cAMP receptor protein(CRP) plays profound roles in many bacteria as a global regulator.In Escherichia coli,CRP E.coli modulates the expression of many operons involved in carbon catabolism,in response to the fluctuation of intracellular cAMP level caused by carbon catabolism.A crp homologue gene has been identified in the genome of Pseudomonas putida,however,little is known about its cellular function.In this work,we investigated ligand response properties of this CRP protein(CRP P.putida).The results showed that in the presence of exogenous cAMP or cGMP,CRP P.putida can activate the lac promoter in E.coli cya crp mutant.In vitro isothermal titration calorimetry(ITC) assays indicated that CRP P.putida could bind cAMP as well as cGMP.Its affinity to cAMP is much higher than CRP E.coli.Sequence alignment of the CRP proteins suggested that the Thr132 of CRP P.putida(analogous to Ser128 of CRP E.coli) could be the key determinant for all ligand responsive properties observed above.When Thr132 of CRP P.putida is mutated to Serine,two phenomena were observed:(i) its affinity to cAMP or cGMP was reduced to a level similar to CRP E.coli ;(ii) its transcriptional activation activity on E.coli lac promoter was diminished.The potential physiological implications of these ligand binding properties are discussed.
The Sinorhizobium meliloti C4-dicarboxylate transport (Dct) system is essential for symbiotic nitrogen fixation. The dctA gene, encoding the C4-dicarboxylate per-mease, is expressed in both free living and symbiotic cells. But in free living cells expression of dctD and dctB is abso-lutely required for the expression of dctA. In this study, in order to investigate the effect of oxygen concentration on the induction of Dct system, E. coli DH5α strain which carries the plasmid-encoded dctABD operon was used in tube assays. It was found that the specific induction of Dct system oc-curred only at a certain depth under the surface of M63- 0.6% agar media, suggesting that Dct system could respond to oxygen concentration during succinate-induced expression. Furthermore, when measured at different oxygen concentra-tions, the highest expression level was observed at oxygen concentration of 2%. Thus, we predict that in addition to dicarboxylates, the induction of Dct system may also regu-lated by oxygen concentration.
When the NifA-mediated activation of Kleb-siella pneumontae nifU promoter is recreated in Escherichia coli, it has been observed that CRP-cAMP has an inhibitory effect on the nifU promoter. Sequence analysis indicates that there is a strong CRP-binding site located upstream of the nifU promoter, overlapping completely with a previously identified Nif A-binding site. In vitro gel retardation analysis indicates that this putative CRP-binding site has similar affinity for CRP, when compared with that at the lac promoter, suggesting that CRP could effectively compete with NifA for such a binding site under physiological conditions. When this putative CRP-binding site on nifU was mutated, in vitro gel retardation analysis indicates that CRP can no longer bind to the mutant promoter. However, when constitutively expressed NifA is used as the activator, CRP-cAMP-mediated inhibitory effect on this mutant nifU promoter has no significant difference when compared with that obtained from its wild-type promoter.
In enteric bacteria, in response to the PTS system, the cAMP receptor protein (CRP) mediates the glucose effect, via regulating σ70-dependent catabolic genes at transcriptional level. In this study, it is observed that the nitrogen fixation capacity of Klebsiella pneumoniae varies strongly when cells are grown on different carbohydrates, and this carbon effect occurs at the level of nif gene expression. Here we show that CRP can repress σ54-dependent nif promoters (nifB, nifE, nifF, nifH, nifJ, nifLA and nifU), in a cAMP dependent fashion, in closed related E. coll background. Sequence analysis of these nif promoters indicates that there is no direct correlation between the fold of CRP-cAMP-mediated inhibition and the upstream cis elements at the promoters. In addition, the crp gene of K. pneumoniae has been isolated and sequenced, which is structural and functional highly homologous to that of E. coli. This suggests that CRP-cAMP-mediated inhibition on the nif promoters could be the reason for
