Tricorder Tech: Genetic Methods Enable The Use Of Fossil Lipids As Biomarkers For Oxygen-producing Primordial Bacteria

Cyanobacteria are a key species in Earth’s history because they first introduced oxygen from the atmosphere. The analysis of their evolution therefore provides important insights into the formation of modern aerobic ecosystems.

For a long time, a certain type of fossil lipid, called 2-methylhopanes, was considered an important biomarker for cyanobacteria in sediments, some of which are hundreds of millions of years old. However, this came into question when it turned out that not only Cyanobacteria but also Alphaproteobacteria are genetically capable of producing these lipids.

An international research team led by Yosuke Hoshino from the GFZ German Research Center for Geosciences and Benjamin Nettersheim from MARUM – Center for Marine Environmental Sciences at the University of Bremen has now studied the phylogenetic diversification and distribution of the genes – including HpnP – responsible for the synthesis of the parent lipids for 2-methylhopanes: The researchers have deciphered when these genes were acquired by certain groups of organisms. They were able to show that HpnP was probably already present in the last common ancestor of Cyanobacteria more than two billion years ago, while the gene only appeared in Alphaproteobacteria about 750 million years ago. For the times before, 2-methylhopanes can therefore serve as a clear biomarker for oxygen-producing Cyanobacteria.

The study, now published in the journal Nature Ecology & Evolution, shows how genetics, in interaction with sedimentology, paleobiology and geochemistry, can improve the diagnostic value of biomarkers and refine the reconstruction of early ecosystems.

Landscape in the Australian Northern Territories during a field trip to the 1.6 billion year old Barney Creek Formation in the McArthur Basin to collect the oldest 2-methylhopane samples for this study. CREDIT Photo: Christian Hallmann, GFZ

Background: The importance of cyanobacteria in Earth’s history

Cyanobacteria played a crucial role in transforming Earth from its initial oxygen-free state to a modern, oxygen-rich system that supports increasingly complex life. Cyanobacteria were probably the only relevant group of organisms that converted inorganic substances into organic substances (so-called primary producers) and produced oxygen during long parts of the Precambrian (the first four billion years of Earth’s history, from the beginning until about 540 years) . million years ago). Therefore, the analysis of their evolution is of great importance for understanding the common history of life and the Earth.

The importance of fossil lipids as biomarkers

In principle, the fossil remains of entire Cyanobacteria can serve as an indicator for the presence of oxygen-rich photosynthesis in the geological past. However, due to conservation biases and ambiguities in recognizing fossil cyanobacterial cells, geochemists prefer to use fossilized diagnostic lipids, such as 2-methylhopanes. 2-Methylhopanoids (non-fossilized parent molecules) are produced by the bacteria and – unlike the bacteria themselves – can be fossilized and detected in sedimentary rocks even after hundreds of millions of years in good quality and in quantities corresponding to their original occurrence.

However, recently there have been doubts about the suitability of 2-methylhopane as a biomarker for cyanobacteria: the discovery of the lipid biosynthesis gene revealed that Alphaproteobacteria are also capable of producing these lipids. This means that temporally tracing oxygen-producing processes on Earth through 2-methylhopanes is no longer possible.

a, Dated phylogeny of Alphaproteobacteria and the emergence of HpnP in the phylum. Both the tree topology and node data were adapted from a recent phylogenomic study (Supplementary Figure 10). Numbers in the tree indicate major evolutionary events for the two phyla Cyanobacteria and Alphaproteobacteria: (1) divergence of cyanobacteria from the crown group; (2) divergence of Alphaproteobacteria from Beta- and Gammaproteobacteria; (3) divergence of Alphaproteobacteria from the crown group; (4) divergence of Hyphombiotices; (5) divergence of HpnP-containing Hyphombiotices families – Beijerinckiaceae (node ​​a), Methylobacteriaceae (node ​​b) and Nitrobacteraceae (node ​​c). Nodes a, b and c indicate the emergence of three HpnP-containing families in Hyphombiotices. Node bars indicate the 95% highest posterior density interval of posterior data. Supplementary Figure 10 provides the dated phylogeny with the species annotation. b, 2-methylhopane index (2-MHI, %C31 2-methylhopane / (C31 2-methylhopane + C30 αβ-hopane)) throughout Earth’s history. The data contains newly analyzed values ​​from this study (open circles) and from the literature (open squares). Each circle and square represents the average of all 2-MHI values ​​from individual geological formations or published studies (Supplementary Table 7). The underlying light green bar graph shows averages binned for geological time units (Supplementary Table 7). Blue-green and light-blue arrows indicate two geological periods when primary marine productivity was dominated by cyanobacteria and algae, respectively. Abbreviations: GOE, Great Oxidation Event; Ma, a million years ago. Figure adapted from ref. 32 under a Creative Commons license CC BY 4.0.

New approach: extensive genetic analysis combined with new, very pure sediment analyses

An international research team led by Yosuke Hoshino and Christian Hallmann, scientists in GFZ Section 3.2 “Organic Geochemistry”, and Benjamin Nettersheim from MARUM at the University of Bremen has now systematically investigated which organisms other than Cyanobacteria possess the genes (abbreviated as the SC and HpnP genes) required for the production of 2-methylhopanoids, and when they acquired those genes during evolution. In this way, the team was able to demonstrate that the fossil lipid 2-methylhopane can still be used as a clear biomarker for the existence of cyanobacteria as far back as 750 million years ago.

Furthermore, the researchers have created an integrated view of 2-methylhopane production throughout Earth’s history. To do this, they combined their molecular data with new sediment analyzes carried out under very pure conditions.

“The method we proposed is in principle applicable to all organic matter in geological archives and has great potential to monitor the evolution of different ecosystems at a much higher temporal and spatial resolution than before,” Hoshino summarizes.

The tree was generated using 135 representative sequences and 534 conserved sites. Filled black circles indicate that node support is >85% for both maximum likelihood inference and Bayesian inference (node ​​support shown only for large clades). Filled gray circles indicate that the node support is above the same threshold for either of the two inferences. The presence/absence of the SC gene is indicated next to the tree. The insert shows major clades of HpnP homologs (HpnP and HpnP-like proteins) that retain three conserved domains; B-12 binding domain, radical SAM domain and DUF4070 domain. The scale bar represents 0.4 amino acid substitutions per site per unit of evolutionary time. Additional figures. 1−3 provide the complete trees with the species annotation.

Methodology I: Computational research for genetic analysis

To analyze the genetic relationships, Hoshino searched publicly available databases, which contain millions of gene and protein sequences, for organisms with the SC and HpnP genes. Based on this genetic data set, he created so-called phylogenetic trees, which provide information about how the SC and HpnP genes were transferred between different organisms and whether the gene transfer occurred vertically through inheritance or horizontally between evolutionarily unrelated organisms. In addition, the researchers were also able to determine when individual gene transfers occurred in the evolutionary history of the genes by comparing previous studies that used the so-called molecular clock technique that takes into account the DNA mutation rate and estimates the timeline for the gene. evolution.

Methodology II: New type of ultraclean sample preparation

In addition, because Precambrian biomarker records are extremely sensitive to contamination, the researchers used an ultraclean method to extract organic material from sediment cores. The geological samples in the form of cores were collected by several co-authors from 16 countries. They represent different geological periods from the Paleoproterozoic (2.5 billion years ago) to the present. The relative abundance of 2-methylhopanes in the organic matter was then measured.

The results in detail

There are many bacteria that possess both SC and HpnP genes, but they are mainly cyanobacteria and alphaproteobacteria. Each group appears to have acquired the two genes independently. This contrasts with previous studies that concluded that Cyanobacteria acquired these genes from Alphaproteobacteria at a late stage in their evolution. The new research also found that the common ancestor of Cyanobacteria already possessed both genes more than 2.4 billion years ago, when oxygen began to accumulate in the atmosphere during the so-called Great Oxidation Event.

In contrast, Alphaproteobacteria acquired the SC and HpnP genes only 750 million years ago at the earliest. Previously, 2-methylhopanoids were only produced by cyanobacteria. The researchers interpret a somewhat delayed increase in sedimentary 2-methylhopanes about 600 million years ago as a sign of the global spread of Alphaproteobacteria, which may have promoted the simultaneous evolutionary emergence of eukaryotic algae.

Summary and prospects

“The individual analytical methods mentioned above are not new, but few researchers have previously attempted to perform comprehensive analyzes for SC and HpnP and integrate genetic data with sedimentary biomarker data, as this requires the combination of two completely different scientific disciplines – molecular biology and organic geochemistry. ” says Hoshino.

“The source of sedimentary 2-methylhopanes has been a subject of long debate,” adds Christian Hallmann. – “This new study not only provides clarity on the diagnosticity of 2-methylhopanes and the role of cyanobacteria in deep time; its methodology provides a new way forward to refine the diagnosticity of, in theory, any biomarker lipid once the biosynthetic genes are known.”

Genetics restores the usefulness of 2-methylhopanes as cyanobacterial biomarkers before 750 million years ago, Nature Ecology & Evolution (open access)

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