Carbon Oxidation State in Microbial Polar Lipids Suggests Adaptation to Hot Spring Temperature and Redox Gradients

The influence of oxidation-reduction (redox) potential on the expression of biomolecules is a topic of ongoing exploration in geobiology. In this study, we investigate the novel possibility that structures and compositions of lipids produced by microbial communities are sensitive to environmental redox conditions. We extracted lipids from microbial biomass collected along the thermal and redox gradients of four alkaline hot springs in Yellowstone National Park (YNP) and investigated patterns in the average oxidation state of carbon (ZC), a metric calculated from the chemical formulae of lipid structures. Carbon in intact polar lipids (IPLs) and their alkyl chains becomes more oxidized (higher ZC) with increasing distance from each of the four hot spring sources. This coincides with decreased water temperature and increased concentrations of oxidized inorganic solutes, such as dissolved oxygen, sulfate, and nitrate. Carbon in IPLs is most reduced (lowest ZC) in the hot, reduced conditions upstream, with abundance-weighted ZC values between −1.68 and −1.56. These values increase gradually downstream to around −1.36 to −1.33 in microbial communities living between 29.0 and 38.1°C. This near-linear increase in ZC can be attributed to a shift from ether-linked to ester-linked alkyl chains, a decrease in average aliphatic carbons per chain (nC), an increase in average degree of unsaturation per chain (nUnsat), and increased cyclization in tetraether lipids. The ZC of lipid headgroups and backbones did not change significantly downstream. Expression of lipids with relatively reduced carbon under reduced conditions and oxidized lipids under oxidized conditions may indicate microbial adaptation across environmental gradients in temperature and electron donor/acceptor supply.

structure and point in the direction of the fragment with the monoisotopic mass, in Da, listed at the end of the arrow. pNLC stands for 'precursor neutral loss chromatogram'. This a chromatogram based on the difference between the mass of a lipid precursor ion before and after the loss of a diagnostic fragment. Usually, in the case of IPLs, this is the headgroup or a piece of the headgroup. M+H and M-H refer to the monoisotopic mass of the displayed structure plus or minus a hydrogen atom, respectively, and assumes the resulting fragment has an overall charge of +1.
Abundance-weighted average properties of IPLs. Table S2 gives abundance-weighted average alkyl chain properties that were calculated using Equation 2 and used in Figures 5 and 6. Table S3 reports abundance-weighted average chemical formulae of IPLs, headgroups, backbones, and alkyl chains for hot spring samples calculated using Equation 2. These average formulae were used in Equation 3 to calculate the average Z C values depicted in Figure 7 and reported in Table S4.

Example workflow for calculating Z C in a hypothetical sample
Here we walk through the process of calculating average properties and Z C values for IPLs, headgroups, backbones, and alkyl chains in a hypothetical sample containing the three IPLs depicted in Figure S9. These IPLs include C80:0 1G-GDGT with no rings, C40:1 APT-DEG, and C38:2 APT-AEG.
First, mole fractions of the three IPLs are calculated using Equation 1. This requires a peak area, monoisotopic mass, and response factor value for each IPL. Peak areas and monoisotopic masses of IPL parent ions are given in Figure S9. Response factor assignments for 1G-GDGT, APT-DEG, and APT-AEG are supplied in Table 1, and values for these response factors are reported in Table S1. Assuming this hypothetical sample was analyzed in the same batch as Bison Pool, Empress Pool, and Octopus Spring (batch 1), 1G-GDGT is assigned a response factor value of 6.72 × 10 4 , and APT-DEG and AEG are both assigned a value of 7.31 × 10 5 . With these parameters, Equation 1 gives mole fractions of 0.661, 0.201, and 0.138 for these three IPLs, respectively.
Next, Equation 2 is used to calculate weighted sample-averaged properties such as nC, nUnsat, x ether , x ester , x GDGT , and abundance of each element in IPLs, headgroups, backbones, and alkyl chains (carbon, hydrogen, nitrogen, oxygen, and phosphorus). This calculation requires IPL mole fractions and the total number of components and properties of interest contained within each IPL. In this case, there are eighty aliphatic carbons among four half-chains in C80 1G-GDGT, forty carbons among two chains in C40 APT-DEG, and thirty-eight carbons among two chains in C38 APT-AEG. Entering these values into Equation 2, along with the mole fractions of each IPL, results in an nC value of 19.9 aliphatic carbons per alkyl chain. One can arrive at an nUnsat value of 0.144 unsaturations per alkyl chain in the same way. Fractions of ether-linked alkyl chains, x ether = 0.958, ester-linked chains, x ester = 0.042, and GDGT half-chains, x GDGT = 0.796, can also be derived assuming there are four ether-linked GDGT half-chains in 1G-GDGT, two ether-linked chains in APT-DEG, and one of each ether-and ester-linked chains in APT-AEG.

Figures
As   Figure S2. Mass spectra at the MS (top panel) and MS/MS (middle panel) levels and putative structure for the C:50 variant of (N)glycosaminyl monoglycosyl phosphatidylarchaeol (NG-G-P-AR). This lipid shares many MS/MS fragments with MeNG-G-P-AR ( Figure S1) due to their structural similarity, differing only by a methyl group on the terminal headgroup moiety.  Figure S3. Mass spectrum at MS/MS level and putative structure and fragments for (6-N,6-N,6-N)trimethyllysine lipid (TM-KL). The headgroup is depicted in its zwitterionic form to indicate how its m/z was interpreted with a proton adduct. The headgroup formula for TM-KL is given in Table 1 as C 9 H 19 NO 2 + for its fully protonated state, which differs from its zwitterionic formula of C 9 H 18 NO 2 . Differences in protonation state do not affect Z C .  Figure S4. Mass spectra at the MS (top panel) and MS/MS (bottom and middle panels) levels for unknown lipid '223'-DAG. The headgroup is indicated by C 7 H 12 NO 6 , our best guess for the chemical formula based on fragmentation patterns.  Figure S7. Mass spectra at the MS and MS/MS levels (top panels) and putative structure and fragments (bottom panels) for triglycosyl (N-acetyl)glycosaminyl glycosyl dietherglycerol (3G-NAcG-G-DEG).  Figure S8. Mass spectra at the MS (top panel) and MS/MS (second panel) levels and putative structure and fragments (bottom panels) for G-GA-DAG.  Figure S9. IPLs, structural divisions, and peak areas used in the workflow example for a hypothetical sample (see text). From top to bottom, the IPLs shown are C80:0 1G-GDGT, C40:1 APT-DEG, and C38:2 APT-AEG. Headgroups (green), backbones (blue), and alkyl chains (orange) are designated according to the IPL component division criteria used in this study. Note that the hydrogen atom on the right side of 1G-GDGT is shown as a headgroup. The dashed black lines indicate where the two membrane-spanning biphytyl chains of 1G-GDGT are divided into four alkyl half-chains during calculation of weighted chain properties, elemental abundance, and Z C .