Periodic Reporting for period 4 - MICROLIPIDS (Microbial lipids: The three domain ‘lipid divide’ revisited)
Reporting period: 2021-04-01 to 2021-09-30
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 (Bacteria, Archaea, and Eukaryotes). 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. The aim of the MICROLIPIDS project was to systematically characterize prokaryotic intact polar lipids (IPLs) with state-of-the-art analytical techniques to bring our knowledge of microbial lipids to the next level. This approach was complemented by the characterization of functional genes for lipid biosynthesis. This involved 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 of the project make a fundamental contribution to our understanding of the evolution of biosynthesis of membrane lipids, their application as microbial markers in the environment, and in the development and application of organic proxies in earth sciences.
A wide range of microbial cultures were analyzed as part of WP1. Through this work many novel lipids have been identified, deepening and broadening our knowledge of the wide diversity of bacterial and archaeal membrane lipids. A wide range of environmental samples were analyzed for the lipid composition as part of WP2. These data sets allowed us to develop a new methodology for environmental lipidomic data workflow using an information theory framework combined with molecular networking based on the similarity of the mass spectra of lipids, enabling the capture of lipidomic diversity and specificity in the environment. As part of WP 3, a range of studies focused on the biosynthetic genes involved in microbial lipid synthesis. These include the biosynthesis of iso-diabolic acid, a major membrane-spanning lipid, and hopanoids in Acidobacteria. We also demonstrated that bacteria of the Fibrobacteres–Chlorobi–Bacteroidetes superphylum encode a putative archaeal pathway for ether-bound isoprenoid membrane lipids in addition to the bacterial fatty acid membrane pathway.
A wide range of microbial cultures were analyzed as part of WP1. Through this work many novel lipids have been identified, deepening and broadening our knowledge of the wide diversity of bacterial and archaeal membrane lipids. A wide range of environmental samples were analyzed for the lipid composition as part of WP2. These data sets allowed us to develop a new methodology for environmental lipidomic data workflow using an information theory framework combined with molecular networking based on the similarity of the mass spectra of lipids, enabling the capture of lipidomic diversity and specificity in the environment. As part of WP 3, a range of studies focused on the biosynthetic genes involved in microbial lipid synthesis. These include the biosynthesis of iso-diabolic acid, a major membrane-spanning lipid, and hopanoids in Acidobacteria. We also demonstrated that bacteria of the Fibrobacteres–Chlorobi–Bacteroidetes superphylum encode a putative archaeal pathway for ether-bound isoprenoid membrane lipids in addition to the bacterial fatty acid membrane pathway.
Nicole Bale (postdoc WP1 and WP2) has been working on intact polar lipid characterization by mass spectrometry, including 200+ microbial cultures, including the only cultured member of the thaumarchaeotal order Nitrosopumilales, 13 strains of halo(alkali)philic euryarchaea from hypersaline lakes and a aerobic psychrotolerant methanotrophic bacteria from a low-temperature environment. Michel Koenen (technician WP1) has been working on biomass extractions and analysis of core lipids by various methods, focusing on the identification of ether and ester bound lipids. Christine Boschman (PhD student WP2; 2017 - 2019) was working on identification of unusual intact polar lipids in the environment. She focused on sample collection (two research cruises) and on improvement of lipid extraction and analysis methods in the laboratory. Su Ding (postdoc WP2) has been developing methods for comprehensive profiling of microbial lipids in marine systems. He has demonstrated this in suspended particulate matter from throughout the water column of a marine euxinic basin (Black Sea) using ultra high-pressure liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS). An information theory framework combined with molecular networking based on the similarity of the mass spectra of lipids enabled us to capture lipidomic diversity and specificity in the environment, identify novel lipids, differentiate microbial sources within a lipid group, and discover potential biomarkers for biogeochemical processes.
Alejandro Abdala Asbun (technician WP2 and WP3) was hired as a bioinformatics research assistant to provide support in bioinformatic approaches. He has contributed to the establishment of bioinformatic pipelines for the analysis of 16S rRNA gene amplicon sequencing, metagenomic analyses and RNAseq analyses, all of them used by the postdocs and PhD students hired in the MICROLIPIDS project. Alexander Westbye (postdoc WP 3; 2017 - 2018). He studied the activity of two ether-lipid biosynthesis genes homologous to those found in the archaeal lipid biosynthetic pathway encoded in a Cloacimonetes bacterium detected in the Black Sea water column. These results supported the synthesis of ‘archaeal/bacterial’ mixed membranes in bacterial groups of the FCB superphylum, missing link in the evolution of lipid membrane acquisition in all domains of life.
The work of Diana Sahonero-Canavesi (postdoc WP3) has focused on the study the hyperthermophilic bacterium Thermotoga maritima. She showed the major physiological and metabolic adaptations to its growth phase and temperature including changes on known lipid pathways. This work has expanded the knowledge on the cellular adaptation to extreme conditions and shed light on the role and plasticity of Thermatoga's membrane-spanning lipids. She also studied the model microorganism Thermoanaerobacter ethanolicus, which synthesizes iso-diabolic acid-based membrane spanning lipids. The results of these studies are key to understand how membrane-spanning lipids are synthesized and by which bacteria.
Ruth Perez Gallego (PhD student WP3) has been working on the identification of genes responsible for the biosynthesis of long chain alkenones and heterocyte glycolipids. Using genomic and transcriptomic techniques on Emiliania huxleyi cultures to determine the genes involved in the synthesis of long chain alkenones, a set of compounds widely used in biogeochemistry to reconstruct past climate environmental factors. Determining the genes involved in this pathway has important consequences in order to understand the biological mechanisms behind the production of these compounds, especially in environments where the proxy is known to be inaccurate, such as in lakes.
Bastiaan von Meijenfeldt (postdoc WP3) performed genomic analysis related to the determination of lipid biosynthetic pathways and assist other members of the group on bioinformatic approaches. Lora Strack van Schijndel (technician WP3) supported the members of the group on molecular and cultivation methods.
Alejandro Abdala Asbun (technician WP2 and WP3) was hired as a bioinformatics research assistant to provide support in bioinformatic approaches. He has contributed to the establishment of bioinformatic pipelines for the analysis of 16S rRNA gene amplicon sequencing, metagenomic analyses and RNAseq analyses, all of them used by the postdocs and PhD students hired in the MICROLIPIDS project. Alexander Westbye (postdoc WP 3; 2017 - 2018). He studied the activity of two ether-lipid biosynthesis genes homologous to those found in the archaeal lipid biosynthetic pathway encoded in a Cloacimonetes bacterium detected in the Black Sea water column. These results supported the synthesis of ‘archaeal/bacterial’ mixed membranes in bacterial groups of the FCB superphylum, missing link in the evolution of lipid membrane acquisition in all domains of life.
The work of Diana Sahonero-Canavesi (postdoc WP3) has focused on the study the hyperthermophilic bacterium Thermotoga maritima. She showed the major physiological and metabolic adaptations to its growth phase and temperature including changes on known lipid pathways. This work has expanded the knowledge on the cellular adaptation to extreme conditions and shed light on the role and plasticity of Thermatoga's membrane-spanning lipids. She also studied the model microorganism Thermoanaerobacter ethanolicus, which synthesizes iso-diabolic acid-based membrane spanning lipids. The results of these studies are key to understand how membrane-spanning lipids are synthesized and by which bacteria.
Ruth Perez Gallego (PhD student WP3) has been working on the identification of genes responsible for the biosynthesis of long chain alkenones and heterocyte glycolipids. Using genomic and transcriptomic techniques on Emiliania huxleyi cultures to determine the genes involved in the synthesis of long chain alkenones, a set of compounds widely used in biogeochemistry to reconstruct past climate environmental factors. Determining the genes involved in this pathway has important consequences in order to understand the biological mechanisms behind the production of these compounds, especially in environments where the proxy is known to be inaccurate, such as in lakes.
Bastiaan von Meijenfeldt (postdoc WP3) performed genomic analysis related to the determination of lipid biosynthetic pathways and assist other members of the group on bioinformatic approaches. Lora Strack van Schijndel (technician WP3) supported the members of the group on molecular and cultivation methods.
The application of information theory framework combined with molecular networking based on the similarity of the mass spectra of lipids enabled us to capture lipidomic diversity and specificity in the environment, identify novel lipids, differentiate microbial sources within a lipid group, and discover potential biomarkers for biogeochemical processes (Ding et al., 2021).
The detection of candidate enzymes involved in the synthesis of specific steps of key membrane lipids to understand the evolution of microbial lipid membranes. This information will aid in the identification of specific lipid biomarker producers in the tree of life, with implications for a better application of these molecules for paleoclimate interpretations, and also to determine the physiological advantage of harboring these lipids.
The detection of candidate enzymes involved in the synthesis of specific steps of key membrane lipids to understand the evolution of microbial lipid membranes. This information will aid in the identification of specific lipid biomarker producers in the tree of life, with implications for a better application of these molecules for paleoclimate interpretations, and also to determine the physiological advantage of harboring these lipids.