Spatiotemporal distribution of chemical signatures exhibited by Myxococcus xanthus in response to metabolic conditions.

Myxococcus xanthus is a common soil bacterium with a complex life cycle, which is known for production of secondary metabolites. However, little is known about the effects of nutrient availability on M. xanthus metabolite production. In this study, we utilize confocal Raman microscopy (CRM) to examine the spatiotemporal distribution of chemical signatures secreted by M. xanthus and their response to varied nutrient availability. Ten distinct spectral features are observed by CRM from M. xanthus grown on nutrient-rich medium. However, when M. xanthus is constrained to grow under nutrient-limited conditions, by starving it of casitone, it develops fruiting bodies, and the accompanying Raman microspectra are dramatically altered. The reduced metabolic state engendered by the absence of casitone in the medium is associated with reduced, or completely eliminated, features at 1140 cm-1, 1560 cm-1, and 1648 cm-1. In their place, a feature at 1537 cm-1 is observed, this feature being tentatively assigned to a transitional phase important for cellular adaptation to varying environmental conditions. In addition, correlating principal component analysis heat maps with optical images illustrates how fruiting bodies in the center co-exist with motile cells at the colony edge. While the metabolites responsible for these Raman features are not completely identified, three M. xanthus peaks at 1004, 1151, and 1510 cm-1 are consistent with the production of lycopene. Thus, a combination of CRM imaging and PCA enables the spatial mapping of spectral signatures of secreted factors from M. xanthus and their correlation with metabolic conditions.

Electrochemical and spectroelectrochemical characterization of bacteria and bacterial systems.

Microbes, such as bacteria, can be described, at one level, as small, self-sustaining chemical factories. Based on the species, strain, and even the environment, bacteria can be useful, neutral or pathogenic to human life, so it is increasingly important that we be able to characterize them at the molecular level with chemical specificity and spatial and temporal resolution in order to understand their behavior. Bacterial metabolism involves a large number of internal and external electron transfer processes, so it is logical that electrochemical techniques have been employed to investigate these bacterial metabolites. In this mini-review, we focus on electrochemical and spectroelectrochemical methods that have been developed and used specifically to chemically characterize bacteria and their behavior. First, we discuss the latest mechanistic insights and current understanding of microbial electron transfer, including both direct and mediated electron transfer. Second, we summarize progress on approaches to spatiotemporal characterization of secreted factors, including both metabolites and signaling molecules, which can be used to discern how natural or external factors can alter metabolic states of bacterial cells and change either their individual or collective behavior. Finally, we address in situ methods of single-cell characterization, which can uncover how heterogeneity in cell behavior is reflected in the behavior and properties of collections of bacteria, e.g. bacterial communities. Recent advances in (spectro)electrochemical characterization of bacteria have yielded important new insights both at the ensemble and the single-entity levels, which are furthering our understanding of bacterial behavior. These insights, in turn, promise to benefit applications ranging from biosensors to the use of bacteria in bacteria-based bioenergy generation and storage.

Redox cycling-based detection of phenazine metabolites secreted from Pseudomonas aeruginosa in nanopore electrode arrays.

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) produces several redox-active phenazine metabolites, including pyocyanin (PYO) and phenazine-1-carboxamide (PCN), which are electron carrier molecules that also aid in virulence. In particular, PYO is an exclusive metabolite produced by P. aeruginosa, which acts as a virulence factor in hospital-acquired infections and is therefore a good biomarker for identifying early stage colonization by this pathogen. Here, we describe the use of nanopore electrode arrays (NEAs) exhibiting metal-insulator-metal ring electrode architectures for enhanced detection of these phenazine metabolites. The size of the nanopores allows phenazine metabolites to freely diffuse into the interior and access the working electrodes, while the bacteria are excluded. Consequently, highly efficient redox cycling reactions in the NEAs can be accessed by free diffusion unhindered by the presence of bacteria. This strategy yields low limits of detection, i.e. 10.5 and 20.7 nM for PYO and PCN, respectively, values far below single molecule pore occupancy, e.g. at 10.5 nM 〈npore〉∼ 0.082 per nanopore - a limit which reflects the extraordinary signal amplification in the NEAs. Furthermore, experiments that compared results from minimal medium and rich medium show that P. aeruginosa produces the same types of phenazine metabolites even though growth rates and phenazine production patterns differ in these two media. The NEA measurement strategy developed here should be useful as a diagnostic for pathogens generally and for understanding metabolism in clinically important microbial communities.

Electrochemical Surface-Enhanced Raman Spectroscopy of Pyocyanin Secreted by Pseudomonas aeruginosa Communities.

Pyocyanin (PYO) is one of many toxins secreted by the opportunistic human pathogenic bacterium Pseudomonas aeruginosa. Direct detection of PYO in biofilms is crucial because PYO can provide important information about infection-related virulence mechanisms in P. aeruginosa. Because PYO is both redox-active and Raman-active, we seek to simultaneously acquire both spectroscopic and redox state information about PYO. The combination of surface-enhanced Raman spectroscopy (SERS) and voltammetry is used here to provide insights into the molecular redox behavior of PYO while controlling the SERS and electrochemical (EC) response of PYO with external stimuli, such as pH and applied potential. Furthermore, PYO secretion from biofilms of different P. aeruginosa strains is compared. Both SERS spectra and EC behavior are observed to change with pH, and several pH-dependent bands are identified in the SERS spectra, which can potentially be used to probe the local environment. Comparison of the voltammetric behavior of wild-type and a PYO-deficient mutant unequivocally identifies PYO as a major component of the secretome. Spectroelectrochemical studies of the PYO standard reveal decreasing SERS intensities of PYO bands under reducing conditions. Extending these experiments to pellicle biofilms shows similar behavior with applied potential, and SERS imaging indicates that secreted PYO is localized in regions approximately the size of P. aeruginosa cells. The in situ spectroelectrochemical biofilm characterization approach developed here suggests that EC-SERS monitoring of secreted molecules can be used diagnostically and correlated with the progress of infection.