K-071. Two-Photon Imaging of Redox-Active Phenazine “Antibiotics” in vivo and in Biofilms

Y. Wang, D. S. Tzeranis, I. Ramos-Solis, P. T. C. So, D. K. Newman;
Massachusetts Inst. of Technology, Cambridge, MA.

Phenazines are redox-active small molecules produced by Pseudomonas species, which often form biofilms on a variety of surfaces, including the lungs of cystic fibrosis patients and the electrodes of microbial fuel cells. Beyond merely serving as antibiotics, recent studies suggest that phenazines may be important in biofilm physiology through their ability to shuttle electrons between bacteria and not easily accessible terminal oxidants (e.g., insoluble Fe(III) minerals and diffusion-limited O2). To test this hypothesis, non-destructive techniques for directly detecting phenazines in vivo and in biofilms are needed. Because they contain multiple aromatic rings, phenazines are candidates for fluorescence. Our observations reveal that reduced but not oxidized phenazines can be excited at wavelengths suitable for live-cell imaging using two-photon fluorescence microscopy. By characterizing the photophysics of these natural fluorophores, imaging the wild type and different mutant strains, and using a robust fitting algorithm, we can differentiate not only phenazines but also two other important intrinsic fluorophores (the major siderophore pyoverdine and the intracellular metabolic indicator NADH) at the single cell level for high-density planktonic P. aeruginosa. Lower levels of pyoverdine and NADH are found in the wild type strain than in a mutant that cannot make phenazines, supporting previous results that suggest phenazines play roles in iron acquisition and in maintaining intracellular redox homeostasis, respectively. With the method developed at the single cell level, we are currently trying to quantify the spatiotemporal distribution of phenazines during P. aeruginosa biofilm development. To link phenazine production with biofilm morphology and spatiometabolic patterning, we will follow expressions of gene products associated with growth activity and metabolism, quantify NADH levels, and test the effects of oxidant gradients.