Negative Effect of Glucose onompAmRNA Stability: a Potential Role of Cyclic AMP in the Repression ofhfqin Escherichia coli

Abstract

Glucose is a carbon source that is capable of modulating the level of cyclic AMP (cAMP)-regulated genes. In the present study, we found that the stability of ompA mRNA was reduced in Escherichia coli when glucose (40 mM) was present in Luria-Bertani (LB) medium. This effect was associated with a low level of cAMP induced by the glucose. The results were confirmed with an adenylyl cyclase mutant with low levels of cAMP that are not modulated by glucose. Northern blot and Western blot analyses revealed that the host factor I (Hfq) (both mRNA and protein) levels were downregulated in the presence of cAMP. Furthermore, we showed that a complex of cAMP receptor protein (CRP) and cAMP binds to a specific P3 hfq promoter region of hfq and regulates hfq expression. The regulation of the hfq gene was confirmed in vivo using an hfq-deficient mutant transformed with an exogenous hfq gene containing the promoter. These results demonstrated that expression of hfq was repressed by the CRP-cAMP complex. The presence of glucose resulted in increased Hfq protein levels, which decreased ompA mRNA stability. An additional experiment showed that cAMP also increased the stability of fur mRNA. Taken together, these results suggested that the repression of Hfq by cAMP may contribute to the stability of other mRNA in E. coli.

Transcriptional Modulation of Enterotoxigenic Escherichia coli Virulence Genes in Response to Epithelial Cell Interactions

“…However, the studies do appear to affirm a central role of cAMP and the cAMP receptor protein (CRP) in modulation (41) of known (22) and putative (42) virulence genes, including genes involved in flagellar biosynthesis (7), whose expression is known to be under the control of crp (43). Interestingly, the crp gene is itself negatively autoregulated by the cAMP-CRP complex (44).…”

High Growth Rate Downregulates fumA mRNA Transcription but Is Dramatically Compensated by Its mRNA Stability in Escherichia coli

“…Northern blot analysis was performed with 10 lg of total RNA according to the standard protocol as previously described [9]. Briefly, RNA isolation was performed according to the procedures of the TRI reagent-RNA kit (Molecular Research Center, Cincinnati, OH).…”

“…The membrane was then hybridized with digoxigenin (DIG)-labeled DNA probes for at least 12 h at 42°C. DNA probes specific to E. coli fumA were amplified with dNTPs and DIG-11-dUTP mixture (Roche Applied Science, Mannheim, Germany) similar to that previously described [9]. After hybridization, the membranes were washed and were detected using a chemiluminescent substrate (Roche Applied Science, Indianapolis, IN).…”

“…The half-life of the mRNA was determined by the initiation of mRNA transcription that was inhibited by the addition of rifampin to a final concentration of 500 lg/mL before harvesting the total cellular RNA. After adding rifampin, the cultured cells were harvested at different time points and chilled in dry ice/ethanol bath for 1 min before RNA extraction [9,19].…”

Positive Effect of Carbon Sources on Natural Transformation in Escherichia coli: Role of Low-Level Cyclic AMP (cAMP)-cAMP Receptor Protein in the Derepression of rpoS

“…The RNA chaperone protein Hfq, coupled with three small RNAs (DsrA, RprA, and ArcZ), positively controls rpoS mRNA stability and stimulates translation by facilitating annealing between the small RNAs and the 5= untranslated region (UTR) of the mRNA to open the hairpin (19). Previous research has shown that the expression of hfq is repressed by cAMP-CRP and that glucose is capable of increasing Hfq protein levels through reduction of cAMP levels (40). Therefore, it is justifiable to speculate that cAMP-CRP suppresses rpoS indirectly by repressing hfq expression and then interfering with rpoS mRNA stability.…”