Functional analysis of the protein machinery required for transport of lipopolysaccharide to the outer membrane of Escherichia coli.
Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.
Sperandeo
Paola
P
Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy.
Lau
Fion K
FK
Carpentieri
Andrea
A
De Castro
Cristina
C
Molinaro
Antonio
A
Dehò
Gianni
G
Silhavy
Thomas J
TJ
Polissi
Alessandra
A
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D001692:Biological Transport / Q000235:genetics / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008070:Lipopolysaccharides / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D046529:Microscopy, Electron, Transmission; D016366:Open Reading Frames / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides