DOI: 10.5281/zenodo.10275589
title: Nitrogen and sulfur for phosphorus: Lipidome adaptation for anaerobic sulfate-reducing bacteria in phosphorus-deprived conditions

description: Abstract 

Understanding how microbial lipidomes adapt to environmental and nutrient stress is crucial for comprehending microbial survival and functionality. Certain anaerobic bacteria can synthesize glycerolipids with ether/ester bonds, yet the complexities of their lipidome remodeling under varying environmental and nutritional conditions remain largely unexplored. In this study, we thoroughly examined the lipidome adaptations of Desulfatibacillum alkenivorans strain PF2803T, a mesophilic anaerobic sulfate-reducing bacterium known for its n-alkene degradation capability, under various cultivation conditions including temperature, pH, salinity, and ammonium and phosphorous concentrations. Employing an extensive analytical and computational lipidomic methodology, we identified nearly 400 distinct lipids for the first time, including a range of glycerol ether/ester lipids and various polar head groups. Information theory-based analysis revealed that temperature fluctuations and phosphate scarcity profoundly influenced the lipidome's composition, leading to enhanced diversity and specificity of novel lipids. Notably, phosphorous limitation led to the creation of novel glucuronosylglycerols and sulfur-containing aminolipids, termed butyramide cysteine glycerols, featuring various ether/ester bonds. This suggests a novel adaptive strategy for anaerobic heterotrophs to thrive in phosphorus-depleted areas of the oceans, characterized by a diverse array of nitrogen- and sulfur-containing polar head groups, moving beyond a reliance on conventional non-phospholipid types.

Repository Contents

1_SRB_lipidome.zip: includes all source data and code scripts used for figures in this study. Files are organized as follows and are associated with the corresponding parts of the manuscript: Figure 2A-F, Figure 4A-E, Figure 5A-C, Figure 6A-C, Supplementary Figures 7.

Figure 2. The impact of culturing conditions on lipidomic variability. A) The number of intact polar lipid species in different lipid classes putatively identified in this study. B) Principal Component Analysis (PCA) based on peak intensity of intact polar lipid species, showcasing the variation in general lipidomic features across individual experimental conditions. C) Information theory analysis showing lipidome diversity and specificity based on the Shannon entropy of the lipidomic frequency distribution. D) Lipid species specificity across the various culturing conditions. E) Hierarchical clustering heatmap depicting the distribution of major lipid classes across all the culturing conditions. F) Cumulative variability of all intact polar lipid species within each range of growth conditions, calculated as the difference in mean abundance between the standard growth condition and the variable conditions. The variability analysis excludes phosphate 0.015 mM as it is under phosphorous-sufficient condition, which showed a similar lipidome composition as the standard growth condition. Each condition analysis is based on three biological replicates. Abbreviations: Polar head groups –phosphatidylethanolamines (PE), phosphatidylglycerols (PG), cardiolipins (CL), novel N-butyramide cysteine (BACys), glucuronosyl (GlcA); Core lipids – diacylglycerols (DAGs), acyl/ether glycerols (AEGs), dietherglycerols (DEGs), tetraetherglycerols (TetraEGs), triether/monoacyl glycerols (TriEGs), diether/diacyl glycerol (DiEGs), monoether/triacyl glycerol (MonoEGs), and tetraacylglycerols (TetraAGs), demethylmenaquinone (DMK).

Figure 4. Variability of major lipid classes across different culturing conditions. A) PG with different ether/ester bond core lipids. B) PE with different ether/ester bond core lipids. C) CL with different ether/ester bond core lipids. D) GlcA with different ether/ester bond core lipids. E) Novel BACys with different ether/ester bond core lipids. Asterisks indicate significant differences between the last condition and the current condition (Student's t tests on pairwise differences, *P < 0.05, **P < 0.01 and ***P < 0.001). The numbers of treatments on the x-axis represent the parameters associated with each condition, ranging from low to high. These parameters include temperature (25°C, 30°C, 40°C), pH levels (6.4, 6.8, 7.8), NaCl concentration (3 g/L, 10 g/L, 25 g/L, 60 g/L), phosphate concentration (0.0005 mM, 0.0015 mM, 0.015 mM, 1.5 mM), and ammonium concentration (0.003 g/L, 0.03g/L, 0.3 g/L).

Figure 5. Adaptation of ether/ester bond lipids and polar headgroups across different culturing conditions. A) The ratio of phospholipids with dialkyl chains and tetraalkyl chains, or the ratio of (PE+PG)/CL, calculated as the summed core lipids within each class. B) The logarithmic ratio of phospholipids/non-phospholipids, phospholipids included both diglyceride phospholipids (PG and PE) and CL. C) The ratio of ether/ester bond lipids. The abundance of ethers in lipids with DEGs is calculated based on their inherent intensity, while the abundance of ethers in lipids containing both ether and ester chains is determined using the ratio of ether% multiplied by the intensity. For instance, in CL-TriEG, which has three ether-bond chains and one ester-bond chain, the abundance of the ether chain is calculated as 0.75 multiplied by the intensity. Asterisks indicate significant differences between the previous condition and the current condition (Student's t tests on pairwise differences, *P < 0.05, **P < 0.01 and ***P < 0.001). The numbers of treatments on the x-axis represent the parameters associated with each condition, ranging from low to high. These parameters include temperature (25°C, 30°C, 40°C), pH levels (6.4, 6.8, 7.8), NaCl concentration (3 g/L, 10 g/L, 25 g/L, 60 g/L), phosphate concentration (0.0005 mM, 0.0015 mM, 0.015 mM, 1.5 mM), and ammonium concentration (0.003 g/L, 0.03g/L, 0.3 g/L). The optimal conditions for growth are temperature 25°C, pH 6.8, [NaCl] 10 g/L, phosphate 1.5 mM, and ammonium  0.3 g/L.

Figure 6. Changes in the averaged carbon chain length and double bond equivalents (DB) of the studied sulfur-reducing bacterial lipidome across different culturing conditions. A) The fractional abundance of lipids with different DBs (0-4). B) The average DBs of total lipids across different culturing conditions. C) The average chain length of two-chain lipids across different culturing conditions. Asterisks indicate significant differences between the last condition and the current condition (Student's t tests on pairwise differences, *P < 0.05, **P < 0.01 and ***P < 0.001). The numbers of treatments on the x-axis of Fig. 4B&C represent the parameters associated with each condition, ranging from low to high. 

Supplementary Figure S7. The fractional abundance of lipids with different carbon chain lengths (26-37, 56-68). The numbers from 26 to 37 represent the summed two-chain carbon atoms, while the numbers from 56 to 68 represent the summed four-chain carbon atoms (from CL).

 

2_Lipidome source data_new.xlsx: includes an original table regarding lipidome identification, abundance, precursor mass, retention time, classification as well as ID (name) in the molecular network.

 

 

 

 

 

 

 
description: We thank M. Koenen for the lipidome extraction and I. Mitteau for help with culturing. We acknowledge A. Mets, D. Dorhout and M. Verweij for technical support. JSSD received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no.694569—MICROLIPIDS) and from a Spinoza award from NWO. VG received funding from the French National Research Agency (ANR-12-BSV7-0003) through the project BAGEL (BActerial production of Glycerol Ether Lipids).