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Trapped lipopolysaccharide and LptD intermediates reveal lipopolysaccharide translocation steps across the Escherichia coli outer membrane.

Li X, Gu Y, Dong H, Wang W, Dong C - Sci Rep (2015)

Bottom Line: LptD/E complex forms a N-terminal LPS transport slide, a hydrophobic intramembrane hole and the hydrophilic channel of the barrel, for LPS transport, lipid A insertion and core oligosaccharide and O-antigen polysaccharide translocation, respectively.However, there is no direct evidence to confirm that LptD/E transports LPS from the periplasm to the external leaflet of the outer membrane.By replacing LptD residues with an unnatural amino acid p-benzoyl-L-phenyalanine (pBPA) and UV-photo-cross-linking in E.coli, the translocon and LPS intermediates were obtained at the N-terminal domain, the intramembrane hole, the lumenal gate, the lumen of LptD channel, and the extracellular loop 1 and 4, providing the first direct evidence and "snapshots" to reveal LPS translocation steps across the outer membrane.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

ABSTRACT
Lipopolysaccharide (LPS) is a main component of the outer membrane of Gram-negative bacteria, which is essential for the vitality of most Gram-negative bacteria and plays a critical role for drug resistance. LptD/E complex forms a N-terminal LPS transport slide, a hydrophobic intramembrane hole and the hydrophilic channel of the barrel, for LPS transport, lipid A insertion and core oligosaccharide and O-antigen polysaccharide translocation, respectively. However, there is no direct evidence to confirm that LptD/E transports LPS from the periplasm to the external leaflet of the outer membrane. By replacing LptD residues with an unnatural amino acid p-benzoyl-L-phenyalanine (pBPA) and UV-photo-cross-linking in E.coli, the translocon and LPS intermediates were obtained at the N-terminal domain, the intramembrane hole, the lumenal gate, the lumen of LptD channel, and the extracellular loop 1 and 4, providing the first direct evidence and "snapshots" to reveal LPS translocation steps across the outer membrane.

No MeSH data available.


Observation of LptD and LPS complexes at the luminal gate, lumen of the barrel and the extracellular loops.The positions of residues where cross-linking with LPS was detected are shown in magenta; residue positions with no LPS cross-linking are shown in yellow. a the residues for the incorporation of pBPA at the lumenal gate. b the residues for incorporating pBPA at the lumen of the LptD barrel and the extracellular loops. c and d the top lanes are the detection of the LptD and LPS complexes at the lumenal gate, lumen of the barrel and the extracellular loops, and the bottom lanes are the protein expression levels of the LptD variants. The LptD/LPS complexes were detected at residues K223, L229 and S765 of the lumenal gate, T236, E773, I779 of the lumen of the LptD barrel, T236, N239 and T351 of the extracellular loops 1 and 4.
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f3: Observation of LptD and LPS complexes at the luminal gate, lumen of the barrel and the extracellular loops.The positions of residues where cross-linking with LPS was detected are shown in magenta; residue positions with no LPS cross-linking are shown in yellow. a the residues for the incorporation of pBPA at the lumenal gate. b the residues for incorporating pBPA at the lumen of the LptD barrel and the extracellular loops. c and d the top lanes are the detection of the LptD and LPS complexes at the lumenal gate, lumen of the barrel and the extracellular loops, and the bottom lanes are the protein expression levels of the LptD variants. The LptD/LPS complexes were detected at residues K223, L229 and S765 of the lumenal gate, T236, E773, I779 of the lumen of the LptD barrel, T236, N239 and T351 of the extracellular loops 1 and 4.

Mentions: The above 39 residues of LptD at various locations along the N-terminal domain, the intramembrane hole, the lumenal gate, the barrel’s lumen, and the extracellular loops of LptD were selected for capturing “snapshots” of LPS transport within LptD and across the outer membrane. Mutagenesis was carried out on plasmid pCDFDuet-HisSenLptD which bears the lptD gene from Salmonella typhimurium LT2, using the primers listed in Supplementary Table 1. Overexpression of mutant LptDs was carried out in E. coli BL21(DE3) co-harbouring the pEVOL-pBpF plasmid, which provides the orthogonal tRNA and aminoacyl-tRNA synthetase. Both full-length and truncated LptDs were His-tagged at their N-termini and could be detected with a His tag antibody15. In the presence of pBPA, the amber codon within the mutant lptD gene was suppressed and the full-length LptD variants were observed (Figs 2, 3), where their protein expression levels were very similar. In contrast, no full length LptD was observed in the absence of pBPA (Supplementary Fig. 2).


Trapped lipopolysaccharide and LptD intermediates reveal lipopolysaccharide translocation steps across the Escherichia coli outer membrane.

Li X, Gu Y, Dong H, Wang W, Dong C - Sci Rep (2015)

Observation of LptD and LPS complexes at the luminal gate, lumen of the barrel and the extracellular loops.The positions of residues where cross-linking with LPS was detected are shown in magenta; residue positions with no LPS cross-linking are shown in yellow. a the residues for the incorporation of pBPA at the lumenal gate. b the residues for incorporating pBPA at the lumen of the LptD barrel and the extracellular loops. c and d the top lanes are the detection of the LptD and LPS complexes at the lumenal gate, lumen of the barrel and the extracellular loops, and the bottom lanes are the protein expression levels of the LptD variants. The LptD/LPS complexes were detected at residues K223, L229 and S765 of the lumenal gate, T236, E773, I779 of the lumen of the LptD barrel, T236, N239 and T351 of the extracellular loops 1 and 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493717&req=5

f3: Observation of LptD and LPS complexes at the luminal gate, lumen of the barrel and the extracellular loops.The positions of residues where cross-linking with LPS was detected are shown in magenta; residue positions with no LPS cross-linking are shown in yellow. a the residues for the incorporation of pBPA at the lumenal gate. b the residues for incorporating pBPA at the lumen of the LptD barrel and the extracellular loops. c and d the top lanes are the detection of the LptD and LPS complexes at the lumenal gate, lumen of the barrel and the extracellular loops, and the bottom lanes are the protein expression levels of the LptD variants. The LptD/LPS complexes were detected at residues K223, L229 and S765 of the lumenal gate, T236, E773, I779 of the lumen of the LptD barrel, T236, N239 and T351 of the extracellular loops 1 and 4.
Mentions: The above 39 residues of LptD at various locations along the N-terminal domain, the intramembrane hole, the lumenal gate, the barrel’s lumen, and the extracellular loops of LptD were selected for capturing “snapshots” of LPS transport within LptD and across the outer membrane. Mutagenesis was carried out on plasmid pCDFDuet-HisSenLptD which bears the lptD gene from Salmonella typhimurium LT2, using the primers listed in Supplementary Table 1. Overexpression of mutant LptDs was carried out in E. coli BL21(DE3) co-harbouring the pEVOL-pBpF plasmid, which provides the orthogonal tRNA and aminoacyl-tRNA synthetase. Both full-length and truncated LptDs were His-tagged at their N-termini and could be detected with a His tag antibody15. In the presence of pBPA, the amber codon within the mutant lptD gene was suppressed and the full-length LptD variants were observed (Figs 2, 3), where their protein expression levels were very similar. In contrast, no full length LptD was observed in the absence of pBPA (Supplementary Fig. 2).

Bottom Line: LptD/E complex forms a N-terminal LPS transport slide, a hydrophobic intramembrane hole and the hydrophilic channel of the barrel, for LPS transport, lipid A insertion and core oligosaccharide and O-antigen polysaccharide translocation, respectively.However, there is no direct evidence to confirm that LptD/E transports LPS from the periplasm to the external leaflet of the outer membrane.By replacing LptD residues with an unnatural amino acid p-benzoyl-L-phenyalanine (pBPA) and UV-photo-cross-linking in E.coli, the translocon and LPS intermediates were obtained at the N-terminal domain, the intramembrane hole, the lumenal gate, the lumen of LptD channel, and the extracellular loop 1 and 4, providing the first direct evidence and "snapshots" to reveal LPS translocation steps across the outer membrane.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

ABSTRACT
Lipopolysaccharide (LPS) is a main component of the outer membrane of Gram-negative bacteria, which is essential for the vitality of most Gram-negative bacteria and plays a critical role for drug resistance. LptD/E complex forms a N-terminal LPS transport slide, a hydrophobic intramembrane hole and the hydrophilic channel of the barrel, for LPS transport, lipid A insertion and core oligosaccharide and O-antigen polysaccharide translocation, respectively. However, there is no direct evidence to confirm that LptD/E transports LPS from the periplasm to the external leaflet of the outer membrane. By replacing LptD residues with an unnatural amino acid p-benzoyl-L-phenyalanine (pBPA) and UV-photo-cross-linking in E.coli, the translocon and LPS intermediates were obtained at the N-terminal domain, the intramembrane hole, the lumenal gate, the lumen of LptD channel, and the extracellular loop 1 and 4, providing the first direct evidence and "snapshots" to reveal LPS translocation steps across the outer membrane.

No MeSH data available.