Functional Identification of Proteus mirabilis eptC Gene Encoding a Core Lipopolysaccharide Phosphoethanolamine Transferase
Abstract
:1. Introduction
2. Results and Discussion
2.1. Identification of Putative LPS PEtN Transferases
2.2. Identification of a Core LPS PEtN Transferase
2.3. eptC Gene Distribution
3. Experimental Section
3.1. Bacterial Strains, Plasmids, and Growth Conditions
3.2. General DNA Methods
3.3. DNA Sequencing and Computer Analysis of Sequence Data
3.4. Plasmid Constructions and Mutant Complementation Studies
3.5. LPS Isolation and SDS-PAGE
3.6. Large-Scale Isolation and Mild-Acid Degradation of LPS
3.7. Mass Spectrometry and NMR Studies
4. Conclusions
Acknowledgments
Conflicts of Interest
Abbreviations
LPS | lipopolysaccharide |
OS | oligosaccharide |
O-PS | O-polysaccharide or O-antigen |
PEtN | phosphoethanolamine |
l,d-Hep | l-glycero-d-manno-heptose |
Kdo | 3-deoxy-d-manno-oct-2-ulosonic acid or 3-deoxyoctulosonic acid |
l-Ara4N | 4-amino-4-deoxy-l-arabinose |
- Author ContributionsAll the authors contributed to experimental data, their analysis, and the writing of the paper.
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Sugar residue | C-1; H-1 | C-2; H-2 | C-3; H-3 (H-3a,3e) | C-4; H-4 | C-5; H-5 | C-6; H-6 (H-6a,6b) | C-7; H-7 (H-7a,7b) | C-8; H-8 (H-8a,8b) |
---|---|---|---|---|---|---|---|---|
β-Glcp-(1→ | 103.7; 4.55 | 74.9; 3.32 | 76.7; 3.52 | 71.2; 3.37 | 77.5; 3.47 | 62.7; 3.97, 3.74 | ||
α-ldHepp6P-(1→ | 102.5; 5.34 | 70.9; 4.15 | 71.5; 3.91 | 67.2; 3.91 | 72.4; 3.79 | 74.6; 4.57 | 62.5; 3.76, 3.85 | |
→3,4)-α-ldHepp-(1→ | 102.2; 5.08 | 71.4; 4.17 | 74.8; 4.15 | 74.9; 4.26 | 72.5; 4.15 | 69.5; 4.13 | 64.4; 3.72, 3.74 | |
→5)-α-Kdop | n.f. | 97.0 | 35.4; 2.11, 1.94 | 67.0; 4.12 | 75.9; 4.12 | 72.3; 3.88 | 70.3; 3.70 | 64.4; 3.81, 3.65 |
NH2CH2CH2O- | 63.3; 4.17 | 41.2; 3.31 |
Strain or plasmid | Relevant characteristics | Reference or source |
---|---|---|
P. mirabilis | ||
HI4320 | Wild type | H.L.T. Mobley a |
S1959 | Wild type, serovar O3 | Z. Sydorckzyk |
R110 | Rough mutant of strain S 1959 | Z. Sydorckzyk b |
51/57 | Serovar O28 | Z. Sydorckzyk b |
50/57 | Serovar O27 | Z. Sydorckzyk b |
14/57 | Serovar O6 | Z. Sydorckzyk b |
TG83 | Serovar O57 | Z. Sydorckzyk b |
K. pneumoniae | ||
52145ΔwabH | Non-polar wabH mutant | [9] |
52145ΔwabG | Non-polar wabG mutant | [16] |
E. coli | ||
DH5α | F− endA hsdR17 (rk− mk−) supE44 thi-1 recA1 gyr-A96 φ80lacZ | [19] |
Plasmid | ||
pGEMT easy | PCR generated DNA fragment cloning vector AmpR | Promega c |
pGEMT-eptC | pGEMT with eptC from strain HI4320, ApR | This study |
pBAD18-Cm | Arabinose-inducible expression vector, CmR | [20] |
pBAD18-Cm-eptC | Arabinose-inducible eptC, CmR | This study |
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Aquilini, E.; Merino, S.; Knirel, Y.A.; Regué, M.; Tomás, J.M. Functional Identification of Proteus mirabilis eptC Gene Encoding a Core Lipopolysaccharide Phosphoethanolamine Transferase. Int. J. Mol. Sci. 2014, 15, 6689-6702. https://doi.org/10.3390/ijms15046689
Aquilini E, Merino S, Knirel YA, Regué M, Tomás JM. Functional Identification of Proteus mirabilis eptC Gene Encoding a Core Lipopolysaccharide Phosphoethanolamine Transferase. International Journal of Molecular Sciences. 2014; 15(4):6689-6702. https://doi.org/10.3390/ijms15046689
Chicago/Turabian StyleAquilini, Eleonora, Susana Merino, Yuriy A. Knirel, Miguel Regué, and Juan M. Tomás. 2014. "Functional Identification of Proteus mirabilis eptC Gene Encoding a Core Lipopolysaccharide Phosphoethanolamine Transferase" International Journal of Molecular Sciences 15, no. 4: 6689-6702. https://doi.org/10.3390/ijms15046689
APA StyleAquilini, E., Merino, S., Knirel, Y. A., Regué, M., & Tomás, J. M. (2014). Functional Identification of Proteus mirabilis eptC Gene Encoding a Core Lipopolysaccharide Phosphoethanolamine Transferase. International Journal of Molecular Sciences, 15(4), 6689-6702. https://doi.org/10.3390/ijms15046689