Fattening Up Microbial Geological Biomarkers

by Paula Welander
 

Figure 1A. The biomarker principle in­volves the burial and preservation of bacterial lipids such as hopanoids in the sedimentary rock over billions of years. Dia­genesis describes the chemical, physi­cal, or biological changes undergone by sediment after its initial de­position. Bac­teria present in the water column die and are deposited and buried in the sediment where they undergo degradation. Hopan­oid molecules lose the majority of their functional groups but the basic hopane structure is preserved over billions of years. Geo­chemists are then able to extract and detect these lipids in ancient sediments. Credit: Paula Welander

First Evolved! Last Extinct! This prokaryotic pride motto was coined by my undergraduate advisor (and good friend) Prof. Mark Martin. As a microbiologist, I love this motto for many reasons, but especially because it alludes to one of the underlying principles of my current re­search. Microbes were indeed the first to evolve and the metabolic inventions of ancient microbes greatly in­fluenced the ancient Earth’s environment and the evo­lution of life. The interaction between the Earth and microbes has been recorded in sedimentary rocks that are billions of years old.
 

Accessing this microbial record continues to be one of the challenges of geomicrobiology. One powerful stra­tegy for understanding the microbial signatures in the rock record is the use of “molecular fossils” or biomark­ers, organic compounds that are produced by select groups of microorganisms and, amazingly, are preserved in both modern and ancient sediments. Geochemists are able to extract these molecules from very old rocks and, based on their distribution in modern organisms, link specific groups of bacteria to ancient environments (Fig. 1A+B).
 

As Tanja Bosak described in her earlier contribution to this blog, both eukaryotic and bacterial lipids are preserved in old rocks, thus both have the potential to be “molecular fossils.” One ex­ample of such is the use of 2-methylhopanoids as biomarkers for cyanobacteria and oxygenic photosynthesis. Hopanoids are pentacyclic triterpenoid lipids produced mainly by bacteria and that are structurally similar to eukaryotic sterols (Fig. 2).
 

Figure 1B. (Legend see Fig. 1A)

As the name implies, 2-methylhopanoids are methylated at the C-2 position. Although seemingly a minor modifi­cation, this chemical alteration endures in ancient rocks. Studies with modern bacteria showed that cyanobacteria are the predominant producers of 2-methylhopanoids. On this basis, 2-methylhopanoids have been used as biomarkers not only for cyanobacteria but also for oxy­genic photosynthesis . The presence of these molecules in rocks provided evidence for the antiquity of oxygenic photosynthesis and for the role of cyanobacteria in the ancient past (click here, here, and here).
 

The link between cyanobacteria and 2-methylhopanoids was brought into question a few years ago when it was discovered that the anoxygenic phototroph Rhodopseudomonas palustris was capable of producing methylated hopanoids under anaerobic conditions and in quantities similar to those made by the cyanobacteria. This finding raised doubts about the previous interpretation of the origin of 2-methylhopanoids in the rock record. It is here where we, as molecular microbio­logists and microbial physiologists, could make a contribution to the biomarker field by “fattening up” the slim amount of phylogenetic and physiological data available for linking hopanoids to specific bacteria and their metabolism.
 

Figure 2. Structure of a 2-methylhopan­oid. Credit: Paula Welander

Currently, our understanding of the taxonomic classi­fi­cation of 2-methylhopanoid producers is based on lipid analysis of cultured bacterial strains. We reasoned that a more constructive approach would be to identify a genet­ic factor required for the production of 2-methylhopan­oids that could be used to survey the vast amount of available genomic and metagenomic data. Utilizing com­parative genomics and genetic deletion analysis, we un­covered the vitamin B-12 binding radical SAM protein (HpnP) in R. palustris responsible for the methylation at the C-2 position. Only 30 bacterial strains (out of approximately 200 hopanoid producers tested) have a copy of the HpnP methylase (Fig. 3). These fall into three phylogenetic groups – the cyanobacteria, the Rhizobiales (an order of the α-proteobacteria that contains R. palustris), and the acidobacteria. Notably, not all cyanobacteria contain HpnP. We found no common thread among the bacteria with the methylase (e.g., they aren’t all phototrophs nor are all found in one type of ecosystem). Clearly just under­standing the phylogenetic distribution of 2-methylhopanoid producers is not sufficient to properly “read” these biomarkers in the rock record. Rather, it would be more useful to identify the cellular function as well as the environmental factors that induce the expression of these compounds.
 

(click to enlarge)
Figure 3. Unrooted tree showing the major clusters of HpnP from different bacterial groups. Source

What to do? We turned once again to the anoxygenic phototroph R. palustris that produces up to six different modified hopanoids. We started out by characterizing a mutant unable to produce hopanoids methylated at the C-2 position, but we found no associated phenotype. It was clear that first we needed to step back and under­stand the function of hopanoids in general. We con­structed an R. palustris mutant that no longer produced any hopanoids by deleting squalene hopene cyclase, an enzyme required for the initial cyclization of squalene to form the basic hopanoid structure. This mutant has simi­lar growth characteristics to the wild type strain under all standard growth conditions including photosynthetic growth, underscoring that hopanoids do not play a role in photosynthesis – and highlighting again that hopanoids are not good biomarkers for any type of photosynthesis! However, the hopanoid deletion mutant showed a growth defect under environmental stress conditions such as high pH or high temperature. Its outer membrane became more permeable, suggesting that enhanced permeability lowers resistance to environmental stress. Interestingly, the mutant deficient in only 2-methylhopanoid synthesis grows normally under stress and its outer mem­brane is as impermeable as the wild type. This means that the methylation at the C-2 position has a unique and distinct purpose, separate from maintaining outer membrane integrity. Seeking some clues as to the function of these specific molecules, we have started work on the biochem­istry of the methylase protein and are investigating the localization of the 2-methylhopan­oids, their possible regulators, and the environmental factors that might stimulate their produc­tion.
 

Although we have made some progress in understanding the function of hopanoid molecules in bacterial cells, we feel we have just begun to scratch the surface. We hope that further investiga­tions into these potentially interesting lipids will reveal novel functions that will not only inform the proper interpretation of hopanoids found in old rocks but will also spark the interest of microbio­logists and cell biologists. Hopanoids have been buried for billions of years and it is time for their mysteries to be unearthed.
 

Reference

Welander PV, Coleman ML, Sessions AL, Summons RE, & Newman DK (2010). Identification of a methylase required for 2-methylhopanoid production and implications for the interpretation of sedimentary hopanes. Proc Natl Acad Sci U.S.A, 107 (19), 8537−42. PMID 20421508

 

Paula is a postdoctoral fellow co-advised by Prof. Roger Summons in the Depart­ment of Earth, Atmospheric and Planetary Sciences at MIT, and Prof. Dianne Newman in the Department of Biology at Caltech.

 
 

 
 

Comments

[Mark O. Martin]

Thank you for the kind words, Paula. I knew you when! What a fantastic article, and I am going to push to my students, to demonstrate that they can become fine scientists---just like you!