El complejo CarD-CarG y su implicación en un nuevo mecanismo de regulación de la expresión de un sistema CRISPR-Cas

Resumen

The bacterium Myxococcus xanthus has a global regulatory protein complex formed by CarD and CarG, which participates in the response to light and starvation-induced multicellular development. CarD contains a C-terminal DNA binding domain similar to that observed in the eukaryotic architectural factors HMGA, and a N-terminal domain (CarDNt), defining the protein family PF02559, which interacts with CarG and with the RNA polymerase (RNAP). CarG does not bind to DNA directly, but can regulate gene expression via its interaction with CarD. Both proteins could act together as a type of bacterial enhanceosome. The CarD-CarG complex is necessary for the action of several ECF-type (ExtraCytoplasmic Function) ? factors that respond to extracellular signals to provide an appropriate physiological response. In this work, it has been shown that CarDNt is essential for CarD function, and the structure of the RNAP interacting segment (CarD1-72) has been determined. CarD1-72 adopts a Tudor-like structure similar to that described for CdnL, another global regulator of M. xanthus belonging to the same family as CarD. Mutational analysis has proved that, although the RNAP interaction surface is conserved in CarD and CdnL, mutations within CarD1-72 do not abolish CarD activity, in contrast to what is observed in CdnL. In addition, it has been shown that mutations in several non-polar and basic amino acids within the C-terminal CarDNt segment disrupt CarD function, whereas its interaction with CarG remains unaffected. Also, two pairs of ECF-?/anti-? factors: CarQ/CarR, which controls the carotenogenic response, and DdvS/DdvA, which drives the expression of a CRISPR-Cas system, have been further analyzed. The topology inside the membrane of the anti-? factors CarR and DdvA has been established, as well as the interacting domain with their respective ECF-? factors. Similarly, other possible ECF-?/anti-? pairs, in which the anti-? factor contains a NZAS domain associated to a eSTK domain, have been studied. In order to understand the mechanism by which the CarD-CarG complex regulates ECF-? factors, CarD and CarG colocalization experiments have been carried out, proving that CarD and CarG colocalize in vivo, which supports the notion that both proteins always act together as a complex and that CarG is recruited to DNA through its interaction with CarD. Moreover, it has been demonstrated that the binding of CarD to the carQ or ddvS promoter regions requires both an active state of the promoter and the presence of CarG. Regarding their action on DdvS/DdvA, this work has revealed that the CarD-CarG complex, together with DdvS/DdvA, regulate the expression of a type III-B CRISPR-Cas system, in which four DdvS-activated CarD-CarG-dependent promoters have been identified, with one of them lying upstream of the cas operon. Consistent with their direct action, DdvS and CarD-CarG localize at these promoters in vivo. The expression of this CRISPR-Cas system generates a long transcript that comprises the cas genes, the CRISPR locus and the leader segment, which has a possible ?A-dependent promoter but with no significant activitity in vivo. It can therefore be concluded that expression of the CRISPR-Cas system, as well as mature CRISPR-RNA (crRNA) production, depend on the ECF-? factor DdvS and the CarD-CarG complex.