Functional diversification despite structural congruence in the HipBST toxin-antitoxin system of Legionella pneumophila.

IMPORTANCE: Toxin-antitoxin (TA) systems are parasitic genetic elements found in almost all bacterial genomes. They are exchanged horizontally between cells and are typically poorly conserved across closely related strains and species. Here, we report the characterization of a tripartite TA system in the bacterial pathogen Legionella pneumophila that is highly conserved across Legionella species genomes. This system (denoted HipBSTLp) is a distant homolog of the recently discovered split-HipA system in Escherichia coli (HipBSTEc). We present bioinformatic, molecular, and structural analyses of the divergence between these two systems and the functionality of this newly described TA system family. Furthermore, we provide evidence to refute previous claims that the toxin in this system (HipTLp) possesses bifunctionality as an L. pneumophila virulence protein. Overall, this work expands our understanding of the split-HipA system architecture and illustrates the potential for undiscovered biology in these abundant genetic elements.

Phosphorylation-mediated regulation of the Bacillus anthracis phosphoglycerate mutase by the Ser/Thr protein kinase PrkC.

Bacillus anthracis Ser/Thr protein kinase PrkC is necessary for phenotypic memory and spore germination, and the loss of PrkC-dependent phosphorylation events affect the spore development. During sporulation, Bacillus sp. can store 3-Phosphoglycerate (3-PGA) that will be required at the onset of germination when ATP will be necessary. The Phosphoglycerate mutase (Pgm) catalyzes the isomerization of 2-PGA and 3-PGA and is important for spore germination as a key metabolic enzyme that maintains 3-PGA pool at later events. Therefore, regulation of Pgm is important for an efficient spore germination process and metabolic switching. While the increased expression of Pgm in B. anthracis decreases spore germination efficiency, it remains unexplored if PrkC could directly influence Pgm activity. Here, we report the phosphorylation and regulation of Pgm by PrkC and its impact on Pgm stability and catalytic activity. Mass spectrometry revealed Pgm phosphorylation on seven threonine residues. In silico mutational analysis highlighted the role of Thr459 residue towards metal and substrate binding. Altogether, we demonstrated that PrkC-mediated Pgm phosphorylation negatively regulates its activity that is essential to maintain Pgm in its apo-like isoform before germination. This study advances the role of Pgm regulation that represents an important switch for B. anthracis resumption of metabolism and spore germination.