Conserved mechanism of cell-wall synthase regulation revealed by the identification of a new PBP activator in <i>Pseudomonas aeruginosa</i>.

Abstract

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 ^EcLpoB was identified as a critical activator of <i>Escherichia coli</i> PBP1b (^EcPBP1b), one of the major PG synthases of this organism. Activation of ^EcPBP1b is mediated through the association of ^EcLpoB with a regulatory domain on ^EcPBP1b called UB2H. Notably, <i>Pseudomonas aeruginosa</i> also encodes PBP1b (^PaPBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potential ^PaPBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity of ^PaPBP1b. We named this protein LpoP and found that it interacts directly with ^PaPBP1b in vitro and is conserved in many Gram-negative species. Importantly, we also demonstrated that ^PaLpoP-^PaPBP1b as well as an equivalent protein pair from <i>Acinetobacter baylyi</i> can fully substitute for ^EcLpoB-^EcPBP1b in <i>E. coli</i> for PG synthesis. Furthermore, we show that amino acid changes in ^PaPBP1b that bypass the ^PaLpoP requirement map to similar locations in the protein as changes promoting ^EcLpoB bypass in ^EcPBP1b. 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.