The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter.

Mattia Benedet,Simone Puccio,Federica A Falchi,Clelia Peano,Gianni Dehò,Cristiano Di Benedetto,Alessandra Polissi,Eric Cascales

doi:10.1371/journal.pone.0161354

Abstract

The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in γ-Proteobacteria. LptBFG constitute the IM ABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable ΔlptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptFSupC). In complementation tests, lptFSupC mutants suppress lethality of both ΔlptC and lptC conditional expression mutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine.

Highlights

Lipopolysaccharide (LPS), the major glycolipid in the outer layer of Gram-negative bacteria outer membrane (OM), is synthesized at the level of the inner membrane (IM) to be transported to its final destination
To stringently assess whether LptC or any LptC domain is strictly essential for E. coli viability, we attempted to isolate mutants lacking lptC from an ectopically complemented lptC-lptA deletion mutant using a double positive selection for the loss of the complementing plasmid coupled to the replacement of the resident plasmid with a chasing plasmid harboring lptA only
The parental strain KG-286.05/pMBM07 harbors on the chromosome the rpsL150 allele and the deletion of the overlapping lptC and lptA genes (ΔlptCA) replaced by a short ORF; the downstream lptB gene expression is driven by the principal yrbGp promoter (Fig 1A) whereas the ΔlptCA deletion is ectopically complemented by the lptCA genes on pMBM07, a thermosensitive-replication plasmid that cannot be maintained at temperatures 37°C

Summary

Introduction

Lipopolysaccharide (LPS), the major glycolipid in the outer layer of Gram-negative bacteria outer membrane (OM), is synthesized at the level of the inner membrane (IM) to be transported to its final destination (reviewed by [1,2,3]). In Escherichia coli, where this process has been best characterized, the LPS transporter (Lpt) exhibits the overall organization of a transenvelope ATP-binding cassette (ABC) transporter [4] composed by seven proteins, LptA through LptG, which co-sediment in a membrane fraction that contains both IM and OM and co-purify as a single complex spanning the cytoplasmic, IM, periplasmic and OM cell compartments [5]. LptAp1 requires σE, this promoter is not activated by several extra-cytoplasmic stress conditions known to induce the σE-dependent promoters, whereas it responds to conditions affecting lipopolysaccharide biogenesis such as depletion of LptC or LptAB, implying a specialized σE-dependent LPS stress signaling pathway [7,8]. Genetic and biochemical evidence indicate that each of the proteins composing the transenvelope complex is essential for cell viability and that the LPS transporter operates as a single device. Depletion of any Lpt protein, using arabinose dependent conditional expression mutants, leads to similar phenotypes, namely cell lethality, LPS accumulation in the periplasmic leaflet of the IM, and abnormal envelope morphology [9,14,15]

Methods

Results

Conclusion