Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase.

Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins has not been studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to the identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein-tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys-224. This residue is situated in a lid region that covers the enzyme's active site. Consequently, acetylation and succinylation negatively regulate the activity of PtpB.

Ser/Thr protein kinase PrkC-mediated regulation of GroEL is critical for biofilm formation in Bacillus anthracis.

PrkC is a conserved Ser/Thr protein kinase encoded in Bacillus anthracis genome. PrkC is shown to be important for B. anthracis pathogenesis, but little is known about its other functions and phosphorylated substrates. Systemic analyses indicate the compelling role of PrkC in phosphorylating multiple substrates, including the essential chaperone GroEL. Through mass spectrometry, we identified that PrkC phosphorylates GroEL on six threonine residues that are distributed in three canonical regions. Phosphorylation facilitates the oligomerization of GroEL to the physiologically active tetradecameric state and increases its affinity toward the co-chaperone GroES. Deletion of prkC in B. anthracis abrogates its ability to form biofilm. Overexpression of native GroEL recovers the biofilm-forming ability of prkC deletion strain. Similar overexpression of GroEL phosphorylation site mutants (Thr to Ala) does not augment biofilm formation. Further analyses indicate the phosphorylation of GroEL in diverse bacterial species. Thus, our results suggest that PrkC regulates biofilm formation by modulating the GroEL activity in a phosphorylation-dependent manner. The study deciphers the molecular signaling events that are important for biofilm formation in B. anthracis.