| Popis: |
CRISPR-Cas (clustered regularly interspaced short palindromic repeats – CRISPR associated genes) is an adaptive immune system in prokaryotes that provides protection against phage infection and foreign DNA such as plasmids. These defence systems consist of cas protein genes and the CRISPR locus that consists of repeat arrays interspaced with sequences originating from invading DNA, which are transcribed and processed into crRNA (CRISPR RNA). In the type IE system, which is found in E. coli, foreign DNA targets are recognized by the crRNA-guided complex called Cascade. Recognition of foreign dsDNA targets results in the formation of an R-loop, in which the crRNA hybridizes with the complementary target DNA strand. The non-complementary strand of the DNA is displaced and this triggers a conformational change in the Cascade complex which leads to the recruitment of the Cas3 protein. Cas3 acts as a nuclease and helicase, and subsequently degrades foreign DNA. It was shown before that E. coli cells are resistant to phage lysis depending on the growth temperature if Cascade, crRNA and Cas3 are expressed while H-NS, a transcriptional repressor of the CRISPR-Cas system, is inactivated. The Cas3 protein was shown to be the limiting factor for resistance to phage infection, but the exact mechanism remained unknown. It has been suggested that the levels of active Cas3 protein could be reduced at elevated temperature of incubation, which was tested in this study. By using a chromosomally inserted hexahistidine tagged cas3 gene fused to an inducible arabinose or native promoter, we determined Cas3 protein levels from different cells (wt, Δhns, ΔhtpG and Δhns ΔhtpG) grown at 30 °C and 37 °C. In strains with an inducible promoter, Western blot analysis showed no significant differences in Cas3 amounts at 30 °C or 37 °C. In strains with the native cas3 promoter, a signal was not detected suggesting that levels of Cas3 protein are very low in the total cell protein lysate. |
