Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.The outer membrane of gram-negative bacteria is decorated with a potent endotoxin (called lipid A), which plays a significant role in bacterial pathogenicity and immune evasion (1). It also acts as a physical barrier protecting the cell from chemical attack and represents a significant obstacle for the effective delivery of numerous antimicrobial agents (2, 3). The outer membrane is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet (4). Phospholipids consist of a glycerol molecule, a phosphate group, and two fatty acid moieties (except for cardiolipins) (5) (see reviews (5, 6) and SI Appendix for the biosynthesis and regulation of phospholipids). LPS, on the other hand, contains three distinct components: lipid A, core oligosaccharides, and O-antigen (7, 8). Lipid A is the sole essential component of LPS, and its biosynthesis involves nine enzyme-catalyzed reactions (8). The lipid A pathway has been widely investigated, and we recently produced a pathway model that incorporates all of the known regulatory mechanisms (9). Briefly, the first reaction step catalyzed by LpxA is highly unfavorable, which makes the proceeding enzyme, LpxC, the first committed enzyme (10). LpxC is regulated by the protease FtsH (11, 12), and we recently postulated that the negative feedback signal arises from lipid A disaccharide, the substrate for LpxK (9). Furthermore, FtsH regulates WaaA (formerly called KdtA), an enzyme downstream of LpxC (13). The exact rationale for WaaA regulation remains unknown.A wealth of research exists for either LPS or phospholipids biosynthesis; however, our current understanding on the crosstalk between both pathways is limited at the moment. Because both pathways are synchronized to ensure a proper balance of membrane components (11, 14), studies underpinning the underlying mechanisms would appear valuable. There are a number of experimental findings that indicate the existence of strong links between both biosynthetic pathways (11, 15, 16). Thus, in the context of outer membrane biogenesis, the role involving phospholipids cannot be ignored in the study of LPS regulation. Furthermore, during membrane synthesis, ∼20 million molecules of fatty acids are synthesized in Escherichia coli (8). Yu et al. (17) reconstituted an in vitro steady-state kinetic system of fatty acid biosynthesis using purified enzymes and observed that the maximum fatty acid production rate obtainable was 100 µM/min. This production rate falls far below the amount of fatty acids required by a cell in vivo if one assumes a cell volume of 6.7 × 10⁻¹⁶ L (18) and a generation time of 30 min (19)]. Therefore, to test the consistency of reported in vitro parameters and investigate the role of the biosynthetic enzymes on fatty acids turnover rate, a “systems” approach is necessary. Similarly, ever since the regulation of WaaA by FtsH was first reported (13), no study has investigated the underlying regulatory mechanism to date. This would also appear important because under wild-type conditions, WaaA catalyzes a step that is required for the endotoxic activity of lipid A (20).In this work, we present a detailed picture of the crosstalk between the LPS and phospholipids biosynthetic machinery. Our work involves a computational kinetic model spanning 81 chemical reactions and involving 90 chemical species. Additionally, we used a series of E. coli fatty acid biosynthesis mutants to investigate the effect of substrate flux into the saturated and unsaturated fatty acid pathway on LpxC stability. Our complete model agrees qualitatively with published datasets and with our own experiments. Our results imply that the catalytic activation of LpxK is dependent on unsaturated fatty acids. Furthermore, our experimental investigations have implicated a secondary protease involved in LpxC regulation. Finally, we have provided experimental evidence to explain the rationale for WaaA regulation.