Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli.

barrier of the outer membrane (OM), preventing the entry of toxic molecules such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperons LPS through the periplasm. We report here the construction of lptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampicin and novobiocin) and the SDS detergent, suggesting that lptA41 affects LPS transport. Indeed, lptA41 is defective in Lpt complex assembly and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin resistant suppressors of lptA41. One mutant, in which only bacitracin sensitivity is suppressed, harbours a small in-frame deletion in mlaA, that codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other one, in which bacitracin, rifampicin and SDS sensitivity is suppressed, harbours an additional amino acid substitution in LptA41 and a nonsense mutation in opgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharides synthesis. Characterization of the suppressor 43 mutants highlights different strategies adopted by the cell to overcome OM defects caused by 44 impaired LPS transport.