A model system for studying protein–lipopolysaccharide synthesis, assembly, and insertion in the outer membrane of Pseudomonas aeruginosa

Abstract

Ethylenediaminetetraacetate (EDTA) has been previously shown to cause the release of a complex of protein and lipopolysaccharide (PrLPS) from the outer membrane of Pseudomonas aeruginosa. In this present work, cells of P. aeruginosa were incubated in a solution of EDTA, phosphate buffer, and hypertonic sucrose to prepare osmoplasts (osmotically sensitive cells). Osmoplasts were able to undergo self-repair of the outer-membrane damage resulting from EDTA treatment and to regain osmotic stability when incubated in growth medium/hypertonic sucrose for 2 h in the absence of exogenous PrLPS and in the absence of cell division. This repair process was inhibited by either chloramphenicol or KCN. Examination of freeze-etched preparations demonstrated that 40% of the PrLPS units in the outer membrane were removed by EDTA; after 2 h in growth medium/hypertonic sucrose, cells were able to repair this damage by synthesizing and inserting new PrLPS units into the outer membrane. Osmoplasts could also be restored to osmotic stability by suspension in osmoplast supernatant fluid containing PrLPS and Mg2+. This latter restoration process, which was not inhibited by chloramphenicol or KCN, was purely physical while the self-repair restoration process was metabolic. These data are consistent with the concept that the outer membrane proteins, especially the PrLPS units, play a role in stabilizing the cell envelope and in maintaining the osmotic stability of P. aeruginosa.