Using the present to inform the past: interpreting potential biomarkers of (an)oxygenic photosynthesis

Time has changed the Earth’s geochemistry substantially, in large part due to bacterial metabolic “inventions”. A classic example is the evolution of the manganese-cofactor of photosystem II, which enabled cells to produce molecular oxygen (O2) from water and thereby oxidize our planet. Prior to this invention, however, microbial life subsisted anaerobically for millions and perhaps billions of years. How did cells cope? What electron acceptors and electron donors did they use for growth? Can we understand how cells transitioned from using these substrates into producing/using O2 for metabolism?

While theories regarding the earliest forms of metabolism abound, if we restrict our focus to the evolution of photosynthesis, it is generally accepted that low-potential electron donors such as hydrogen, sulfide and Fe(II) were the most important fuels for ancient primary production. Of these, Fe(II) may well have been the most abundant.  Because Fe(II) has an intermediate redox potential relative to other electron donors used in photosynthesis, it has been suggested that Fe(II)-phototrophy may represent a transitional form of photosynthetic metabolism. Just as water is oxidized to O2 by plants, anoxygenic phototrophs oxidize Fe(II) to ferric iron [Fe(III)] minerals. Although the evolutionary importance of Fe(II)-phototrophy was first postulated in 1984, it was not until 1993 that bacteria capable of using Fe(II) in photosynthesis were isolated. When our group started working with these bacteria, nothing was known about how they catalyzed Fe(II) oxidation, nor whether their activities might leave a specific fingerprint in the rock record.  A tantalizing hypothesis we sought to test was that an enigmatic class of ancient iron ore deposits, known as “Banded Iron Formations” (BIFs), could have accreted from the Fe(III) minerals produced by anoxygenic phototrophs.  Conventionally, it has been thought that BIFs formed due to Fe(II) oxidation to Fe(III) by O2, the byproduct of oxygenic photosynthesis. Thus BIFs likely record a complex story of the evolution of photosynthesis; the challenge is how to read them.

To address this problem, we began by culturing diverse Fe(II)-oxidizing phototrophs and measuring their rates of Fe(II) oxidation.   By making conservative assumptions about how these rates might apply to an ancient environment, we made a quantitative case that Fe(II)-oxidizing phototrophs could have accounted for BIF deposition without needing to invoke the presence of O2.  If two completely different pathways can result in the same output (e.g. Fe(III) minerals), how can we tell them apart?  One way might be to look for differences in the Fe isotopic composition of the minerals themselves. By measuring the 56Fe/54Fe ratios of the Fe(III) products relative to aqueous Fe[II] for a variety of Fe(II)-oxidizing bacteria, we determined that the Fe(III) products are consistently enriched in the heavier isotope and similar to those observed in nature. This fractionation is independent of the rate of Fe(II)-oxidation, and comparable in magnitude to that observed for Fe-isotope fractionation by Fe(III)-reducing bacteria, suggesting that cell-associated ligands common to both types of organisms might control the fractionation through an equilibrium process.  This motivated us to investigate the path of Fe in the cell, as not only would this better enable us to interpret our results, but it was interesting in its own right. Unlike the majority of organisms for which Fe acquisition is a challenge, anoxygenic phototrophs must solve the reverse problem—how to avoid toxicity in the presence of abundant Fe. To gain insight into this, we took two genetic approaches, resulting in the identification of different operons in two anoxygenic phototrophs that catalyze Fe(II) oxidation, both of which contain novel c-type cytochromes. One of these operons also encodes an intriguing b-barrel protein that is probably involved in Fe transport, but appears distinct from known Fe-transport proteins. Through detailed structural/functional studies of these cytochromes and transporters, we hope to uncover novel mechanisms of Fe uptake and homeostasis.

Given that anoxygenic phototrophs could have been responsible for producing sedimentary features that are conventionally attributed to oxygenic phototrophs, we are left with the challenge of identifying a biomarker that could distinguish one metabolic type from the other. Towards this end, we have recently begun to study a class of organic molecules that are preserved in ancient rocks called 2-methylhopanes. Due to their unique carbon skeleton, these molecules can unambiguously be recognized as the molecular fossils of 2-methylbacterialhopanoids (2-MeBHPs), functionalized isoprenoids found in select modern bacteria. Cyanobacteria – the only bacteria that engage in oxygenic photosynthesis – have been considered the only quantitatively important source of 2-MeBHPs in the modern environment. Accordingly, the finding of 2-methylhopanes in 2.7 billion year-old sediments has been taken as evidence that photosynthetically-derived O2 first appeared on Earth at least that long ago. But does this make sense? A number of independent proxies indicate that a major global redox transition did not occur until roughly 400 million years later. If cyanobacteria were engaging in oxygenic photosynthesis at 2.7 Ga, why did it take so long to alter the surface redox state of the Earth? There may well be a good explanation for this lag, but if we are incorrect in the assumption that 2-methylhopanes are biomarkers for oxygenic photosynthesis, then this paradox may be artificial.

A key question, therefore, is whether 2-MeBHPs and oxygenic photosynthesis are functionally related. Surprisingly, given the importance of this assumption, no such evidence exists. To test this model, we have begun to study 2-MeBHP production by genetically-tractable cyanobacteria with the aims of identifying the conditions that elicit their production, determining where in the cell they reside, elucidating how they are made, and probing the phenotypes of mutant cells that no longer make them. In Rhodopseudomonas palustris TIE-1, one of our “negative” control Fe(II)-oxidizing phototrophs (previous surveys of related strains had failed to detect 2-MeBHPs), we were surprised and excited to discover that it could produce 2-MeBHPs under strictly anaerobic conditions in amounts equivalent to those made by cyanobacteria. Not only has this caused us to seriously question the use of 2-MeBHPs as biomarkers for oxygenic photosynthesis, it has motivated us further to understand their biological function.

Although nothing is known about the specific function of 2-MeBHPs, something is known about the general functions of hopanoids. Like eukaryotic sterols, hopanoids are thought to influence membrane fluidity and permeability. Unlike sterols, however, hopanoid biosynthesis does not require molecular oxygen. Were 2-MeBHPs “invented” in an anaerobic world to serve a purpose related to membrane properties and then later co-opted by cyanobacteria with similar cell biological needs? Intriguingly, R. palustris and several cyanobacteria that produce 2-MeBHPs have lamellar membranes. It is now well established that structural modifications of sterols, including methylation of the polycyclic domain, can have a dramatic impact on their biological function in higher organisms as well as influence membrane curvature. Recently, it has become apparent that sterols are capable of organizing heterogeneous microdomains within lipid bilayers. These microdomains, or lipid rafts, tend to sort proteins into clusters of functional significance. Specific structurally-mediated lipid-lipid and lipid-protein interactions may be critical in determining the composition and subcellular localization of these rafts. While the existence of lipid rafts is not yet well documented in bacteria it seems possible that methylation of C-2 on BHPs might be involved in the localization and activation of transmembrane proteins with a specific function. Regardless of whether 2-MeBHPs are functionally related to oxygenic photosynthesis, understanding their role in modern organisms will greatly improve our interpretation of what their fossilized ancestors mean. Perhaps 2-MeBHPs will be a marker for the evolution of a particular type of membrane fold, rather than a particular type of metabolism. In either case, the answer is equally interesting. 

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