Analysis of the acquired microbial defence mechanism against foreign DNA by the CRISPR system

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Microbial life is threatened constantly by the invasion of foreign genetic elements, such as phages, transposons or plasmids. Recently, a new defence system has been discovered, which confers inheritable immunity to cells that have survived the invasion of foreign DNA. The system, termed CRISPR, consists of chromosomal arrays of short spacer DNA sequences, originating from the foreign genetic elements, which are separated by palindromic repeats. A siRNA-like mechanism has been assumed to explain resistance to foreign DNA but the molecular details of this defence mechanism are not known. Here we propose the analysis of the basic steps of the CRISPR defence system and its regulation in E. coli. Experiments are suggested to analyze the transcription and processing of the CRISPR elements to short crRNAs and to study the involvement of several Cas proteins in this process. Our main objective is to understand the molecular mechanisms of the crRNA-dependent inactivation of foreign DNA. In our first results we could identify new promoters and characterize several elements of their regulation. The results expected from this proposal are of direct importance for biotechnology and food industries, which use microbial cultures and will have the very useful potential for applications against the spreading of antibiotic resistance.