Remodeling of Lipid A in Pseudomonas syringae pv. phaseolicola In Vitro
Abstract
:1. Introduction
2. Results
2.1. Orthologues of Lipid A-Modifying Genes Can Be Found in Pseudomonas spp.
2.2. Mass Spectrometric Analysis of Pph 1448A Lipid A Reveals the Activity of PagL, EptA, and LpxO
2.3. Lack of Lipid A-Modifying Enzymes Does Not Affect Growth and LPS Formation in Pph 1448A
3. Discussion
4. Materials and Methods
4.1. Strains and Growth Conditions
4.2. Analysis of Predicted Proteomes of Pseudomonas spp.
4.3. Gene Knockout in Pph 1448A
4.4. Preparation of Lipopolysaccharide from Pph 1448A Strains
4.5. Lipid A Preparation
4.6. Mass Spectrometric Analysis of Lipid A via ESI-MS
4.7. Analysis of Fatty Acids via GC-MS
4.8. Crude LPS Extraction
4.9. Mass Spectrometric Analysis of Crude Lipid A Extracts via MALDI-TOF
4.10. SDS-PAGE and Silver Staining of Bacterial LPS
4.11. Bacterial Growth Curve
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Calculated Mono-Isotopic Mass | Substituting Acyl or Acyloxy-Acyl Chains/Additional Residues on Lipid A Backbone | |||||
---|---|---|---|---|---|---|
[Da] | C-4′ | C-3′ | C-2′ | C-3 | C-2 | C-1 |
1322.872 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) # | H | 10:0((R)-3-O(12:0)) # | |
1338.867 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) # | H | 10:0((R)-3-O(12:0((S)-2-OH))) # | |
1350.903 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | H | 12:0((R)-3-O(12:0)) | |
1366.898 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | H | 12:0((R)-3-O(12:0((S)-2-OH))) | |
1402.838 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) # | H | 10:0((R)-3-O(12:0)) # | P |
1418.833 § | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) # | H | 10:0((R)-3-O(12:0((S)-2-OH))) # | P |
1418.833 § | P | 10:0((R)-3-OH) | 12:0((R)-3-OH) ## | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) ## | P |
1430.870 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | H | 12:0((R)-3-O(12:0)) | P |
1446.865 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | H | 12:0((R)-3-O(12:0((S)-2-OH))) | P |
1473.912 | PP-EtN | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | H | 12:0((R)-3-O(12:0)) | |
1489.907 | PP-EtN | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | H | 12:0((R)-3-O(12:0((S)-2-OH))) | |
1521.034 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | |
1537.029 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0((S)-2-OH))) | |
1541.842 §§ | P * | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) # | H | 10:0((R)-3-O(12:0((S)-2-OH))) # | PP-EtN * |
1541.842 §§ | P * | 10:0((R)-3-OH) | 12:0((R)-3-OH) ## | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) ## | PP-EtN * |
1553.878 | P * | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | H | 12:0((R)-3-O(12:0)) | PP-EtN * |
1569.873 | P * | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | H | 12:0((R)-3-O(12:0((S)-2-OH))) | PP-EtN * |
1572.969 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) # | 10:0((R)-3-OH) | 10:0((R)-3-O(12:0)) # | P |
1588.964 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) # | 10:0((R)-3-OH) | 10:0((R)-3-O(12:0((S)-2-OH))) # | P |
1601.000 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | P |
1616.995 | P | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0((S)-2-OH))) | P |
1676.887 | PP-EtN | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | H | 12:0((R)-3-O(12:0)) | PP-EtN |
1692.882 | PP-EtN | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | H | 12:0((R)-3-O(12:0((S)-2-OH))) | PP-EtN |
1724.009 | P * | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | PP-EtN * |
1740.004 | P * | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0((S)-2-OH))) | PP-EtN * |
1847.017 | PP-EtN | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | PP-EtN |
1863.012 | PP-EtN | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0)) | 10:0((R)-3-OH) | 12:0((R)-3-O(12:0((S)-2-OH))) | PP-EtN |
Strain (Figure Number of Respective MS1 Spectrum) | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
WT (2a) | ΔlpxO (2b) | ΔpagL (2c) | WT (3a) | ΔeptA (3b) | ΔphoPQ (3c) | |||||||
Mcal | Mexp* | Error (Δppm) | Mexp* | Error (Δppm) | Mexp* | Error (Δppm) | Mexp* | Error (Δppm) | Mexp* | Error (Δppm) | Mexp* | Error (Δppm) |
1322.872 | 1322.872 | 0.0 | 1322.872 | 0.0 | n.d. | - | 1322.872 | 0.0 | n.d. | - | 1322.871 | −0.8 |
1338.867 | 1338.867 | 0.0 | n.d. | - | 1338.866 | −0.7 | 1338.868 | 0.7 | 1338.869 | 1.5 | 1338.866 | −0.7 |
1350.903 | 1350.904 | 0.7 | 1350.904 | 0.7 | n.d. | - | 1350.904 | 0.7 | 1350.905 | 1.5 | 1350.904 | 0.7 |
1366.898 | 1366.899 | 0.7 | n.d. | - | 1366.898 | 0.0 | 1366.899 | 0.7 | 1366.900 | 1.5 | 1,366.898 | 0.0 |
1402.838 | n.d. ** | - | 1402.841 | 2.1 | n.d. | - | 1402.842 | 2.9 | 1402.842 | 2.9 | n.d. | - |
1418.833 $ | 1418.836 | 2.1 | 1418.837 | 2.8 | 1418.836 | 2.1 | 1418.836 | 2.1 | 1,418.836 | 2.1 | 1418.836 | 2.1 |
1430.870 | 1430.873 | 2.1 | 1430.872 | 1.4 | 1430.872 | 1.4 | 1430.872 | 1.4 | 1,430.873 | 2.1 | 1430.872 | 1.4 |
1446.865 | 1446.867 | 1.4 | n.d. | - | 1446.867 | 1.4 | 1446.867 | 1.4 | 1,446.867 | 1.4 | 1446.867 | 1.4 |
1473.912 | 1473.914 | 1.4 | 1473.914 | 1.4 | n.d. | - | 1473.914 | 1.4 | n.d. | - | 1473.913 | 0.7 |
1489.907 | 1489.909 | 1.3 | n.d. | - | n.d. | - | 1489.909 | 1.3 | n.d. | - | 1489.908 | 0.7 |
1521.034 | 1521.034 | 0.0 | 1521.035 | 0.7 | 1521.035 | 0.7 | 1521.034 | 0.0 | n.d. | - | 1521.033 | −0.7 |
1537.029 | 1537.029 | 0.0 | n.d. | - | 1537.029 | 0.0 | 1537.030 | 0.7 | 1537.031 | 1.3 | 1537.029 | 0.0 |
1541.842 $ | 1541.845 | 2.0 | 1541.846 | 2.6 | 1541.845 | 2.0 | 1541.845 | 2.0 | n.d. | - | n.d. | - |
1553.878 | 1553.881 | 1.9 | 1553.881 | 1.9 | 1553.882 | 2.6 | 1553.881 | 1.9 | n.d. | - | 1553.880 | 1.3 |
1569.873 | 1569.876 | 1.9 | n.d. | - | 1569.877 | 2.5 | 1569.876 | 1.9 | n.d. | - | 1569.875 | 1.3 |
1572.969 | n.d. | - | n.d. | - | 1572.972 | 1.9 | 1572.973 | 2.5 | 1572.973 | 2.5 | n.d. | - |
1588.964 | n.d. | - | n.d. | - | 1588.967 | 1.9 | 1588.968 | 2.5 | 1588.968 | 2.5 | n.d. | - |
1601.000 | 1601.004 | 2.5 | 1601.003 | 1.9 | 1601.003 | 1.9 | 1601.004 | 2.5 | 1601.004 | 2.5 | 1601.003 | 1.9 |
1616.995 | 1616.999 | 2.5 | n.d. | - | 1616.997 | 1.2 | 1616.998 | 1.9 | 1616.998 | 1.9 | 1616.998 | 1.9 |
1676.887 | 1676.891 | 2.4 | 1676.890 | 1.8 | n.d. | - | 1676.890 | 1.8 | n.d. | - | 1676.890 | 1.8 |
1692.882 | 1692.886 | 2.4 | n.d. | - | n.d. | - | 1692.886 | 2.4 | n.d. | - | 1692.885 | 1.8 |
1724.009 | 1724.013 | 2.3 | 1724.012 | 1.7 | 1724.012 | 1.7 | 1724.012 | 1.7 | n.d. | - | 1724.012 | 1.7 |
1740.004 | 1740.008 | 2.3 | n.d. | - | 1740.007 | 1.7 | 1740.007 | 1.7 | n.d. | - | 1740.007 | 1.7 |
1847.017 | 1847.022 | 2.7 | 1847.021 | 2.2 | 1847.020 | 1.6 | 1847.021 | 2.2 | n.d. | - | 1847.021 | 2.2 |
1863.012 | 1863.017 | 2.7 | n.d. | - | 1863.015 | 1.6 | 1863.016 | 2.1 | n.d. | - | 1863.016 | 2.1 |
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Gerster, T.; Wröbel, M.; Hofstaedter, C.E.; Schwudke, D.; Ernst, R.K.; Ranf, S.; Gisch, N. Remodeling of Lipid A in Pseudomonas syringae pv. phaseolicola In Vitro. Int. J. Mol. Sci. 2022, 23, 1996. https://doi.org/10.3390/ijms23041996
Gerster T, Wröbel M, Hofstaedter CE, Schwudke D, Ernst RK, Ranf S, Gisch N. Remodeling of Lipid A in Pseudomonas syringae pv. phaseolicola In Vitro. International Journal of Molecular Sciences. 2022; 23(4):1996. https://doi.org/10.3390/ijms23041996
Chicago/Turabian StyleGerster, Tim, Michelle Wröbel, Casey E. Hofstaedter, Dominik Schwudke, Robert K. Ernst, Stefanie Ranf, and Nicolas Gisch. 2022. "Remodeling of Lipid A in Pseudomonas syringae pv. phaseolicola In Vitro" International Journal of Molecular Sciences 23, no. 4: 1996. https://doi.org/10.3390/ijms23041996
APA StyleGerster, T., Wröbel, M., Hofstaedter, C. E., Schwudke, D., Ernst, R. K., Ranf, S., & Gisch, N. (2022). Remodeling of Lipid A in Pseudomonas syringae pv. phaseolicola In Vitro. International Journal of Molecular Sciences, 23(4), 1996. https://doi.org/10.3390/ijms23041996