In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability 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 chaperones 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, rifampin, and novobiocin) and the detergent SDS, 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, harbors a small in-frame deletion in mlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation in opgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport. IMPORTANCE Lipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective in lptA that exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.
|Titolo:||Mutation and suppressor analysis of the essential lipopolysaccharide transport protein LptA reveals strategies to overcome severe outer membrane permeability defects in Escherichia coli|
|Parole Chiave:||Lipopolysaccharide transport; MlaA; OpgH; Outer membrane biogenesis; Suppressor analysis; Amino Acid Substitution; Bacitracin; Bacterial Outer Membrane Proteins; Carrier Proteins; Escherichia coli; Escherichia coli Proteins; Glycosyltransferases; Lipid A; Lipopolysaccharides; Membrane Proteins; Rifampin; Sodium Dodecyl Sulfate; Cell Membrane Permeability; Suppression, Genetic; Microbiology; Molecular Biology|
|Settore Scientifico Disciplinare:||Settore BIO/19 - Microbiologia Generale|
|Data di pubblicazione:||2018|
|Data ahead of print / Data di stampa:||2017|
|Digital Object Identifier (DOI):||10.1128/JB.00487-17|
|Appare nelle tipologie:||01 - Articolo su periodico|