Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - MICROLIPIDS (Microbial lipids: The three domain ‘lipid divide’ revisited)

Teaser

Tremendous progress has been made in the last decade in the genetic characterization of microorganisms, both in culture and in the environment. However, our knowledge of microbial membrane lipids, essential building blocks of the cell, has only marginally improved. This is...

Summary

Tremendous progress has been made in the last decade in the genetic characterization of microorganisms, both in culture and in the environment. However, our knowledge of microbial membrane lipids, essential building blocks of the cell, has only marginally improved. This is remarkable since there exists a dichotomy in the distribution of lipids between the three Domains of Life. Diacyl glycerols based on straight-chain fatty acids are produced by bacteria and eukaryotes, whereas archaea synthesize isoprenoidal glycerol ether lipids. From a microbial evolutionary perspective, this ‘lipid divide’ is enigmatic since it has recently become clear that eukaryotes evolved from the archaea. Previous work in our research group showed that when novel analytical methodology is used, there is a large hidden diversity in microbial lipid composition that may resolve this fundamental question. The aim of the MICROLIPIDS project is to systematically characterize prokaryotic intact polar lipids (IPLs) with state-of-the-art analytical techniques based on liquid chromatography and high-resolution mass spectrometry to bring our knowledge of microbial lipids to the next level. This approach will be complemented by the characterization of functional genes for lipid biosynthesis. This will involve both mapping of known genes, based on the analysis of published whole (meta)genome data, as well as the identification of as yet unknown genes in selected groups of prokaryotes. The results are expected to make a fundamental contribution to (i) our understanding of the evolution of biosynthesis of membrane lipids, (ii) their application as microbial markers in the environment, and (iii) in the development and application of organic proxies in earth sciences.

Work performed

As part of WP 1 we have been working on IPL characterization in microbial cultures. These cultures include halophilic euryarchaea isolated from hypersaline lakes, cold-tolerant methanotrophic bacteria from Siberia and a thermophilic Thaumarchaeota. Among the wide range of lipids identified, a range of novel lipids have been structurally elucidated. Ongoing work as part of WP1 is examining the lipid composition of groups of bacteria in order to examine specific questions about the microbial lipidome. These questions include the methylation of hopanoid lipids in the α-proteobacteria, the range of bacteria that can form ether-bonds and the phylogenetic distribution of iso-diabolic acid production. As part of WP 2 we have developed an exciting data handling method for accurate mass lipid data from the environment. This utilizes software to produce visual representations of the full range of lipids, the so called lipidome, such as two-dimensional heatmap dendrograms.

In WP3 we have been studying the hyperthermophilic bacterium Thermotoga maritima since it possesses ether-bound long chain di-carboxylic acids (diabolic acid) as part of its membrane. To understand how these membrane spanning lipids are formed she has been analyzing the composition of diabolic acid under distinct growth conditions. Through RNA-sequencing she has been study those genes that are upregulated at different growth phases and temperature that correlate with the increase of diabolic acid content to better understand which genes may be involved in this process. Additionally our WP3 research has been focusing on biosynthetic pathway of long chain alkenones (LCAs). These lipids produced by Haptophyte algae form the basis of the paleotemperature proxy UK’37 which is used to reconstruct past sea surface temperatures (SST). Despite its wide use, several studies have pointed out factors other than temperature that can cause uncertainty in the SST estimated by UK’37 such as salinity, light intensity, genetic diversity and ecology of the producers. In order to tackle these issues a better connection between the LCAs and their biological producers we have been investigating its regulation in different physicochemical conditions with the aim of determining the genes responsible for the synthesis of the carbon chain as well as other genes potentially involved in other desaturation steps in the LCA synthesis. Also within WP3 we have studied the activity of two ether-lipid biosynthesis enzymes encoded in a bacterium by recombinant expression in Escherichia coli, as well as performing biochemical assays and analyses of lipids by LC/MS. He established cultivation procedures for thermo-acidophilic and ammonium oxidizing archaea and investigated effects of inhibitors on their lipid production by LC-MS.

Final results

We expect to continue our high output of lipid composition analysis which forms the basis of the MICROLIPIDS project. This will lead to a wide range of publications on top of 17+ publications already published. We will fully develop the Lipidomic method developed in WP2 and apply it to a wide range of environmental samples. This will lead to publications that go well beyond the state-of-the-art for environmental lipid analysis. We will determine the biosynthesis of diabolic membrane spanning lipids in Thermotoga maritima and of long chain alkenones. Both of which will significantly improve our understanding of these compounds that underpin environmental proxies.