Dokument

Summary:
Legume crops undergo a symbiotic relationship with nitrogen-fixing bacteria in order to make atmospheric nitrogen accessible to them. The interaction is mainly controlled by the plant, however the symbiotic bacterium Sinorhizobium meliloti mediates its infection behaviour using cAMP-mediated transcription regulation. To that end, its genome encodes an extraordinary number of 28 adenylyl or guanylyl cyclases, three of which have been linked with suppression of secondary infection. CyaD1, CyaD2 and CyaK each contain a CHASE2 regulatory domain of unknown function. In the proximity of the gene coding for CyaD1 a few other genes were shown to be involved in the regulatory mechanism via overexpression or deletion mutants. One of those is cAMP receptor protein-like (Crp) transcription factor Clr, that has been shown to be regulated by both cAMP and cGMP. Downstream from CyaD1 a gene coding for a CpdA-like phosphodiesterase was identified. Clr is the first bifunctional Crp ortholog to be reported and the underlying mechanism of this feature remains to be revealed. This is of particular interest as cGMP signalling in bacteria is a largely unexplored field. Additionally, CpdA has been reported to be specific for 2’,3’-cAMP. How it is linked to the rest of the regulon was therefore unclear. To support the understanding of function of this novel cAMP and cGMP regulated process of secondary infection repression and downstream gene regulation, the biochemical and structural features of the CyaD1 locus components were investigated in this work. Affinity measurements for the formation of the Clr-effector-DNA complex reveal similar target DNA binding strengths in the presence of cAMP and cGMP. The crystal structure of the complex bound to each nucleotide shows that they elicit the same active conformation, but bind the effector molecules in different conformers. The differences from other Crp orthologs are not sufficient to explain the basis of Clr bifunctionality. Instead, a modified dynamic network likely results in the ability to be activated by cGMP as well. The crystal structure of the Clr ∙ cNMP ∙ DNA complex also shows how the transcription factor directly interacts with the conserved DNA binding motif. HDX-MS measurements give indications on the regions involved in the apo to holo transition. The phylogenetic analysis of Crp-like proteins indicates that Clr belongs to an unexplored subclass of these transcription factors, potentially differentiated by their ability to be activated by cGMP. The structure of CpdA from S. meliloti combined with a characterization of its activity gives new insights in its potential biological role. Contrary to previous reports, CpdA is very promiscuous in its substrate utilization and able to degrade 3’,5’-cAMP with high activity. Aside from that, it also is capable to hydrolyse 2’,3’-cAMP, 2’,3’-cGMP and 3’,5’-cGMP with slightly efficiency. It is the first class III phosphodiesterase to mainly produce 3’-AMP or 2’-AMP from the hydrolysis of 3’,5’-cAMP or 2’,3’-cAMP respectively. Its activity is amplified by manganese addition, which is likely its native cofactor (alone or heteronuclear with iron). The crystal structure of CpdA is only the second class III PDE structure to be reported and nicely reveals a catalytic hydroxide within the active site. With this it provides convincing evidence for a catalytic mechanism involving a SN2-like attack of the bimetallically coordinated hydroxide molecule on the cNMP phosphate, as opposed to the asymmetric transition state involving an additional hydroxide found in E. coli UshA. Additionally, this work provides a first bioinformatic analysis of the CHASE2 regulatory domain structure found in CyaD1D2K and gives a general classification of the aforementioned structural aspects in the cAMP-mediated regulon of Sinorhizobium meliloti.

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