The numbers also indicate the nucleotide positions upstream the transcriptional start sites. We also show the amounts of His-OmpR and His-CRP used in each lane. Discussion Autoregulation of CRP-cAMP In E. coli, CRP acts as both repressor and activator for its own gene [28, 29], while also repressing the cyaA expression [30]. Enteric bacteria catabolize other sugars only when the supply of glucose has become depleted, whereas the presence of glucose prevents the bacteria from catabolizing alternative sugars, which is referred to as catabolite repression mainly mediated by CRP-cAMP for positively

controlling the metabolism Romidepsin price of alternative sugars [13, 14]. A mode for the regulation of the CRP-cAMP machinery during catabolite repression could be established in E. coli as follows [28, 29, 31, 32]: i) the presence of glucose (catabolite Napabucasin clinical trial repression) reduces the cAMP level by decreasing the phosphorylated form of enzyme IIAGlc, which is involved in the activation of CyaA, after which the reduction of cAMP can affect the positive autoregulatory mechanism of crp (see below) to cause a further decrease of crp expression; and ii) once at cAMP-rich conditions (e.g., the replacement of glucose by mannitol), CRP-cAMP

activates the crp transcription by occupying the CRP binding site II, after whichthe elevated expression of CRP-cAMP enables its recognition of the CRP binding site I located 40 bp downstream the crp transcription start site (thereby preventing the occupation of RNA polymerase at the crp promoter), while repressing the cyaA transcription; and finally, a return to basal levels of CRP and cAMP is induced. It is noteworthy that transcriptional regulatory association between CRP and its own gene can be detected in Y. pestis. However, CRP bound to a DNA region that overlapped the promoter -10 region of cyaA, can block the entry of the RNA polymerase Ribonucleotide reductase for repressing the transcription of cyaA in Y. pestis (data unpublished). Since the cyaA -encoding

adenylyl cyclase is a key enzyme catalyzing the synthesis of cAMP, which is the sole essential cofactor of CRP, repression of cAMP production by CRP represents a mechanism for negative modulation of cellular CRP function. CRP-cAMP and osmoregulation The cellular cAMP levels are significantly increased at high osmolarity relative to low osmolarity in E. coli; this osmoregulation requires the cAMP molecule, and is mainly exerted at the transcriptional level although the control at the posttranscriptional level cannot be excluded [33]. The replacement of glucose by other catabolites in the medium triggers the elevation of both cAMP and CRP levels in E. coli [32, 34], resulting in the increase and decrease of OmpF and OmpC levels, respectively [8]. OmpF allows a higher number of compounds to enter the cell than the more restrictive OmpC channel, thereby contributing to the transport of amino acids as a secondary carbon/energy source for E.