An essential membrane protein modulates the proteolysis of LpxC to control lipopolysaccharide synthesis in Escherichia coli

Abstract

Gram-negative bacteria are surrounded by a complex cell envelope that includes two membranes. The outer membrane prevents many drugs from entering these cells and is thus a major determinant of their intrinsic antibiotic resistance. This barrier function is imparted by the asymmetric architecture of the membrane with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The LPS and phospholipid synthesis pathways share a common intermediate. Proper membrane biogenesis therefore requires that the flux through each pathway be balanced. In Escherichia coli, a major control point in establishing this balance is the committed step of LPS synthesis mediated by LpxC. Levels of this enzyme are controlled through its degradation by the inner membrane protease FtsH and its presumed adaptor protein LapB(YciM). How turnover of LpxC is controlled has remained unclear for many years. Here, we demonstrate that the essential protein of unknown function YejM(PbgA), which we have renamed ClxD (control of LpxC degradation), participates in this regulatory pathway. Suppressors of ClxD essentiality were identified in lpxC and lapB, and LpxC overproduction was shown to be sufficient to allow survival of {Delta}clxD mutants. Furthermore, the half-life of LpxC was shown to be reduced in cells lacking ClxD, and genetic and physical interactions between LapB and ClxD were detected. Taken together, our results are consistent with a model in which ClxD directly modulates LpxC turnover by FtsH-LapB to regulate LPS synthesis and maintain membrane homeostasis. SIGNIFICANCEThe outer membrane is a major determinant of the intrinsic antibiotic resistance of Gram-negative bacteria. It is composed of both lipopolysaccharide (LPS) and phospholipid, and the synthesis of these lipid species must be balanced for the membrane to maintain its barrier function in blocking drug entry. In this report, we identify an essential protein of unknown function as a key new factor in maintaining LPS/phospholipid balance in the model bacterium Escherichia coli. Our results provide novel insight into how this organism and most likely other Gram-negative bacteria maintain membrane homeostasis and their intrinsic resistance to antibiotics.