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, what factors control their metabolic state and its dynamics are very important to understand. 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 was 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. 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 strictly anaerobic conditions (acylated versions of these compounds are important electron carriers in the membranes of some methanogenic archaea) 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 the past few years, we have tested this hypothesis in several ways using P. aeruginosa strain PA14. We have shown that: (i) phenazines function effectively as electron shuttles to Fe(III) minerals (25, 60), which may aid in Fe(II) acquisition; (ii) phenazines help modulate intracellular redox homeostasis as oxidants for NADH and/or by affecting carbon flux through central metabolic pathways; (iii) phenazines are signaling molecules, influencing the expression of a limited set of genes during the transition from exponential growth into stationary phase; (iv) 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 (v) phenazines play an important role in biofilm development, dramatically affecting the morphology of multicellular communities. 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 (e.g., transcription factors that directly sense phenazines, oxidoreductases that reduce them, and membrane complexes that transport them). We have also begun to develop specific analytical tools—both electrochemical and spectroscopic—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.

One of our next goals is to extend these findings. An exciting implication of our work is that the functions we are finding for phenazines may apply to a broad class of redox-active natural products produced by many different types of bacteria. While much remains to be done, we are encouraged that many “secondary” metabolites may be of primary importance for the development and maintenance of diverse microbial communities. In addition to determining the generality our findings, we hope to pursue the potential clinical relevance of our work by focusing on the pathology of P. aeruginosa. In collaboration with other investigators in the Boston area studying P. aeruginosa in the context of infection, we would like to test the hypothesis that phenazine production and cycling (or that of other redox-active compounds) is important for the survival of P. aeruginosa in the CF lung, where P. aeruginosa is thought to exist in a biofilm-like state. If the hypothesis proves true that redox active small molecules help biofilms survive and/or develop in vivo, it would suggest a new class of targets for rational drug design to control infection. Future research would then be aimed at developing specific methodologies to control their trafficking in this context.

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