Mutation and suppressor analysis of the essential lipopolysaccharide transport protein LptA reveals strategies to overcome severe outer membrane permeability defects in Escherichia coli

Federica A. Falchi, Elisa A. Maccagni, Simone Puccio, Clelia Peano, Cristina De Castro, Angelo Palmigiano, Domenico Garozzo, Alessandra M. Martorana, Alessandra Polissi, Gianni Dehò, Paola Sperandeo

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Article numbere00487-17
JournalJournal of Bacteriology
Volume200
Issue number2
DOIs
Publication statusPublished - Jan 1 2018
Externally publishedYes

Keywords

  • Lipopolysaccharide transport
  • MlaA
  • OpgH
  • Outer membrane biogenesis
  • Suppressor analysis

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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