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. 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. 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 co-evolving 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.

 

      Using the past to inform the present: reconsidering the function of redox-active “secondary” metabolites and the biology of stasis