The regulation of outer membrane biogenesis and its role in cell morphology

Abstract

The Gram-negative bacterial cell envelope consists of three layers: the inner membrane (IM), which is symmetric and composed of phospholipids (PL) on both the inner and outer leaflets, the cell wall which is composed of peptidoglycan (PG), and the outer membrane (OM) which is asymmetric and composed of phospholipids in the inner leaflet and a glycolipid called lipopolysaccharide (LPS) in the outer leaflet. The cell envelope is required for viability, and has proved to be a promising target for antibiotic development. While many of the proteins required for synthesizing and assembling the individual layers of the cell envelope have been identified, how these components are coordinated to ensure uniform cell envelope expansion during growth remains unknown. Here, we investigate the regulation of outer membrane biogenesis. First, we discuss our work characterizing the essential protein YejM as a regulator of LPS synthesis. It has long been established that LpxC, the enzyme that catalyzes the first committed step in LPS synthesis, is regulated by the protease FtsH and the adapter protein LapB. It is unclear how proteolysis is tuned to allow for sufficient LPS synthesis to build the OM while preventing against the toxic intracellular accumulation of LPS. Our work demonstrates that YejM functions as an antagonist to FtsH/LapB and prevents aberrant degradation of LpxC. Next, we describe a genetic analysis of a cell wall synthesis machine called the Rod complex. This study revealed that Rod complex mutants have reduced LPS synthesis in addition to reduced PG synthesis and mutations in genes encoding regulators of LPS synthesis can partially suppress Rod complex phenotypes. Our investigation of these suppressors revealed that the OM has a role in cell morphology. Further analysis revealed a genetic connection between PL synthesis and Rod complex activity. Using transposon insertion sequencing (Tn-seq), we found that insertions in the gene that encodes PlsX, which works together with PlsY to generate precursors of PL synthesis, can rescue some cell wall defects. Previous work revealed that PlsY interacts with YejM, although the significance of this interaction is unknown. These observations lead us to examine possible connections between the PlsXY biosynthetic pathway and the regulation of LPS. Although the mechanistic details are unclear, we suggest that PlsY functions to modulate the activity of YejM in a substrate-dependent manner. Taken together, the work presented here advances our understanding of the biogenesis of the Gram-negative cell envelope and reveals a previously unknown role for the OM in cell shape determination.