Time has changed the Earth's geochemistry substantially, in large part through 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. The advent of oxygenic photosynthesis and the subsequent accumulation of O2 in the atmosphere forever changed biogeochemical cycling on Earth. While my group has contributed to understanding diverse respiratory and photosynthetic processes involving metal(loids), in recent years we have focused our attention on two questions: (1) Can we utilize certain biomarkers in ancient rocks to trace when cells began producing or utilizing O2? (2) What strategies did cells evolve to survive in the absence of readily accessible O2 or other inorganic oxidants to fuel respiration?
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. Guiding our approach has been the assumption that studying how modern microorganisms catalyze reactions of geochemical interest is vital to understanding the history of life. Moreover, because many biological microenvironments are hypoxic or anoxic, including those in chronic 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 problems my group has been pursuing, and the new directions in which they are taking us.
Using the Present to Inform the Past: Interpreting Molecular Fossils in Ancient Rocks
Steranes and hopanes are organic compounds found in ancient rocks that have been used to date the rise of oxygenic photosynthesis. Because of their unique carbon skeletons, these molecules can unambiguously be recognized as molecular fossils of steroids and hopanoids (steroid analogs in bacteria), important constituents of cell membranes (Figure 1). While key steps in the biosynthesis of steroids require O2, hopanoid biosynthesis does not. Modern steroids and hopanoids are structurally diverse, yet only their carbon skeletons are preserved after diagenesis. Remarkably, the total amount of hopanes trapped within ancient rocks is thought to be roughly equivalent to the amount of organic carbon present on Earth today. One of the most important geostable hopanoid modifications is methylation at C-2, and molecular fossils of this type are called 2-methylhopanes (deriving from 2-methylbacteriohopanepolyols, 2-MeBHPs, in modern cells). Cyanobacteria—bacteria that engage in oxygenic photosynthesis—used to be considered the only quantitatively important source of 2-MeBHPs; accordingly, the occurrence of 2-methylhopanes in sediments that are 2.7 billion years old was taken as evidence that photosynthetically derived O2 first appeared on Earth at least that long ago. But because several independent geochemical proxies indicate that a major global redox transition did not occur until several hundred million years later, we decided, in collaboration with organic geochemists, to examine key assumptions underpinning the use of hopanes and steranes as O2 biomarkers.
When we began, although a considerable amount was known about steroid cell biology, what the O2 threshold necessary for steroid biosynthesis is—and the impact this value has on models of atmospheric oxygenation—was unclear. By carefully controlling the O2 available to our cultures, we found that steroid biosynthesis can occur with dissolved O2 concentrations in the nanomolar range. This low requirement helps explain the temporal decoupling between the sterane biomarker record of O2 utilization and the dating of a global redox transition: models of atmospheric oxygenation are consistent with the hypothesis that O2 could have cycled as a trace gas in the marine environment for millions of years prior to its atmospheric accumulation. Key to this discovery was our investment in the ability to culture diverse bacteria in hypoxic and anoxic environments where O2 could be precisely measured. This ability also enabled the isolation of Rhodopseudomonas palustris TIE-1, an anoxygenic phototroph that we serendipitously discovered could produce 2-MeBHPs in as great abundance as cyanobacteria under certain conditions.
Because R. palustris grows quickly and is metabolically versatile, we developed it into a model system in which to study hopanoid cell biology. We elucidated the biosynthetic pathway for diverse hopanoids, the transporter responsible for localizing hopanoids to the outer membrane, and the mechanism and conditions responsible for regulating 2-MeBHP biosynthesis. Our discovery that the C-2 hopanoid methylase (HpnP) is well conserved among all 2-MeBHP–producing bacteria allowed us to circumvent the problem of conditional 2-MeBHP production by using the hpnP gene to identify 2-MeBHP production capacity in other microbial genomes and metagenomes. This survey not only revealed that only a minority of cyanobacteria make 2-MeBHPs but also revealed that a statistically significant correlation exists in modern environments between 2-MeBHP production capacity and an ecological niche defined by low O2, high osmolytes, and sessile microbial communities. In modern environments, this tracks with microenvironments found in microbial mats, stromatolites, and the rhizosphere; relevant to the latter, the occurrence of hpnP is significantly enriched in the genomes of well-characterized plant symbionts.
Motivated by this new correlation, we have expanded our model system set to include Nostoc punctiforme and Bradyrhizobium japonicum, genetically tractable 2-MeBHP–producing bacteria with well-characterized plant partners. In parallel with our work in R. palustris, we are exploring the regulation of hopanoid production by these strains and how hopanoid production affects diverse phenotypes. This has required us to develop novel methods to detect and quantify hopanoids both in single cells and from lipid mixtures extracted from bulk cultures. Using these methods, we are systematically characterizing the membrane composition of diverse hopanoid-producing wild-type and mutant strains grown in vitro and in planta. These results are informing biophysical studies to test the effects of hopanoids on membrane fluidity, permeability, and curvature. Finally, in collaboration with chemical biologists, we are building a molecular toolkit to identify proteins and other biomolecules that interact with hopanoids.
It is now clear that while the O2 requirement for sterane biosynthesis is compatible with other proxies for dating the rise of O2, 2-methylhopanes cannot be used as biomarkers of O2 photosynthesis. Our new goal is to provide a better interpretation of sedimentary hopanes by gaining a deeper understanding of their modern counterparts. Do hopanoids facilitate plant-microbe symbioses in specific ways? With which other membrane components do they interact? What explains their phylogenetic distribution? Unlike steroids in eukaryotes, hopanoid production by bacteria is only essential under certain conditions, offering the possibility of using bacterial systems to explore fundamental questions of membrane homeostasis that are not as readily addressed in eukaryotes.
Using the Past to Inform the Present: Reconsidering the Function of Redox-Active "Secondary" Metabolites
While ancient rocks have motivated us to study the cell biology of hopanoids, they have also shaped our thinking about other small molecules and biological processes. 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 habitats. Indeed, if we penetrate only a few microns below the surfaces of most biofilms, we encounter hypoxic and anoxic worlds. 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 better understanding anaerobic modes of energy generation?
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 commonly acquired in hospitals) can function as extracellular electron shuttles. We went on to show that this is indeed the case, and that they can be exchanged between diverse bacterial species.
Because of the rich history of Pseudomonas research, and the fact that it offered a well-defined and experimentally tractable system in which to study electron shuttling, we decided to focus on the phenazine molecules it produces (Figure 2). Most current literature emphasizes the role of phenazines as virulence factors that generate toxic byproducts (e.g., reactive oxygen species) when oxidized in an oxic 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 most phenazines can be synthesized under anoxic 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 more basic functions.
In recent years, we have used P. aeruginosa strain PA14 to test this hypothesis in several ways. We have shown that (1) phenazines function effectively as electron shuttles to Fe(III), be it trapped in a mineral state or bound to proteins of the innate immune system, facilitating Fe(II) acquisition and signaling; (2) phenazines are signaling molecules, influencing the expression of a limited set of genes during the transition from exponential growth into stationary phase; (3) when respiratory oxidants (O2 or nitrate) are limited, phenazines modulate intracellular redox homeostasis; (4) phenazines permit survival under anoxic conditions by enabling flux through a fermentation pathway that produces ATP, enabling the generation of a proton motive force across the inner membrane; and (5) phenazines play a dramatic role in defining the habitable zone and morphology of biofilm communities, consistent with their other functions (Figure 3). We are working out the molecular pathways that underpin these phenomena by identifying and characterizing proteins that interact with phenazines intracellularly, as well as those that respond to changes in the extracellular environment stimulated by phenazines, such as the specific sensing of extracellular Fe(II) once it rises to low micromolar concentrations.
Motivated by these findings, we have become increasingly curious about whether phenazine redox cycling helps sustain Pseudomonas and other pathogens in complex chronic infections. To explore this, we chose the mucus accumulating on the lungs of CF patients as our test environment because it is expectorated daily and can be readily collected from patients. In collaboration with clinicians at Boston Children's Hospital and Children's Hospital Los Angeles, we have measured phenazine and iron concentrations (ferric and ferrous) in a cross-section of CF patients. Both phenazine and Fe(II) abundance exhibit significant positive correlations with disease progression. We now seek to understand how pathogens are coevolving with phenazine-mediated and other environmental changes in CF sputum, how quickly they are growing, and which metabolic programs are most important for survival. As we characterize the host environment and microbial physiology in situ, we can better design mechanistic experiments to gain insight into the specific cellular factors that promote survival as infections progress. This knowledge may one day enable the design of novel antimicrobial therapeutics that will be effective over a wider range of CF disease states. The approach we are taking is conceptually generic, and we hope to expand our work into other realms of chronic infections.
Grants from the National Institutes of Health, the National Science Foundation, and the National Aeronautics and Space Administration provided partial support for these projects.
As of March 23, 2016