Abstract:
Pseudomonas aeruginosa enhances alginate synthesis in response to oxygen limitation by a mechanism that is currently not fully understood. It was previously found that the membrane-bound diguanylate cyclase (DGC) SadC stimulates alginate synthesis in the lack of oxygen. The C-terminal cytoplasmic part of SadC (SadCcyt) containing the GGEEF domain has substantially higher DGC activity when purified from bacteria grown under anaerobic conditions than aerobic conditions. sadC is located in a predicted and highly conserved operon with genes encoding a mono-functional enoyl-CoA isomerase (odaA, PA4330) and putative dioxygenase reductase (odaI, PA4331), which function as a positive and negative regulator of alginate synthesis under oxygen limitation, respectively. It is vital to notice that SadC does not appear to contain oxygen sensing domains. It suggests that SadC and possibly other proteins regulate alginate synthesis in an oxygen-dependent manner. mRNA levels of those genes are not influenced by changes in oxygen tensions. Bacterial Adenylate Cyclase Two-Hybrid assay was performed to study interactions that occur between SadC, SadCcyt, OdaA, and OdaI. Results indicate that OdaA interacts with SadC and itself in the presence and absence of oxygen. No interaction between OdaI and SadC was observed, suggesting that the inhibitory effect of OdaI on alginate synthesis is not based on direct interaction with SadC. Moreover, we show that a transmembrane domain of SadC is critical for its functionality.
In this work, cysteine residues (C390, C420, C421, and C478) of SadCcyt were substituted with alanine. It was found that in the case of mutant SadCcyt C420A C421A, the binding efficiency of GTP was enhanced. That can mean that oxygen may modify cysteine residues of SadC and influence in that way its functionality. It was hypothesized that the activity of the SadC may be altered in aerobic and anaerobic compared to microaerophilic conditions, which were previously shown to be optimal for the growth of P. aeruginosa.
Through mRNA sequencing, significant transcriptional changes across numerous genes in P. aeruginosa were identified, particularly highlighting impact of deletion of SadC on alginate biosynthesis genes. This global analysis underscored the intricate regulatory network mediated by SadC, suggesting its pivotal role in bacterial adaptation to varying oxygen conditions and its influence on alginate production pathways.
SadC