The outer membrane (OM) of Gram-negative bacteria, such as Escherichia coli, is essential for their viability. Lipopolysaccharide (LPS) constitutes the major component of OM, providing the permeability barrier, and a tight balance exists between LPS and phospholipids amounts as both of these essential components use a common metabolic precursor. Hence, checkpoints are in place, right from the regulation of the first committed step in LPS biosynthesis mediated by LpxC through its turnover by FtsH and HslUV proteases in coordination with LPS assembly factors LapB and LapC. After the synthesis of LPS on the inner leaflet of the inner membrane (IM), LPS is flipped by the IM-located essential ATP-dependent transporter to the periplasmic face of IM, where it is picked up by the LPS transport complex spanning all three components of the cell envelope for its delivery to OM. MsbA exerts its intrinsic hydrocarbon ruler function as another checkpoint to transport hexa-acylated LPS as compared to underacylated LPS. Additional checkpoints in LPS assembly are: LapB-assisted coupling of LPS synthesis and translocation; cardiolipin presence when LPS is underacylated; the recruitment of RfaH transcriptional factor ensuring the transcription of LPS core biosynthetic genes; and the regulated incorporation of non-stoichiometric modifications, controlled by the stress-responsive RpoE sigma factor, small RNAs and two-component systems.
Lipopolysaccharide (LPS) constitutes the major component of the outer membrane and is essential for bacteria, such as Escherichia coli. Recent work has revealed the essential roles of LapB and LapC proteins in regulating LPS amounts; although, if any additional partners are involved is unknown. Examination of proteins co-purifying with LapB identified LapD as a new partner. The purification of LapD reveals that it forms a complex with several proteins involved in LPS and phospholipid biosynthesis, including FtsH-LapA/B and Fab enzymes. Loss of LapD causes a reduction in LpxC amounts and vancomycin sensitivity, which can be restored by mutations that stabilize LpxC (mutations in lapB, ftsH and lpxC genes), revealing that LapD acts upstream of LapB-FtsH in regulating LpxC amounts. Interestingly, LapD absence results in the substantial retention of LPS in the inner membranes and synthetic lethality when either the lauroyl or the myristoyl acyl transferase is absent, which can be overcome by single-amino acid suppressor mutations in LPS flippase MsbA, suggesting LPS translocation defects in ΔlapD bacteria. Several genes whose products are involved in cell envelope homeostasis, including clsA, waaC, tig and micA, become essential in LapD’s absence. Furthermore, the overproduction of acyl carrier protein AcpP or transcriptional factors DksA, SrrA can overcome certain defects of the LapD-lacking strain.
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