Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Plasmids used in this study.
Figure 1.
Neisserial LptA::Hisx6 transfers PEA to lipid A of E. coli LPS.
Lipid A profiles of LPS extracted from E. coli strains JCB571 expressing EcDsbA (CKEC272) (Panel A), E. coli JCB571 expressing LptA::Hisx6 (CKEC543) (Panel B) and JCB571 expressing LptA::Hisx6 and EcDsbA (CKEC564) (Panel C) as determined by MALDI-TOF MS. bis-Phosphorylated hexaacylated lipid A (m/z = 1796), the mono-phosphorylated derivative (m/z = 1716), and the heptaacylated version due to the addition of a palmitic acyl residue (m/z = 2034) were detected in all strains. bis-Phosphorylated tetraacylated lipid A (m/z = 1360) was found abundantly in the MALDI spectra of all three strains, which was likely produced from bis-phosphorylated hexaacylated lipid A (m/z = 1796) during the ionization step on MALDI. The lipid A preparations from CKEC543 expressing LptA (Panel B) and CKEC564 co-expressing LptA and EcDsbA (Panel C) also contained ions consistent with one PEA added to the bis-phosphorylated structure (such as m/z 1919; i.e. 1796+123) and the heptaacylated structure (such as m/z = 2157, i.e. 2034+123).
Figure 2.
LptA::Hisx6 stability is dependent upon oxidoreductase activity in E. coli.
Standardised whole cell lysates were separated by SDS-PAGE. A Western immunoblot was developed using anti-His tag antibody to detect the presence of LptA::Hisx6 in the cellular extracts. Lanes were: Lane 1, ColorPlus pre-stained protein molecular weight marker (New England Biolabs); Lane 2: E. coli JCB571 expressing EcDsbA (CKEC272); Lane 3: E. coli JCB571 carrying pTrc99A (CKEC288); Lane 4: E. coli JCB571 expressing LptA::Hisx6 (CKEC543); Lane 5: E. coli JCB571 expressing LptA::Hisx6 and EcDsbA (CKEC564); Lane 6: CKEC564 treated with DTT and alkylated with AMS; and Lane 7: CKEC564 alkylated with AMS. Molecular weights (kDa) are indicated on the left.
Figure 3.
Oxidation status of LptA::Hisx6 in oxidoreductase mutants of N. meningitidis.
Standardised cell lysates were separated by SDS-PAGE, followed by transfer to a membrane and western immunoblot using anti-Hisx6 HRP conjugate antibody to detect the presence of LptA::Hisx6. Panel A. Lane 1, protein molecular weight standard (New England Biolabs, Cat-2-212); Lane 2: NMBΔdsbA1/dsbA2 expressing LptA::Hisx6 from pCMK1001 (CKNM221) untreated; Lane 3: CKNM221 treated with DTT and alkylated with AMS; Lane 4: CKNM221 alkylated with AMS, Lane 5: NMBΔdsbA3 expressing LptA::Hisx6 (CKNM222) untreated; Lane 6: CKNM222 treated with DTT and alkylated with AMS; Lane 7: CKNM222 alkylated with AMS. Panel B. Lane 1, protein molecular weight standard (New England Biolabs, Cat-2-212); Lane 2: NMB expressing LptA::Hisx6 (CKNM216) untreated; Lane 3: CKNM216 treated with DTT and alkylated with AMS; Lane 4: CKNM216 alkylated with AMS; Lane 5: NMBΔdsbA1/NmdsbA2/dsbA3 expressing LptA::Hisx6 (CKNM755); Lane 6: CKNM755 treated with DTT and alkylated; Lane 7: CKNM755 alkylated with AMS.
Figure 4.
Lipid A substitution profiles of meningococcal oxidoreductase mutants.
Lipid A profiles of LOS extracted from N. meningitidis strain NMB (Panel A), NMBΔlptA::aadA (Panel B), NMBΔNmdsbA1/NmdsbA2 (Panel C), NMBΔNmdsbA3 (Panel D) and NMBΔdsbA1/dsbA2/dsbA3 (Panel E) as determined by MALDI-TOF MS. bis-Phosphorylated hexaacylated lipid A (m/z = 1712), the mono-phosphorylated (m/z = 1632) and the tri-phosphorylated derivative (m/z = 1792) were detected in all strains. Strain NMB and the oxidoreductase mutants all expressed the mono-phosphorylated, bis-phosphorylated and tri-phosphorylated hexaacylated lipid A with a single PEA addition (m/z = 1755, m/z = 1835 and m/z = 1915). Consistent with the loss of LptA activity, NMBΔlptA::aadA lacked these ions.