Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

Martorana, Alessandra M.; Benedet, Mattia; Maccagni, Elisa A.; Sperandeo, Paola; Villa, Riccardo; Dehò, Gianni; Polissi, Alessandra

doi:10.1128/jb.00329-16

Cited by 18 publications

(25 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Since LptA m is still proficient in LptC interaction we speculate that the truncated protein can still take contact with the LptDE translocon although with a lower efficiency compared with the wild type protein. This result nicely parallels the finding that cells carrying a C-terminally truncated LptC missing the last 53 amino acids are viable 31 . It has been proposed that in these mutants cells the unstable C-terminally truncated LptC protein, stabilized by LptB overexpression, can still be recruited in the Lpt system and interact with LptA to build functional LPS export machineries 31 .…”

Section: Discussionsupporting

confidence: 84%

See 1 more Smart Citation

Interaction of lipopolysaccharides at intermolecular sites of the periplasmic Lpt transport assembly

Laguri

Sperandeo

Pounot

et al. 2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

Transport of lipopolysaccharides (LPS) to the surface of the outer membrane is essential for viability of Gram-negative bacteria. Periplasmic LptC and LptA proteins of the LPS transport system (Lpt) are responsible for LPS transfer between the Lpt inner and outer membrane complexes. Here, using a monomeric E. coli LptA mutant, we first show in vivo that a stable LptA oligomeric form is not strictly essential for bacteria. The LptC-LptA complex was characterized by a combination of SAXS and NMR methods and a low resolution model of the complex was determined. We were then able to observe interaction of LPS with LptC, the monomeric LptA mutant as well as with the LptC-LptA complex. A LptC-LPS complex was built based on NMR data in which the lipid moiety of the LPS is buried at the interface of the two β-jellyrolls of the LptC dimer. The selectivity of LPS for this intermolecular surface and the observation of such cavities at homo- or heteromolecular interfaces in LptC and LptA suggests that intermolecular sites are essential for binding LPS during its transport.

show abstract

Section: Discussionsupporting

confidence: 84%

“…In E. coli and in absence of mutation in LptF, assembly of LptC and LptA is essential for the LPS transport 31 and presents a stronger affinity than the LptA-LptA complex 26 . This complex was proposed to be stabilized by a head-to-tail interaction between the two jellyroll protein structures.…”

Section: Discussionmentioning

confidence: 99%

Interaction of lipopolysaccharides at intermolecular sites of the periplasmic Lpt transport assembly

Laguri

Sperandeo

Pounot

et al. 2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…LPS assembly and translocation are intricate ATP-dependent processes. In E. coli and Salmonella Typhimurium, an inner-membrane-spanning protein, MsbA, and the LptB 2 FGC protein complex play central roles in LPS transport that are energized by ATP hydrolysis (74)(75)(76). MsbA flips newly synthesized LPS from the cytoplasm to the periplasmic inner membrane leaflet, where LPS interacts with LptB 2 FGC (59).…”

Section: Discussionmentioning

confidence: 99%

More than Rotating Flagella: Lipopolysaccharide as a Secondary Receptor for Flagellotropic Phage 7-7-1

et al. 2018

View full text Add to dashboard Cite

Bacteriophage 7-7-1, a member of the family, infects the soil bacterium sp. H13-3. Infection requires attachment to actively rotating bacterial flagellar filaments, with flagellar number, length, and rotation speed being important determinants for infection efficiency. To identify secondary receptor(s) on the cell surface, we isolated motile, phage-resistant sp. H13-3 transposon mutants. Transposon insertion sites were pinpointed using arbitrary-primed polymerase chain reaction and bioinformatics analyses. Three genes were recognized, whose corresponding proteins had the following computationally predicted functions: AGROH133_07337, a glycosyl transferase, AGROH133_13050, a UDP-glucose 4-epimerase, and AGROH133_08824, an integral cytoplasmic membrane protein. The first two gene products are part of the lipopolysaccharide (LPS) synthesis pathway, while the latter is predicted to be a relatively small (13.4 kDa) cytosolic membrane protein with up to four transmembrane helices. Phenotypes of transposon mutants were verified by complementation and site-directed mutagenesis. Additional characterization of motile, phage resistant mutants is also described. Given these findings, we propose a model for sp. H13-3 infection by bacteriophage 7-7-1 where the phage initially attaches to the flagellar filament and is propelled down towards the cell surface by clockwise flagellar rotation. The phage then attaches to and degrades the LPS to reach the outer membrane, and ejects its DNA into the host using its syringe-like contractile tail. We hypothesize that the integral membrane protein plays an important role in events following viral DNA ejection or in LPS processing and/or deployment. The proposed two-step attachment mechanism may be conserved among other flagellotropic phages infecting Gram-negative bacteria. Flagellotropic bacteriophages belong to tailed phage order , which are the most abundant in the virome. While it is known that these viruses adhere to the bacterial flagellum and use flagellar rotation to reach the cell surface, their infection mechanisms are poorly understood. Characterizing flagellotropic phage-host interactions is crucial to understanding how microbial communities are shaped. Using a transposon mutagenesis approach combined with a screen for motile, phage-resistant mutants, we identified lipopolysaccharides as the secondary cell surface receptor for phage 7-7-1. This is the first cell surface receptor identified for flagellotropic phages. One hypothetical membrane protein was also recognized as essential for infection. These new findings, together with previous results, culminated in an infection model for phage 7-7-1.

show abstract

“…Further questions such as how and where LPS binds and how the proteins interact with each other and change conformation during function are also being resolved . In vitro studies on the soluble domain of LptC (amino acids 24–191) show that LPS co‐elutes with 6xHis‐tagged LptC from affinity resin and suggest that the transfer of LPS from LptC to LptA is unidirectional .…”

Section: Introductionmentioning

confidence: 99%

Characterization of and lipopolysaccharide binding to the E. coliLptC protein dimer

Schultz

Klug

2017

Protein Science

View full text Add to dashboard Cite

Lipopolysaccharide (LPS, endotoxin) is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium. LPS is a large lipid containing several acyl chains as its hydrophobic base and numerous sugars as its hydrophilic core and O-antigen domains, and is an essential element of the organisms' natural defenses in adverse environmental conditions. LptC is one of seven members of the lipopolysaccharide transport (Lpt) protein family that functions to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of the bacterium. LptC is anchored to the IM and associated with the IM LptFGB complex. It is hypothesized that LPS binds to LptC at the IM, transfers to LptA to cross the periplasm, and is inserted by LptDE into the outer leaflet of the outer membrane. The studies described here comprehensively characterize and quantitate the binding of LPS to LptC. Site-directed spin labeling electron paramagnetic resonance spectroscopy was utilized to characterize the LptC dimer in solution and monitor spin label mobility changes at 10 sites across the protein upon addition of exogenous LPS. The results indicate that soluble LptC forms concentration-independent N-terminal dimers in solution, LptA binding does not change the conformation of the LptC dimer nor appreciably disrupt the LptC dimer in vitro, and LPS binding affects the entire LptC protein, with the center and C-terminal regions showing a greater affinity for LPS than the N-terminal domain, which has similar dissociation constants to LptA.

show abstract

Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

Cited by 18 publications

References 43 publications

Interaction of lipopolysaccharides at intermolecular sites of the periplasmic Lpt transport assembly

Interaction of lipopolysaccharides at intermolecular sites of the periplasmic Lpt transport assembly

More than Rotating Flagella: Lipopolysaccharide as a Secondary Receptor for Flagellotropic Phage 7-7-1

Characterization of and lipopolysaccharide binding to the E. coliLptC protein dimer

Contact Info

Product

Resources

About