## 5 Regulation of CRISPR/Cas Systems  
As CRISPR/Cas systems function as a defense mechanism their expression might be expected to respond to invasion by extrachromosomal elements. This was recently proven in a shotgun proteomics approach in _S. thermophilus_, which showed an increase in Cas protein expression following phage infection [94]. Likewise, CRISPR and Cas expression were increased by phage infection in _Thermus thermophilus_[39]. Furthermore, CRISPR/Cas systems can be modulated in response to UV and play a role in sensitivity to DNA damage, hinting towards other possible roles besides the neutralization of invading elements [63, 67]. However, to date little is known about how CRISPR/Cas systems are regulated in response to those external stimuli or during periods in which the system is not required [63, 67]. Under certain conditions it could be viewed as favorable to down-regulate CRISPR/Cas activity when beneficial elements are available. However, most evidence demonstrates up-regulation of CRISPR/Cas upon exposure to phage. Since CRISPR/Cas systems are not 100% effective, some beneficial elements will be acquired and when they provide a selective advantage they are likely to be maintained. An overview of the regulatory inputs is shown in Figure 4.  
Most information about CRISPR/Cas regulation is available for the type I-E systems in _E. coli_ and _Salmonella enterica_ serovar Typhi backgrounds. The Histone-like nucleoid structuring protein (H-NS) is a global regulator involved in compacting the bacterial chromosome, via high affinity binding to AT-rich, curved DNA (reviewed by [PERSON] _et al_. [95]). Many promoters are located in close proximity to curved DNA. Binding of H-NS to these promoters prevents RNA polymerase (RNAP) binding, which results in gene-silencing [95]. In _E. coli_, H-NS negatively regulates _cas_ gene expression, due to H-NS binding sites near the _cas_ operon promoters [41, 96]. It is hypothesized that  
Figure 4: **Regulation of CRISPR/Cas activity. ** Network model summarizing the regulation of CRISPR/Cas activity. Triangular and flat arrowheads indicate positive and negative effects on CRISPR/Cas activity, respectively for _E. coli_ (red), _Salmonella_ (cyan), _M. xanthus_ (green), _Sulfolobus_ (black), _T. thermophilus_ (purple) and _S. thermophilus_ (blue). For details see text. H-NS will bind to invading nucleic acids when they enter the cell, due to the higher AT-content [97, 98]. Sequestration of H-NS is predicted to free the _cas_ and/or LeuO promoter (see below) for RNAP recognition and thus activate expression, allowing effective CRISPR/Cas-mediated defense [41, 96, 99]. LRP (lysine-responsive regulatory protein) is a negative regulator for _cas_ expression in _S. typhi_[100]. LRP functions in a similar fashion to H-NS by binding to the _cas_ promoter and competitively excluding binding of RNAP. This appears to be independent of H-NS. However, LRP is able to bind simultaneously alongside H-NS, indicating that the two proteins interact to generate a nucleosome structure for _cas_ gene repression [100]. Interestingly, LRP does not regulate the _cas_ operon in _E. coli_, suggesting that the type I-E CRISPR/Cas systems in these two closely-related organisms function differently at the regulatory level [41, 100]. LeuO, a LysR-type transcriptional regulator that also responds to amino acid starvation, affects _cas_ gene expression via competition with H-NS in \(S\). Typhi and _E. coli_[96, 100, 101]. LeuO binds to a region flanking the _cas_ promoter and the H-NS binding site. The binding of LeuO to the DNA competes with H-NS binding, and relieves the inhibition of _cas_ expression by enabling promoter recognition by RNAP [96, 100, 101]. During amino acid starvation expression of LeuO is increased in response to accumulation of the small molecules guanosine 3'-diphosphate 5'-triphosphate and guanosine 3',5'-bis(diphosphate), collectively called (p)ppGpp [102]. Interestingly, accumulation of (p)ppGpp does not occur during phage lambda infection in _E. coli_[103].