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. 2023 Oct 11;14:6368. doi: 10.1038/s41467-023-42007-w

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a The envelope fractions of wild-type (WT, lptM⁺) and ΔlptM transformed with pLptDE^His cells were solubilized using the mild detergent DDM and subjected to Ni-affinity purification. The purified OM LPS translocon was analyzed by SDS-PAGE (lanes 1 and 2) and BN-PAGE (lanes 3 and 4) followed by Coomassie Brilliant Blue staining. Data are representative of three independent experiments. b DDM-solubilized LptM^His purified from ΔlptM cells transformed with pLptDEM^His was analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. Data are representative of three independent experiments. c Native mass-spectrometry analysis of LptDE^His and of LptDEM^His purified as in (a) and (b), respectively. A schematic diagram of each complex is shown for each spectrum; LptD in gray, LptE in yellow, LptM in purple. Top: LptDE^His formed a heterodimeric complex of 108,425 ± 11 Da (LptD, theoretical mass of the mature protein = 87,080 Da; LptE^His, theoretical mass of the mature and tri-acylated protein = 21,348 Da). Bottom: LptDEM^His formed a heterotrimeric complex of 114,325 ± 14 Da (LptD, theoretical mass of the mature protein = 87,080 Da; LptE, theoretical mass of the mature and tri-acylated protein = 20,248 Da; LptM^His, theoretical mass of the mature and tri-acylated protein = 6,988 Da). The mass difference between the two complexes is 5,900 Da ± 25 Da, corresponding to the mass of mature tri-acylated LptM 5888 Da. d ΔlptM cells transformed with pLptDEM^His derivative (*) plasmids that carry pBpa at distinct positions in LptD were subjected to UV irradiation as indicated prior to DDM-solubilization and Ni-affinity purification of LptM^His. Upon SDS-PAGE of the elution fractions, proteins were revealed by Coomassie Brilliant Blue staining or Western blotting using an antiserum against LptA. Data are representative of three independent experiments. e Logoplot representation of the LptM amino acid sequence in Enterobacteriaceae. The plot was obtained from the multiple alignment of 37 amino acid sequences of LptM in representative genomes of a restricted Enterobacteriaceae family reported in Supplementary Fig. 4 (see also Supplementary Information). The location of PF13627 is indicated above the logoplot.