Conserved mechanism of cell-wall synthase regulation revealed by the identification of a new PBP activator in Pseudomonas aeruginosa

Penicillin-binding proteins (PBPs) are synthases required to build the essential peptidoglycan (PG) cell wall surrounding most bacterial cells. The mechanisms regulating the activity of these enzymes to control PG synthesis remain surprisingly poorly defined given their status as key antibiotic targets. Several years ago, the outer-membrane lipoprotein (Ec)LpoB was identified as a critical activator of Escherichia coli PBP1b ((Ec)PBP1b), one of the major PG synthases of this organism. Activation of (Ec)PBP1b is mediated through the association of (Ec)LpoB with a regulatory domain on (Ec)PBP1b called UB2H. Notably, Pseudomonas aeruginosa also encodes PBP1b ((Pa)PBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potential (Pa)PBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity of (Pa)PBP1b. We named this protein LpoP and found that it interacts directly with (Pa)PBP1b in vitro and is conserved inmany Gram-negative species. Importantly, we also demonstrated that (Pa)LpoP-(Pa)PBP1b as well as an equivalent protein pair from Acinetobacter baylyi can fully substitute for (Ec)LpoB-(Ec)PBP1b in E. coli for PG synthesis. Furthermore, we show that amino acid changes in (Pa)PBP1b that bypass the (Pa)LpoP requirement map to similar locations in the protein as changes promoting (Ec)LpoB bypass in (Ec)PBP1b. Overall, our results indicate that, although different Gram-negative bacteria activate their PBP1b synthases with distinct lipoproteins, they stimulate the activity of these important drug targets using a conserved mechanism.