engleski

Activation of antiviral defense at low temperature of incubation in Escherichia coli

CRISPR (clustered regularly interspaced short palindromic repeats) and its associated Cas proteins is a recently discovered defence mechanism against invading genetic elements in many bacteria and archaea. CRISPR loci comprise arrays of sequence repeats separated by spacers that are homologous to sequences of some phage and plasmids. Escherichia coli have a type IE CRISPR/Cas system that targets invader DNA with CRISPR RNA (crRNA) bound within a "Cascade" nucleoprotein complex. Cas3 helicase-nuclease is thought to then degrade invader DNA. The most efficient protection of cells depends on 5’-AWG-3’ PAM (protospacer adjacent motifs) sequences that are immediately next to a protospacer on the target DNA (e.g. phage) genome. Mutation or variations in PAM result in lower binding affinity of Cascade to the target DNA and consequently absence of resistance to phage infection. Co-expression of Cascade and Cas3 proteins with crRNA is effective at protecting E. coli cells from propagation of bacteriophage into lytic plaques, but the transcriptional repressor H-NS has been shown to repress Cascade and crRNA expression from their chromosomal loci. Factors that control transcription of E. coli cas3 gene have not been identified. Previous studies showed that cells lacking H-NS have elevated levels of Cascade and crRNA transcripts and are resistant to infection by phage vir if they contain appropriate anti-lambda spacer. Surprisingly, resistance was strongly dependent on post-infection temperature of incubation: at 30°C E. coli hns cells containing anti-lambda spacer were fully resistant to phage attack but were sensitive if incubated at 37°C. In this work we wanted to understand the mechanism responsible for temperature dependent resistance of E. coli cells to the phage attack. Our genetic analysis showed that efficient resistance to the phage attack at 37C depended on the correct PAM sequence and expression of the cas3 gene. Surprisingly, using qPCR we provide evidence that the expression of cas3 gene is induced in hns cells at both temperatures. This indicates that the temperature of incubation might regulate the expression of cas3 gene at the post-transcription level.

E. coli; CRISPR; Cas; lambda phage; temperature

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