Outer Membrane-Peptidoglycan Anchoring in Pseudomonas aeruginosa

Gram-negative bacteria have an elaborate envelope that is composed of an outer membrane, the cell wall, and the inner membrane, which collectively encase the cytoplasm. The outer membrane is covalently anchored to the peptidoglycan, the major constituent of the cell wall. We document that the gene product of PA2854 is the catalyst that performs this transformation between the cell wall and the outer-membrane lipoprotein OprI in live Pseudomonas aeruginosa. Furthermore, we reconstitute this reaction in vitro with the use of purified recombinant PA2854, OprI, and synthetic samples of the cell-wall peptidoglycan, in each case documenting the attachment of the side-chain ε-amino group of Lys83 of OprI to the peptide stem of the peptidoglycan. OprI forms a trimeric structure. The X-ray structure of the trimeric state of OprI was solved to 2.1 Å resolution, which reveals it as an extended 82 Å helix bundle. The X-ray structure of PA2854 was also solved at 2.63 Å resolution. The enzyme is composed of three domains, which were documented to bind to the synthetic peptidoglycan and to OprI. The enzyme turns over both non-cross-linked and cross-linked peptidoglycan as its substrate. A model for the ternary assembly of the complex of PA2854-OprI-peptidoglycan is proposed based on the collective evidence. We document that anchoring of the outer membrane to the cell wall catalyzed by PA2854 does not appear to be a redundant reaction (product of more than one enzyme), in the absence of which the bacterium shows a weakened envelope, prone to disruption.