Evolución de la red fotorreguladora en la bacteria "Myxococcus xanthus" y plasticidad en el modo de acción molecular de los fotorreceptores dependientes de la vitamina B12

Resumen

Light signals diverse biological processes but can also damage cells. Myxococcus xanthus has two pathways to sense light and control carotenogenesis, a photoprotective response. One uses 5’- deoxyadenosylcobalamin (AdoCbl), a B12 form to which CarH (prototype of a new photoreceptor family) binds. The other requires CarA (a B12-independent CarH paralog) and other proteins, some of which are eukaryotic-like. CarH and CarA repress carotenogenesis in the dark and have a DNA-binding (DBD) and a B12-binding (CBD) domain, but only CarH requires B12. Previous work with CarH from M. xanthus (CarHMx) and Thermus thermophilus (CarHTt) showed that AdoCbl provokes transition from a monomer to an AdoCbl-bound oligomer (a tetramer for CarHTt) that binds DNA, and whose light-induced disassembly to monomers relieves repression. Crystal structures of CarHTt revealed how AdoCbl-CarHTt is used as a light sensor, and how three of its four DBDs contact three direct repeats (DRs). Whether the same mode of action applies to other CarH must be addressed to better understand these photoreceptors. Here, Bacillus megaterium CarH homolog (CarHBm) was analysed. ApoCarHBm is a molten globule tetramer, rather than a monomer, that forms DNA-binding tetramers when bound to AdoCbl. Light disrupts CarHBm tetramers to dimers, rather than to monomers. CarHBm uses its four DBDs to bind four DRs at its promoter or three DRs and one pseudorepeat (dr) at PcrtI, but can also bind just three, revealing a plasticity that may be related to the linker between its two domains. The CarHBm operator design is conserved in an operator controlling SpxA2, a global redox-response regulator. Analyses with AdoRhbl (a photostable synthetic AdoCbl homolog with rhodium replacing cobalt) showed that it binds to CarH, though with lower affinity than AdoCbl, promoting formation of oligomers that bind DNA but are unresponsive to light. CarH is widely distributed among bacteria and often coexists with a standalone CBD of unknown function. Within myxobacteria, CarH exists in all three suborders, but CarA and its associated factors only in Cystobacterineae, with carA and carH invariably in tandem in a syntenic carotenogenic operon. The CarA pathway possibly evolved via gene duplication and divergence of an ancestral B12-dependent CarH, de novo gene emergence, and horizontal gene transfer, perhaps driven by necessity to cope with limited B12. Intriguingly, Cystobacter/Mellitangium species have CarH and the proteins of the CarA pathway except CarA. Cystobacter CarH is AdoCbl-dependent but, unlike other characterized homologs, it transitions from an inactive dimer to active AdoCbl-bound dimers, which become inactive monomers upon photolysis. Moreover, Cystobacter CarH exhibits CarA-like properties in its stepwise DNA binding (to three tandem DRs and one dr) and its abrogation by CarS (the CarA antirrepresor). Analyses with Sorangium cellulosum (Sorangiineae) and Haliangium ochraceum (Nannocystineae), which repress carotenogenesis in the dark with B12, revealed that even operators with one or two DRs (with flanking drs) are suitable for CarH binding. Altogether, the results highlight a remarkable plasticity in the behavior of B12-based photoreceptors, which can be helpful for its use as an optogenetic tool. We also analysed the phylogenetic distribution of CarF, the protein known to act earliest in the B12-independent pathway and recently unmasked as the desaturase involved in plasmalogen biosynthesis. CarF bacterial homologs are present only in myxobacteria, Leptospiraceae and Alphaproteobacteria, but they are widespread in animals (including humans) and plants. Homologs from animals and Leptospira are significantly more similar to those in myxobacteria than those from Alphaproteobacteria and plants. Mutation of the conserved histidines in the human CarF homolog had the same effect as mutation of the equivalent histidines in CarF, thus reinforcing that they are closely related.