To my parents, Amy and Ed Saunders, thank you for giving me every opportunity to succeed. To Elena Perry, thank you for your insightful comments and for our wonderful trip to Brazil. To Renee Wang, thank you for filling the lab with your sunny disposition and genuine curiosity.
To Chelsea VanDrisse, thank you for your infectious enthusiasm, especially regarding lab organization and genetics. To Kristy Nguyen, thank you for supporting me in the lab and always lending a kind ear. To Will DePas, thank you for bringing both levity and Ruth to our side of the lab.
To Liz Ayala and Raina Beaven, thank you for all your support over the years. Paul McElhany, thank you for introducing me to the world of experiments in all their messy glory.
Introduction
These molecules can serve as extracellular electron boats (EES), allowing microbes to access substrates at a distance. In this review, we argue that the written world of EES has been neglected for too long. Here, we describe the chemical diversity and potential distribution of EES producers and users, discuss the costs associated with their biosynthesis, and critically evaluate strategies for their economical use.
We hope that this review will inspire efforts to identify and explore the importance of EES cycling by a wide range of microorganisms, so that their contributions to the formation of microbial communities can be better assessed and exploited. It is the cycle of EES and their facilitation of electron transfer inside and outside the cell that supports their important physiological functions. Cycling of EES represents a strategy by which microbes can facilitate extracellular electron transfer, but it is by no means the only one.
Thus, it is important to consider the possible involvement of EES in any context where extracellular electron transfer is important – be it the rhizopher soil or the inflamed tissues of chronic infections. We critically discuss the costs of EES biosynthesis, as well as bioenergetic concerns related to the cell biology of their reduction and potential loss to the environment.
Diversity of Endogenous EES and Organisms
A few other observations in the literature suggest the production of EES by various organisms, including Geothrix and Geobacter species, but in most cases the molecular nature of the putative EES is unknown. Although many secreted redox-active natural products have been well known for decades, such as indigoidin, their physiological functions have only recently begun to be investigated (25). Unlike intracellular redox-active metabolites, endogenous EES described in the literature to date generally lack modifications such as adenylation or lipidation.
We expect that a convergence of metabolomics and computational chemistry, together with the ongoing detailed study of select molecules such as phenazines, will implicate many of these redox-active natural products as ubiquitous and diverse EES. Studies outside the Enterobacteriacae then showed that SoxR did not upregulate the oxidative stress response in P . Strikingly, SoxR homologs are also identified in the emerging and established pathogens of the Mycobacteria (~400 genomes), the agriculturally important Rhizobia (493 genomes) and Frankia (33 genomes), and recently discovered phyla (6 genomes).
First, not all secreted redox-active molecules can serve as EES due to redox potential limitations (see Box 2). Our bioinformatics analysis only suggests the potential for widespread production and use of EES in the microbial world, which we hope will stimulate experimental follow-up (sidebar 1).
Costs of EES biosynthesis
We expect that metabolically prudent regulation could further reduce the actual cost of EES under oxidant limitation.
Cell biology of electron shuttling
This activity may allow phenazines to promote ATP synthesis during glucose oxidation by increasing the flux through pyruvate dehydrogenase and acetate kinase (47) (Fig. 3b). Invoking cytoplasmic EES reduction has interesting implications regarding the proton motive force. Upon reduction, phenazines, quinones and flavins all take on two protons at circumneutral pH (Fig. 1c).
The subsequent secretion and oxidation of the reduced shuttle would therefore release these two protons outside the cell, essentially translocating two protons across the inner membrane (Fig. 3b). If this shuttle does not require active transport into or out of the cell, redox cycling of a shuttle can drive the generation of a proton motive force. Conversely, shuttles that accept electrons from the cytoplasmic surface of NADH dehydrogenase can consume the driving force of the proton, such as the artificial compound paraquat (18) (Fig. 3c).
Finally, while we have focused here on the mechanisms of EES reduction, electron transfer involves a more subtle point with respect to the terminal electron acceptor. Moreover, mineral reduction is accompanied by alkalization (Fig. 3c), which further inhibits the available free energy and can even negatively affect the proton motive force if it occurs close to the cell.
EES in the extracellular environment
Regardless of the mechanism of electron transfer, sufficient concentrations of EES must be maintained for viability. EES is viewed as a public good because molecules are secreted that benefit nearby individuals and the producing cell (124). Therefore, microbes are more likely to produce public goods when they are closely related to their neighbors, when direct and indirect benefits are high, and production costs are low (78).
By dividing rapidly and producing an extracellular matrix, many bacteria can establish highly related microcolonies that favor the development of public goods (78). A good example of QS regulation of EES production can be found in how Pseudomonas species regulate phenazine production (32). Other strategies that may facilitate the viability of EES utilization concern privatization mechanisms, such as physical retention or a requirement for special machinery to exploit the secreted molecule (124).
Physical retention can be achieved through non-covalent interactions between small molecules and biofilm components, which in the case of EES, may also provide a scaffold for electron hopping. This may help to rationalize co-regulation of EES and exopolymeric substances (EPS), as in the case of phenazines, eDNA and specific types of EPS.
Outlook
The phenazine pyocyanin is a terminal signaling factor in the quorum sensing network of Pseudomonas aeruginosa. The low conductivity of Geobacter uraniireducens pili indicates a diversity of extracellular electron transfer mechanisms in the genus Geobacter. By replacing NAD+ in the pyruvate dehydrogenase complex (PDH), phenazines enable the synthesis of acetyl-CoA, which can drive ATP synthesis by the enzymes phosphate transacetylase and acetate kinase (46, 47).
Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by binding to eDNA. Reduction of PYO by PCA or PCN continued to completion in the presence of eDNA ( Fig. 3B ). We observed only ruthenium luminescence in the presence of biofilm suspension, consistent with ruthenium luminescence being associated with binding to eDNA.
Equilibration of PYO (from the IDA biofilm) with fresh medium was monitored by square wave voltammetry (SWV) (Fig. 5B), in which the peak current (Iswv) is proportional to the concentration of PYO remaining in the biofilm at each time interval (Bard et al., 1980). Iswv for each time point is expected to give a linear dependence with slope (m) proportional to $𝐷!” (Figure 5D) when Dap is independent of PYO concentration in the biofilm (Akhoury et al., 2013; White). et al., 1982a) Considering that the heterogeneity of the biofilm matrix allows different electron transfer pathways to occur in these complex systems, we favor two eDNA-mediated mechanisms for the action of phenazine EET in the matrix (Fig. 6B).
In the second model (Fig. 6B bottom), reduced phenazines (probably PCN) intercalate in eDNA and reduce PYOox via DNA CT (ii). The Pel polysaccharide may serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix.
The color map is the same as I. I) Background subtracted data from biofilm plate H. In both models, oxidized PYO is mostly bound and retained in the oxic region of the biofilm. Electron transfer reactions between phenazines under anoxic conditions were monitored in the anaerobic plate reader described above.
A freshly polished Pt wire used as an auxiliary electrode was also immersed in the buffer from the top of the well. Rough edges were smoothed with a razor blade, and two holes were poked in the bottom of the vial. Bottom – the oligomer sequences used to assemble the PCN and thiol-modified ds DNA monolayers.
It established that certain phenazines are retained in the biofilm matrix through a binding interaction with extracellular DNA.
