Bioelectronic Tools for Microbiology
For studying biological systems, optical microscopy techniques in which photons act as an intermediary between the biological system and the solid-state world remain paramount. In this project, we seek to develop new tools for microbiology based on electrochemical sensing of metabolites. These tools give us new insight into microbial communities, including those that may be pathogenic and those that may benefit human health (such as in the gut microbiome).
In one project, we are developing electrochemical imaging arrays, which are capable of performing electrochemical analysis on a massively parallel scale with integrated electrodes. We have so far applied these techniques to multiplexed biosensing but are considering other applications in which direct electrochemical sensing provides advantages over optical techniques such as in probing the gut microbiome.
Hassan Sakhtah, Leslie Koyama, Yihan Zhang, Diana K. Moralesa, Blanche L. Fields, Alexa Price-Whelan, Deborah A. Hogan, Kenneth Shepard, and Lars E. P. Dietricha The Pseudomonas aeruginosa efflux pump MexGHIOpmD transports a natural phenazine that controls gene expression and biofilm development PNAS, Early Edition, June 6, 2016.
Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHIOpmD in the opportunistic pathogen Pseudomonas aeruginosa. Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHIOpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We also show that 5-Me-PCA is sufficient to fully induce MexGHI-OpmD expression and that it is required for wild-type colony biofilm morphogenesis. These physiological effects are consistent with the high redox potential of 5-Me-PCA, which distinguishes it from other well-studied P. aeruginosa phenazines. Our observations highlight the importance of this compound, which was previously overlooked due to the challenges associated with its detection, in the context of P. aeruginosa gene expression and multicellular behavior. This study constitutes a unique demonstration of efflux-based selfresistance, controlled by a simple circuit, in a Gram-negative pathogen.
D. L. Bellin, H. Sakhtah, Y. Zhang, A. Price-Whelan, L. E.P. Dietrich & K. L. Shepard Electrochemical camera chip for simultaneous imaging of multiple metabolites in biofilms. Nat. Commun. 7:10535 doi: 10.1038/ncomms10535 (2016).
Monitoring spatial distribution of metabolites in multicellular structures can enhance understanding of the biochemical processes and regulation involved in cellular community development. Here we report on an electrochemical camera chip capable of simultaneous spatial imaging of multiple redox-active phenazine metabolites produced by Pseudomonas aeruginosa PA14 colony biofilms. The chip features an 8mm8mm array of 1,824 electrodes multiplexed to 38 parallel output channels. Using this chip, we demonstrate potential-sweepbased electrochemical imaging of whole-biofilms at measurement rates in excess of 0.2 s per electrode. Analysis of mutants with various capacities for phenazine production reveals distribution of phenazine-1-carboxylic acid (PCA) throughout the colony, with 5-methylphenazine-1-carboxylic acid (5-MCA) and pyocyanin (PYO) localized to the colony edge. Anaerobic growth on nitrate confirms the O2-dependence of PYO production and indicates an effect of O2 availability on 5-MCA synthesis. This integrated-circuit-based technique promises wide applicability in detecting redox-active species from diverse biological samples.
D.L. Bellin, H. Sakhtah, J.K. Rosenstein, P.M. Levine, J. Thimot, K. Emmet, L.E.P. Dietrich, and K.L. Shepard Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms, Nature Communications 5:3256 (2014) doi:10.1038/ncomms4256
Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. A technique for spatially resolved detection of small molecules would benefit the study of redox-active metabolites that are produced by microbial biofilms and can affect their development. Here we present an integrated circuit-based electrochemical sensing platform featuring an array of working electrodes and parallel potentiostat channels. ‘Images’ over a 3.250.9mm2 area can be captured with a diffusion-limited spatial resolution of 750 μm. We demonstrate that square wave voltammetry can be used to detect, identify and quantify (for concentrations as low as 2.6 μm) four distinct redox-active metabolites called phenazines. We characterize phenazine production in both wild-type and mutant Pseudomonas aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine biosynthetic gene expression.