Alkyl quinolones mediate heterogeneous colony biofilm architecture that improves community-level survival.

Bacterial communities exhibit complex self-organization that contributes to their survival. To better understand the molecules that contribute to transforming a small number of cells into a heterogeneous surface biofilm community, we studied acellular aggregates, structures seen by light microscopy in Pseudomonas aeruginosa colony biofilms using light microscopy and chemical imaging. These structures differ from cellular aggregates, cohesive clusters of cells important for biofilm formation, in that they are visually distinct from cells using light microscopy and are reliant on metabolites for assembly. To investigate how these structures benefit a biofilm community we characterized three recurrent types of acellular aggregates with distinct geometries that were each abundant in specific areas of these biofilms. Alkyl quinolones (AQs) were essential for the formation of all aggregate types with AQ signatures outside the aggregates below the limit of detection. These acellular aggregates spatially sequester AQs and differentiate the biofilm space. However, the three types of aggregates showed differing properties in their size, associated cell death, and lipid content. The largest aggregate type co-localized with spatially confined cell death that was not mediated by Pf4 bacteriophage. Biofilms lacking AQs were absent of localized cell death but exhibited increased, homogeneously distributed cell death. Thus, these AQ-rich aggregates regulate metabolite accessibility, differentiate regions of the biofilm, and promote survival in biofilms.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen with the ability to cause infection in the immune-compromised. It is well established that P. aeruginosa biofilms exhibit resilience that includes decreased susceptibility to antimicrobial treatment. This work examines the self-assembled heterogeneity in biofilm communities studying acellular aggregates, regions of condensed matter requiring alkyl quinolones (AQs). AQs are important to both virulence and biofilm formation. Aggregate structures described here spatially regulate the accessibility of these AQs, differentiate regions of the biofilm community, and despite their association with autolysis, correlate with improved P. aeruginosa colony biofilm survival.

Metabolic and Oxidative Stress Effects on the Spectroelectrochemical Behavior of Single Pseudomonas aeruginosa Cells.

Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a wide range of diseases in immunocompromised patients. In order to better understand P. aeruginosa behavior and virulence and to advance drug therapies to combat infection, it would be beneficial to understand how P. aeruginosa cells survive stressful conditions, especially environmental stressors. Here, we report on a strategy that measures potential-dependent fluorescence of individual P. aeruginosa cells, as a sentinel, for cellular response to starvation, hunger, and oxidative stress. This is accomplished using a micropore electrode array capable of trapping large numbers of isolated, vertically oriented cells at well-defined spatial positions in order to study large arrays of single cells in parallel. We find that conditions promoting either starvation or oxidative stress produce discernible changes in the fluorescence response, demonstrated by an increase in the prevalence of fluorescence transients, one of three canonical spectroelectrochemical behaviors exhibited by single P. aeruginosa cells. In contrast, more modest nutrient limitations have little to no effect on the spectroelectrochemical response when compared to healthy cells in the stationary phase. These findings demonstrate the capabilities of micropore electrode arrays for studying the behavior of single microbial cells under conditions where the intercellular spacing, orientation, and chemical environment of the cells are controlled. Realizing single-cell studies under such well-defined conditions makes it possible to study fundamental stress responses with unprecedented control.

Nanopore-Enabled Dark-Field Digital Sensing of Nanoparticles.

In this study, we use nanopore arrays as a platform for detecting and characterizing individual nanoparticles (NPs) in real time. Dark-field imaging of nanopores with dimensions smaller than the wavelength of light occurs under conditions where trans-illumination is blocked, while the scattered light propagates to the far-field, making it possible to identify nanopores. The intensity of scattering increases dramatically during insertion of AgNPs into empty nanopores, owing to their plasmonic properties. Thus, momentary occupation of a nanopore by a AgNP produces intensity transients that can be analyzed to reveal the following characteristics: (1) NP scattering intensity, which scales with the sixth power of the AgNP radius, shows a normal distribution arising from the heterogeneity in NP size, (2) the nanopore residence time of NPs, which was observed to be stochastic with no permselective effects, and (3) the frequency of AgNP capture events on a 21 × 21 nanopore array, which varies linearly with the concentration of the NPs, agreeing with the frequency calculated from theory. The lower limit of detection (LOD) for NPs was 130 fM, indicating that the measurement can be used in applications in which ultrasensitive detection is required. The results presented here provide valuable insights into the dynamics of NP transport into and out of nanopores and highlight the potential of nanopore arrays as powerful, massively parallel tools for nanoparticle characterization and detection.

Spectroelectrochemical behavior of parallel arrays of single vertically oriented Pseudomonas aeruginosa cells.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen responsible for a number of healthcare-associated infection. It is currently difficult to assess single cell behaviors of P. aeruginosa that might contribute to acquisition of antibiotic resistance, intercellular communication, biofilm development, or virulence, because mechanistic behavior is inferred from ensemble collections of cells, thus averaging effects over a population. Here, we develop and characterize a device that can capture and trap arrays of single P. aeruginosa cells in individual micropores in order to study their behaviors using spectroelectrochemistry. Focused ion beam milling is used to fabricate an array of micropores in a Au/dielectric/Au/SiO2-containing multilayer substrate, in which individual micropores are formed with dimensions that facilitate the capture of single P. aeruginosa cells in a predominantly vertical orientation. The bottom Au ring is then used as a working electrode to explore the spectroelectrochemical behavior of parallel arrays of individual P. aeruginosa cells. Application of step-potential or swept-potential waveforms produces changes in the fluorescence emission that can be imaged and correlated with applied potential. Arrays of P. aeruginosa cells typically exhibit three characteristic fluorescence behaviors that are sensitive to nutritional stress and applied potential. The device developed here enables the study of parallel collections of single bacterial cells with well-defined orientational order and should facilitate efforts to elucidate methods of bacterial communication and multidrug resistance at the single cell level.

Effect of Micro-Patterned Mucin on Quinolone and Rhamnolipid Profiles of Mucoid Pseudomonas aeruginosa under Antibiotic Stress.

Pseudomonas aeruginosa (P. aeruginosa) is commonly implicated in hospital-acquired infections where its capacity to form biofilms on a variety of surfaces and the resulting enhanced antibiotic resistance seriously limit treatment choices. Because surface attachment sensitizes P. aeruginosa to quorum sensing (QS) and induces virulence through both chemical and mechanical cues, we investigate the effect of surface properties through spatially patterned mucin, combined with sub-inhibitory concentrations of tobramycin on QS and virulence factors in both mucoid and non-mucoid P. aeruginosa strains using multi-modal chemical imaging combining confocal Raman microscopy and matrix-assisted laser desorption/ionization-mass spectrometry. Samples comprise surface-adherent static biofilms at a solid-water interface, supernatant liquid, and pellicle biofilms at an air-water interface at various time points. Although the presence of a sub-inhibitory concentration of tobramycin in the supernatant retards growth and development of static biofilms independent of strain and surface mucin patterning, we observe clear differences in the behavior of mucoid and non-mucoid strains. Quinolone signals in a non-mucoid strain are induced earlier and are influenced by mucin surface patterning to a degree not exhibited in the mucoid strain. Additionally, phenazine virulence factors, such as pyocyanin, are observed in the pellicle biofilms of both mucoid and non-mucoid strains but are not detected in the static biofilms from either strain, highlighting the differences in stress response between pellicle and static biofilms. Differences between mucoid and non-mucoid strains are consistent with their strain-specific phenology, in which the mucoid strain develops highly protected biofilms.

Potential Dependent Spectroelectrochemistry of Electrofluorogenic Dyes on Indium-Tin Oxide.

Indium-tin oxide (ITO) is used in a variety of applications due to its electrical conductivity and optical transparency. Moreover, ITO coated glass is a common working electrode for spectroelectrochemistry. Thus, the ITO substrates should exhibit well-understood spectroscopic characteristics. Here, we report anomalous potential-dependent luminescence emission from three structurally-dissimilar electrofluorogenic probe on ITO coated glass. The three probes, flavin mononucleotide, resorufin, and Nile blue, show the expected fluorescence modulation between their oxidized, emissive forms and their reduced, non-fluorescent forms at low laser irradiance and/or high concentrations. However, at high irradiance and/or low concentration, the emission intensity increases at reducing potentials, contrary to expectations. In addition, a strong interplay between probe molecule concentration and laser irradiance is observed. We attribute the anomalous behavior to a combination of (1) irradiance-dependent ITO carrier dynamics, and (2) interaction of the fluorescent probe with ITO at reducing potentials resulting in a charge transfer state with altered emission behavior. Thus, the potential- and irradiance-dependent behavior of ITO and the resulting charge transfer state may not only interfere with the observation of potential-dependent fluorescence from redox probes but can completely reverse the polarity of the potential-dependent luminescence, especially at high irradiance and low concentration.

Actively Controllable Solid-Phase Microextraction in a Hierarchically Organized Block Copolymer-Nanopore Electrode Array Sensor for Charge-Selective Detection of Bacterial Metabolites.

Pseudomonas aeruginosa produces a number of phenazine metabolites, including pyocyanin (PYO), phenazine-1-carboxamide (PCN), and phenazine-1-carboxylic acid (PCA). Among these, PYO has been most widely studied as a biomarker of P. aeruginosa infection. However, despite its broad-spectrum antibiotic properties and its role as a precursor in the biosynthetic route leading to other secondary phenazines, PCA has attracted less attention, partially due to its relatively low concentration and interference from other highly abundant phenazines. This challenge is addressed here by constructing a hierarchically organized nanostructure consisting of a pH-responsive block copolymer (BCP) membrane with nanopore electrode arrays (NEAs) filled with gold nanoparticles (AuNPs) to separate and detect PCA in bacterial environments. The BCP@NEA strategy is designed such that adjusting the pH of the bacterial medium to 4.5, which is above the pKa of PCA but below the pKa of PYO and PCN, ensures that PCA is negatively charged and can be selectively transported across the BCP membrane. At pH 4.5, only PCA is transported into the AuNP-filled NEAs, while PYO and PCN are blocked. Structural characterization illustrates the rigorous spatial segregation of the AuNPs in the NEA nanopore volume, allowing PCA secreted from P. aeruginosa to be quantitatively determined as a function of incubation time using square-wave voltammetry and surface-enhanced Raman spectroscopy. The strategy proposed in this study can be extended by changing the nature of the hydrophilic block and subsequently applied to detect other redox-active metabolites at a low concentration in complex biological samples and, thus, help understand metabolism in microbial communities.

MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics.

Mitochondrial Ca(2+) Uniporter (MCU)-dependent mitochondrial Ca(2+) uptake is the primary mechanism for increasing matrix Ca(2+) in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1) have severely impaired [Ca(2+)]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca(2+)-dependent mitochondrial metabolism.