CRISPR-Cas, DNA Supercoiling, and Nucleoid-Associated Proteins.

In this opinion article we highlight links between the H-NS nucleoid-associated protein, variable DNA topology, the regulation of CRISPR- cas locus expression, CRISPR-Cas activity, and the recruitment of novel genetic information by the CRISPR array. We propose that the requirement that the invading mobile genetic element be negatively supercoiled limits effective CRISPR action to a window in the bacterial growth cycle when DNA topology is optimal, and that this same window is used for the efficient integration of new spacer sequences at the CRISPR array. H-NS silences CRISPR promoters, and we propose that antagonists of H-NS, such as the LeuO transcription factor, provide a basis for a stochastic genetic switch that acts at random in each cell in the bacterial population. In addition, we wish to propose a mechanism by which mobile genetic elements can suppress CRISPR- cas transcription using H-NS homologues. Although the individual components of this network are known, we propose a new model in which they are integrated and linked to the physiological state of the bacterium. The model provides a basis for cell-to-cell variation in the expression and performance of CRISPR systems in bacterial populations. A novel proposal is made for the operation of CRISPR-Cas systems based on the abilities of physiologically responsive DNA topology, nucleoid-associated proteins, and conventional transcription factors to operate in combination to set and to reset the bacterial transcriptome. Our model exploits knowledge that CRISPR-Cas systems are dependent on DNA supercoiling for efficient operation, and that their expression and expansion are sensitive to the activities of nucleoid-associated proteins, especially H-NS and integration host factor (IHF). We describe the mechanisms by which DNA supercoiling and nucleoid-associated proteins influence the adaptation, the expression, and the interference stages of CRISPR-Cas function. A stochastic regulatory switch, based on the mutually antagonistic activities of the H-NS and LeuO proteins, drives cell-to-cell variation in CRISPR- cas transcription within bacterial populations. [ABSTRACT FROM AUTHOR]