Research Summary
Dianne Newman's research focuses on understanding the coevolution of microbial metabolism and the geochemistry of the Earth.
Electron-transfer reactions are fundamental to metabolism. Whether an organism is autotrophic or heterotrophic, free living or an obligate parasite, every cell must solve the energy-generation problem to survive. At the cellular level, most of our knowledge of electron transfer comes from mechanistic studies of oxygenic photosynthesis and aerobic respiration in prokaryotic and eukaryotic systems. While we know in exquisite detail the structure and function of various membrane-bound proteins involved in electron-transfer processes (e.g., cytochrome c oxidase in mitochondria), we know far less about the electron-transfer agents of more ancient forms of metabolism.
As a geobiologist interested in the origin and evolution of the biochemical functions that sustain modern life, my work has focused on probing the coevolution of metabolism with Earth's near-surface environments. Understanding how modern microorganisms with archaic metabolisms function is a step toward this end. Moreover, because many biological microenvironments are anaerobic, including those in most bacterial infections, this path of inquiry leads inexorably to insights about cellular electron-transfer mechanisms that potentially have profound biomedical implications. To illustrate this, I will describe two examples of problems that my group is pursuing, their broader implications, and how we propose to explore them further.
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 or using O2 to sustain their metabolism?
2-Methylhopanes are a class of organic molecules that are preserved in ancient rocks. 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 (Figure 1). 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 sediments that are 2.7 billion years old 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 recently 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. InRhodopseudomonas palustris TIE-1, one of our "negative" anoxygenic controls (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 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 (Figure 1). 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.
Although 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 interpretations 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.
Using the Past to Inform the Present: Reconsidering the Function of Redox-Active "Secondary" Metabolites
While the rock record provides an incentive to study the function of certain biomolecules by demonstrating their evolutionary importance, it also affects our thinking about biological processes in other ways. For example, many bacteria live together in biofilms, communities of cells attached to surfaces. Despite their ubiquity—from the lungs of cystic fibrosis (CF) patients, to medical implants, to the surfaces of rocks in sediments—we know very little about the rules of metabolism that sustain life in these environments. Indeed, if we penetrate only a few microns below the surfaces of most biofilms, we encounter an anaerobic world, similar in some important respects to conditions on Earth billions of years ago. Bacteria living in these environments face the challenge of sustaining their metabolism under conditions where oxidants for cellular-reducing power are limited. Because the effectiveness of antibiotic treatment depends significantly on the physiological state of biofilm cells, it is important to understand how these cells sustain their metabolism. Can we gain insights into how biofilm communities survive today by considering the evolutionary origins of their metabolism?
Our entry into this problem came from considering how bacteria respire Fe(III) minerals, probably the most abundant and important terminal electron acceptors for ancient cellular respiration. Working first with the metabolically versatile bacterium Shewanella oneidensis, we demonstrated that it excretes small organic molecules that mediate electron transfer from the cell to mineral surfaces. Our results suggested that self-produced electron shuttles might be an important mechanism for mineral transformation by many different types of bacteria. By looking at their chemical structures, we inferred that certain redox-active antibiotics (e.g., phenazines and some glycopeptides) produced by common soil bacteria (e.g., Pseudomonas chlororaphis and Streptomyces coelicolor) and clinical isolates (e.g., Pseudomonas aeruginosa, an opportunistic pathogen) could function as extracellular electron shuttles. We went on to show that this is indeed the case, and that they could be exchanged between diverse bacterial species.
Due to the rich history of Pseudomonas research, we decided to focus on the phenazine molecules it produces (Figure 2). Most current literature emphasizes the role of phenazines as virulence factors, which generate toxic byproducts (e.g., O2– and H2O2) when oxidized in an aerobic environment. For this reason, phenazines are conventionally thought to be toxic to other organisms and are believed to provide the producer with a competitive advantage. However, because phenazines can be synthesized under anaerobic conditions and are often produced at concentrations below their toxic threshold, we hypothesized that their "antibiotic" activity might be a consequence of the geochemical conditions prevalent on Earth today, but not a reflection of their original function.
In recent years, we have tested this hypothesis in several ways using P. aeruginosa strain PA14. We have shown that: (1) phenazines function effectively as electron shuttles to Fe(III) minerals, which may aid in Fe(II) acquisition; (2) phenazines help modulate intracellular redox homeostasis as oxidants for NADH and/or by affecting carbon flux through central metabolic pathways; (3) phenazines are signaling molecules, influencing the expression of a limited set of genes during the transition from exponential growth into stationary phase; (4) phenazines are produced in biofilms, as expected—given that phenazine biosynthesis had previously been shown to be up-regulated by quorum sensing and low oxygen tension; and (5) phenazines play an important role in biofilm development, dramatically affecting the morphology of multicellular communities (Figure 3). We are beginning to work out the molecular pathways that underpin these phenomena by identifying and characterizing the proteins that respond to phenazines as well as those required for phenazine trafficking within and between cells.
We have also begun to develop specific analytical tools to localize and quantify phenazine distribution in multicellular communities and at the single-cell level. Ultimately, we seek to understand the trafficking of the various phenazines that P. aeruginosa produces, as we have preliminary evidence that they have different functions. Regardless of which phenazine does what, our results demonstrate that phenazines are much more than "antibiotics," profoundly affecting the producing organism metabolically and developmentally. In collaboration with colleagues at Harvard Medical School and Children's Hospital Boston, we hope to determine whether phenazine production and cycling is important for the survival of P. aeruginosa in the CF lung, where P. aeruginosa is thought to exist in a biofilm-like state.
As of February 26, 2010
