Abstract
MtrA is an essential response regulator (RR) protein in M. tuberculosis, and its activity is modulated after phosphorylation from its sensor kinase MtrB. Interestingly, many regulatory effects of MtrA have been reported to be independent of its phosphorylation, thereby suggesting alternate mechanisms of regulation of the MtrAB two-component system in M. tuberculosis. Here, we show that RR MtrA undergoes non-enzymatic acetylation through acetyl phosphate, modulating its activities independent of its phosphorylation status. Acetylated MtrA shows increased phosphorylation and enhanced interaction with SK MtrB assessed by phosphotransfer assays and FRET analysis. We also observed that acetylated MtrA loses its DNA-binding ability on gene targets that are otherwise enhanced by phosphorylation. More interestingly, acetylation is the dominant post-translational modification, overriding the effect of phosphorylation. Evaluation of the impact of MtrA and its lysine mutant overexpression on the growth of H37Ra bacteria under different conditions along with the infection studies on alveolar epithelial cells further strengthens the importance of acetylated MtrA protein in regulating the growth of M. tuberculosis. Overall, we show that both acetylation and phosphorylation regulate the activities of RR MtrA on different target genomic regions. We propose here that, although phosphorylation-dependent binding of MtrA drives its repressor activity on oriC and rpf, acetylation of MtrA turns this off and facilitates division in mycobacteria. Our findings, thus, reveal a more complex regulatory role of RR proteins in which multiple post-translational modifications regulate the activities at the levels of interaction with SK and the target gene expression.
Highlights
One of the primary signaling systems found in bacteria is two-component signaling (TCS), which contains distinct sensory and regulatory components or proteins
In a typical two-component signaling cascade, phosphorylationmediated gene expression changes are the most well-characterized adaptive response associated with them. It is by far the most widely studied adaptive response for TCS, primarily on account of changes recorded in the DNA binding activity of the RR proteins through changes in their phosphorylation status (Stock et al, 2000; Gao et al, 2007; Gao and Stock, 2009)
Similar to the eukaryotic regulator proteins, in which acetylation-dependent gene expression modulation is more prevalent (Verdin and Ott, 2015), the same has been recorded for RR proteins (Christensen et al, 2019a)
Summary
One of the primary signaling systems found in bacteria is two-component signaling (TCS), which contains distinct sensory and regulatory components or proteins. The communication relay between these proteins occurs via a unique phosphorylation design wherein external stimulus causes the autophosphorylation of the sensor kinase (SK) protein, followed by phosphorylation of the response regulator (RR) protein by a phosphotransfer reaction In this reaction, the Acetylation Regulates Repressor Activity of MtrA phosphoryl group from the SK is transferred to the RR without involving fresh ATP hydrolysis. The phosphorylation of RR has been shown to alter its downstream activity, which generally involves changes in its DNA binding activity (Bourret and Silversmith, 2010; Bretl et al, 2011). This stimulus-toresponse cascade, essentially relies on the changes in the phosphorylation status of the RR protein. Similar to phosphorylation (Gao et al, 2007), acetylation has been shown to affect transcriptional activities of RR proteins (Kim and Yang, 2011; Thao et al, 2011; van Noort et al, 2012; Hu et al, 2013), thereby linking signaling changes to metabolic perturbations in the bacterium (Singh et al, 2019)
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