1. Structural Biology and Molecular Biophysics
  2. Evolutionary Biology
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Methylation at the C-2 position of hopanoids increases rigidity in native bacterial membranes

  1. Chia-Hung Wu
  2. Maja Bialecka-Fornal
  3. Dianne K Newman  Is a corresponding author
  1. Howard Hughes Medical Institute, California Institute of Technology, United States
  2. California Institute of Technology, United States
Research Article
Cite this article as: eLife 2015;4:e05663 doi: 10.7554/eLife.05663
8 figures and 4 tables

Figures

Structures of selected hopanoids, cholesterol, and squalene.
https://doi.org/10.7554/eLife.05663.003
Whole cell membrane fluidity.

Error bars represent the standard deviation from three biological replicates (total 21–26 technical replicates).

https://doi.org/10.7554/eLife.05663.005
Membrane fractionation and hopanoids analysis using GC–MS.

(A) Three distinct bands were formed after ultracentrifugation in a Percoll gradient. (B) The bands were recovered and resuspended. (C) Samples were ultracentrifuged to pellet down the purified membranes, which sat on top of a transparent solid Percoll layer. (D) GC–MS of fractionated membranes of R. palustris TIE-1 WT and ΔhpnP.

https://doi.org/10.7554/eLife.05663.006
Molar percentage of hopanoids in the inner membrane (IM) and outer membrane (OM) of WT and ΔhpnP determined by GC–MS.

Error bars represent the standard deviation from three biological replicates. Total hopanoids = sum of (2Me)-Dip and (2Me)-BHT.

https://doi.org/10.7554/eLife.05663.008
Partitioning of hopanoids in the inner membrane (IM) and outer mebraane (OM) of R. palustris TIE-1.

(A) Molar ratio between short (Dip and 2Me-Dip) and long (BHT and 2Me-BHT) hopanoids in WT and ΔhpnP. (B) Molar ratio between methylated and desmethylated hopanoid in WT. Error bars represent the standard deviation from three biological replicates. *p = 0.015; **p < 0.01.

https://doi.org/10.7554/eLife.05663.009
Figure 6 with 1 supplement
Membrane rigidity measurements at 25°C using model lipids.

(A) Dioleoyl phosphatidylcholine (DOPC) and (B) E. coli polar lipid extract (PLE) were mixed with different mol% of cholesterol, squalene, and hopanoids. Error bars represent the standard deviation from three biological replicates (total 21 technical replicates).

https://doi.org/10.7554/eLife.05663.010
Figure 6—figure supplement 1
Membrane rigidity measurements at 25°C and 40°C using model lipids.

(A) DOPC and (B) E. coli polar lipid extract (PLE) were mixed with different mol% of cholesterol, squalene, and hopanoids. Error bars represent standard deviation from three biological replicates (total 21 technical replicates).

https://doi.org/10.7554/eLife.05663.011
LC-MS profiles of the inner membrane (IM) and outer membrane (OM) of R. palustris TIE-1 WT and ΔhpnP.
https://doi.org/10.7554/eLife.05663.012
Figure 8 with 1 supplement
Membrane rigidity measurements at 25°C using total lipid extract from R. palustris TIE-1 inner membrane (IM) and outer membrane (OM).

The IM (A) or OM (B) from ΔhpnP was mixed with different mol% of hopanoids. Error bars represent the standard deviation from three biological replicates (total 21 technical replicates). *p < 0.001 (relative to ΔhpnP).

https://doi.org/10.7554/eLife.05663.015
Figure 8—figure supplement 1
Membrane rigidity measurements at 40°C using total lipid extract from R. palustris TIE-1 inner membrane (IM) and outer membrane (OM).

The IM (A) or OM (B) from ΔhpnP was mixed with different mol% of hopanoids. Small unilamellar vesicles from the lipid mixtures were prepared, and the membrane rigidity was measured by fluorescence polarization of a reporter dye diphenyl hexatriene. Error bars represent standard deviation from three biological replicates (total 21 technical replicates).

https://doi.org/10.7554/eLife.05663.016

Tables

Table 1

Mutant strains used for the whole cell membrane rigidity measurements

https://doi.org/10.7554/eLife.05663.004
GeneFunctionDeletion effect
shc (hpnF)Cyclization of squalene to form C30 hopanoids (diploptene and diplopterol)No hopanoid production and accumulation of squalene
hpnHAddition of adenosine to diploptene to generate adenosylhopane, a precursor for extended hopanoid productionNo extended hopanoid production, accumulation of C30 hopanoids
hpnGRemoval of adenine from adenosyl hopaneNo BHT and aminoBHT production, accumulation of adenosylhopane
hpnNAn IM transporter that transports hopanoids to the outer membraneAbsence of hopanoids in the OM and accumulation of hopanoids in the IM
hpnOProduction of aminoBHTNo aminoBHT production
hpnPMethyl transfer to A ring at C-2No hopanoid methylation
  1. The function of the gene and the effect of its deletion are listed.

Table 2

Purification yields of membrane fractionation using Percoll gradient

https://doi.org/10.7554/eLife.05663.007
Weight % membranes in dry cellsTotalWeight % TLE in membranes
WTinner4.7 ± 0.512.4 ± 0.8
mix3.7 ± 1.25.6 ± 0.1
outer3.3 ± 0.411.6 ± 1.44.9 ± 0.6
ΔhpnPinner4.2 ± 1.315 ± 2.3
mix2.3 ± 0.88.3 ± 2.3
outer2.9 ± 0.69.4 ± 2.25.3 ± 1.4
  1. The yields in wt% of membrane fractionation. Errors represent standard deviation from three biological replicates.

Table 3

Annotation of phospholipids identified by LC-MS analyses (see Figure 7)

https://doi.org/10.7554/eLife.05663.013
CompoundRT (min)[M+H]+[M−C3H7O2HPO4]+[M+NH4]+
PG32:14.39549.4895
PG34:24.89575.505
PC34:25.94758.5694
PG34:16.11577.5208
PG36:26.2603.5364
PE34:26.38716.5225
PC_cyc35:16.76772.5851
PG_cyc356.95591.5364
PG_cyc37:17.05617.5521
PC(35:2)7.06772.5851
PE_cyc35:17.28730.5381
PC34:17.51760.5851
PC36:27.65786.6007
PG(17:0/17:0)7.86579.5364
PG36:17.99605.5521
PE34:18.14718.5381
PE36:28.27744.5538
PC_cyc358.58774.6007
PC_cyc37:18.72800.6164
PE_cyc359.28732.5538
PE_cyc37:19.42758.5694
PC(17:0/17:0)9.75762.6007
PC36:19.91788.6164
PE(17:0/17:0)10.53720.5538
PE36:110.71746.5694
PC_cyc3711.23802.632
PE_cyc3712.1760.5851
PC36:012.79790.634
PE3612.98748.5851
PC36:413.39782.569
CL70:415.091447.0373
CL68:315.11421.0217
CL72:415.311475.0686
CL70:315.341449.053
CL68:215.351423.0373
CL72:315.581477.0843
CL70:215.61451.0686
  1. The types of lipids (PC: phosphatidylcholine, PE: phosphotidylethanolamine, PG: phosphatidylglycerol, CL: cardiolipin, cyc: cyclopropanation; the first number indicates the total number of carbon of the fatty acid chains and the second number indicates the number of double bonds in these chains) and their retention time (RT, min) and m/z value of the base peak are shown. For PC and PE, the base peak is the proton adduct and for CL, the base peak is the ammonium adduct. For PG, the base peak indicates a loss of glycerophosphate (−171 m/z).

Table 4

Phospholipid compositions in the inner membrane (IM) and outer membrane (OM) of R. palustris TIE-1 WT and ΔhpnP analyzed by LC-MS

https://doi.org/10.7554/eLife.05663.014
Weight % of TLE
CompoundRT (min)WT IMWT OMΔhpnP IMΔhpnP OM
PC36:27.655.15 ± 0.475.34 ± 0.265.44 ± 0.575.94 ± 0.48
PC_cyc37:18.724.33 ± 0.454.01 ± 0.484.07 ± 1.133.96 ± 1.24
PC36:19.912.90 ± 0.31.84 ± 0.522.59 ± 0.932.15 ± 1.08
PC34:17.512.66 ± 0.222.23 ± 0.332.53 ± 0.582.38 ± 0.57
PC34:25.941.56 ± 0.121.24 ± 0.441.41 ± 0.511.44 ± 0.48
PC_cyc358.580.23 ± 0.010.14 ± 0.050.20 ± 0.070.17 ± 0.1
PC_cyc35:16.760.12 ± 0.010.07 ± 0.020.11 ± 0.040.09 ± 0.06
PC(35:2)7.060.09 ± 0.010.05 ± 0.030.08 ± 0.030.06 ± 0.05
PC_cyc3711.230.07 ± 0.010.04 ± 0.010.06 ± 0.020.05 ± 0.03
PC36:012.790.02 ± 00.01 ± 00.02 ± 0.010.01 ± 0.01
Sum17.1214.9616.5016.25
PE_cyc37:19.424.92 ± 0.363.36 ± 0.944.68 ± 1.54.18 ± 1.64
PE34:18.142.49 ± 0.191.44 ± 0.492.28 ± 0.841.82 ± 0.92
PE36:110.712.29 ± 0.281.08 ± 0.382.05 ± 0.851.43 ± 0.95
PE36:28.271.88 ± 0.211.22 ± 0.461.74 ± 0.651.59 ± 0.72
PE34:26.380.43 ± 0.020.23 ± 0.080.42 ± 0.160.33 ± 0.21
PE_cyc359.280.20 ± 0.020.11 ± 0.040.19 ± 0.070.15 ± 0.09
PE_cyc35:17.280.12 ± 0.010.06 ± 0.020.11 ± 0.050.09 ± 0.06
PE_cyc3712.10.07 ± 0.010.04 ± 0.010.07 ± 0.020.05 ± 0.03
PE3612.980.04 ± 00.03 ± 0.010.04 ± 0.010.03 ± 0.01
Sum12.447.5811.589.67
PG36:26.22.59 ± 0.241.68 ± 0.542.25 ± 0.671.87 ± 0.8
PG36:17.991.13 ± 0.080.69 ± 0.190.98 ± 0.310.81 ± 0.35
PG34:16.110.77 ± 0.170.5 ± 0.20.6 ± 0.240.51 ± 0.19
PG_cyc37:17.050.11 ± 0.020.07 ± 0.050.08 ± 0.020.07 ± 0.04
PG34:24.890.05 ± 00.03 ± 0.010.04 ± 0.010.03 ± 0.02
PG_cyc356.950.02 ± 00.01 ± 00.02 ± 0.010.01 ± 0.01
PG32:14.390.00 ± 00.01 ± 00 ± 00.01 ± 0
Sum4.6733.973.31
PC + PE + PGTotal % of TLE34.2325.5432.0629.23

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