPR-Cas system independently of the StpA protein, while the excess of the Cas3 protein at 37°C requires RNA chaperone StpA to preserve the negative effect. Increased expression of antiCas transcripts in mutants with inducible genes cas causes cell lysis at both 31°C and 37°C