TABLE 3.
Negative ion CE-ES-MS data and proposed compositions of O-deacylated LPS (LPS-OH) from the P. multocida parent (AL435), the hptA mutant (AL836), in vivo-derived kdkA and hptA double mutants, the kdkA mutant (AL721), and complemented kdkA mutant strains
| Description, strain | Observed ions (m/z) |
Molecular mass (Da)a |
Relative intensity | Proposed composition | ||
|---|---|---|---|---|---|---|
| (M-2H)2− | (M-3H)3− | Observed | Calculated | |||
| Parent, AL435 | 1,488.6 | 991.9 | 2,978.9 | 2,977.6 | 0.1 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH |
| 1,499.4 | 999.2 | 3,000.7 | 2,999.5 | 0.1 | 2PCho, 4Hex, 4Hep, Kdo-P, lipid A-OH | |
| 1,548.9 | 1,032.9 | 3,100.7 | 3,100.6 | 0.2 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn | |
| 1,560.0 | 1,040.1 | 3,122.6 | 3,122.6 | 0.6 | 2PCho, 4Hex, 4Hep, Kdo-P-PEtn, lipid A-OH | |
| 1,621.5 | 1,081.2 | 3,245.8 | 3,245.6 | 1.0 | 2PCho, 4Hex, 4Hep, Kdo-P-PEtn, lipid A-OH-PEtn | |
| hptA mutant, AL836 | 616.8 | 1,235.6 | 1,252.2 | 0.1 | Kdo-P, lipid A-OH | |
| 678.4 | 451.6 | 1,358.8 | 1,375.2 | 0.2 | Kdo-P-PEtn, lipid A-OH | |
| 739.8 | 492.6 | 1,481.2 | 1,498.3 | 0.4 | Kdo-P-PEtn, lipid A-OH-PEtn | |
| 1,487.4 | 992.1 | 2,978.1 | 2,977.6 | 0.3 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH | |
| 1,549.9 | 1,033.4 | 3,102.6 | 3,100.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn | |
| In vivo-derived kdkA hptA double mutants | ||||||
| A112V KdkA, AL1774 | 1,487.4 | 992.1 | 2,978.1 | 2,977.6 | 0.3 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH |
| (AL1775),b bird 18 | 1,548.9 | 1,032.9 | 3,100.7 | 3,100.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn |
| R123P KdkA, AL1777 | 1,487.7 | 992.1 | 2,978.3 | 2,977.6 | 0.4 (0.3)c | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH |
| (AL1778), bird 18 | 1,549.5 | 1,032.6 | 3,100.9 | 3,100.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn |
| D193N KdkA, AL1780 | 1,487.7 | 991.5 | 2,977.5 | 2,977.6 | 0.4 (0.5) | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH |
| (AL1781), bird 18 | 1,549.2 | 1,032.3 | 3,100.1 | 3,100.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn |
| H168Y KdkA, AL1782 | 1,487.7 | 991.5 | 2,977.5 | 2,977.6 | 0.4 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH |
| (AL1783), bird 21 | 1,549.2 | 1,032.3 | 3,100.1 | 3,100.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn |
| KdkA complementation data | ||||||
| kdkA mutant, AL721 | 992.1 | 2,979.3 | 2,977.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH | |
| 1,032.9 | 3,101.7 | 3,100.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn | ||
| kdkA mutant + vector, | 1,487.7 | 991.5 | 2,977.5 | 2,977.6 | 0.4 (0.3) | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH |
| AL839 (AL1807) | 1,548.9 | 1,032.9 | 3,100.7 | 3,100.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn |
| kdkA mutant + wild-type | 991.8 | 2,978.4 | 2,977.6 | 0.1 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH | |
| KdkA, AL840 (AL1808) | 1,032.9 | 3,101.7 | 3,100.6 | 0.3 (0.2) | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn | |
| 1,560.0 | 1,040.1 | 3,122.6 | 3,122.6 | 0.6 (0.5) | 2PCho, 4Hex, 4Hep, Kdo-P-PEtn, lipid A-OH | |
| 1,621.5 | 1,081.2 | 3,245.8 | 3,245.6 | 1.0 | 2PCho, 4Hex, 4Hep, Kdo-P-PEtn, lipid A-OH-PEtn | |
| kdkA mutant + A112V | 1,488.0 | 991.5 | 2,977.7 | 2,977.6 | 0.6 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH |
| KdkA, AL1799 (AL1800) | 1,549.2 | 1,032.6 | 3,100.6 | 3,100.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn |
| 1,040.1 | 3,123.3 | 3,122.6 | 0.3 (0.2) | 2PCho, 4Hex, 4Hep, Kdo-P-PEtn, lipid A-OH | ||
| 1,621.5 | 1,080.9 | 3,245.4 | 3,245.6 | 0.4 (0.5) | 2PCho, 4Hex, 4Hep, Kdo-P-PEtn, lipid A-OH-PEtn | |
| kdkA mutant+R123P | 1,488.0 | 991.5 | 2,977.7 | 2,977.6 | 0.3 (0.4) | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH |
| KdkA, AL1801 (AL1802) | 1,548.9 | 1,032.6 | 3,100.4 | 3,100.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn |
| kdkA mutant + H168Y | 1,487.7 | 991.5 | 2,977.5 | 2,977.6 | 0.4 (0.5) | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH |
| KdkA, AL1803 (AL1804) | 1,549.2 | 1,032.6 | 3,100.6 | 3,100.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn |
| kdkA mutant + D193N | 1,487.7 | 991.2 | 2,977.0 | 2,977.6 | 0.4 (0.3) | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH |
| KdkA, AL1805 (AL1806) | 1,548.9 | 1,032.6 | 3,100.4 | 3,100.6 | 1.0 | 2PCho, 3Hex, 4Hep, 2Kdo, lipid A-OH-PEtn |
a
Molecular mass was determined from the observed ions or calculated from the proposed composition. Average mass units were used for calculation of molecular weight, based on the proposed composition, as follows: lipid A-OH, 952.00; hexose (Hex), 162.15; heptose (Hep), 192.17; Kdo-P, 300.13; Kdo, 220.18; PEtn, 123.05; phosphocholine (PCho), 165.05.
b
Duplicate strains were analyzed, but data for only one strain are shown.
c
Where the relative intensity of each glycoform differed between duplicate strains, the value for the second strain is shown in parentheses.