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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.


Detection of LptD and LPS intermediates at the N-terminal domain and the hydrophobic intramembrane hole.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 residues for incorporating pBPA at the N-terminal domain. b residues are selected for the incorporation of pBPA at the intramembrane hole. c the top lanes are western blots for detecting LptD and LPS complexes at the N-terminal domain, while the bottom lanes are the protein expression levels of the LptD variants. LptD/LPS complexes were observed at residues V52, V104, A126, L128 and Y112. d the top lanes are the detection of the LptD and LPS complexes at the intramembrane hole, and the bottom lanes are the protein expression levels of the LptD variants. The protein and LPS complexes are captured at residues A196, I198, F203, L218, V220 and F228.
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f2: Detection of LptD and LPS intermediates at the N-terminal domain and the hydrophobic intramembrane hole.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 residues for incorporating pBPA at the N-terminal domain. b residues are selected for the incorporation of pBPA at the intramembrane hole. c the top lanes are western blots for detecting LptD and LPS complexes at the N-terminal domain, while the bottom lanes are the protein expression levels of the LptD variants. LptD/LPS complexes were observed at residues V52, V104, A126, L128 and Y112. d the top lanes are the detection of the LptD and LPS complexes at the intramembrane hole, and the bottom lanes are the protein expression levels of the LptD variants. The protein and LPS complexes are captured at residues A196, I198, F203, L218, V220 and F228.

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)

Detection of LptD and LPS intermediates at the N-terminal domain and the hydrophobic intramembrane hole.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 residues for incorporating pBPA at the N-terminal domain. b residues are selected for the incorporation of pBPA at the intramembrane hole. c the top lanes are western blots for detecting LptD and LPS complexes at the N-terminal domain, while the bottom lanes are the protein expression levels of the LptD variants. LptD/LPS complexes were observed at residues V52, V104, A126, L128 and Y112. d the top lanes are the detection of the LptD and LPS complexes at the intramembrane hole, and the bottom lanes are the protein expression levels of the LptD variants. The protein and LPS complexes are captured at residues A196, I198, F203, L218, V220 and F228.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Detection of LptD and LPS intermediates at the N-terminal domain and the hydrophobic intramembrane hole.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 residues for incorporating pBPA at the N-terminal domain. b residues are selected for the incorporation of pBPA at the intramembrane hole. c the top lanes are western blots for detecting LptD and LPS complexes at the N-terminal domain, while the bottom lanes are the protein expression levels of the LptD variants. LptD/LPS complexes were observed at residues V52, V104, A126, L128 and Y112. d the top lanes are the detection of the LptD and LPS complexes at the intramembrane hole, and the bottom lanes are the protein expression levels of the LptD variants. The protein and LPS complexes are captured at residues A196, I198, F203, L218, V220 and F228.
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.