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Determination of Cas3 protein levels in Escherichia coli hns and htpG cells under different growth conditions

CRISPR-Cas systems provide adaptive immunity against foreign genetic elements in many prokaryotes by targeting invader DNA with CRISPR RNA (crRNA) bound within a "Cascade" nucleoprotein complex. Cascade enables immunity by an interference reaction between the short crRNA and its complementary target DNA strand, while displacing the non-target strand. Cas3 helicase-nuclease is then recruited onto formed R-loop to degrade both invader DNA strands. The protection of E. coli cells depends on co-expression of all components, i.e. Cascade, Cas3 and crRNA. Previous studies showed that cells lacking the transcriptional repressor H-NS have elevated levels of Cascade and crRNA transcripts, but not of cas3. We observed previously that cas3 is also under transcriptional control by H-NS, but that this is exerted only in stationary phase cells. Furthermore, we also observed that Type I-E CRISPR-Cas mediated resistance to phage λ was strongly temperature dependent, showing that the expression levels of Cas3 protein became limiting for CRISPR-Cas immunity. In this work we wanted to confirm our genetic observations by directly measuring the levels of the Cas3 protein in wt, Δhns, ΔhtpG, Δhns htpG cells. Cells with chromosomally engineered His-tagged cas3 under inducible pBad promoter were grown to log and stationary phase at two different growth temperatures (30°C and 37°C), and the levels of Cas3 protein were determined using anti-His antibody. The obtained results are in agreement with our genetic observations, showing that Cas3 protein levels are increased in stationary phase cells grown at 30°C in comparison to cells grown at 37°C. In addition, the levels of Cas3 protein varied depending on the Δhns or ΔhtpG mutation, but only at 37°C. Interestingly, Cas3 levels were similar in all log phase cells at both temperatures of incubation suggesting that Cas3 protein levels are stable in log phase cells. Further research will be required to better understand the reasons of reduced Cas3 protein levels at 37°C.

CRISPR-Cas; Cas3; H-NS; HtpG; E. coli

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