Fine scale sampling reveals early differentiation of rhizosphere microbiome from bulk soil in young Brachypodium plant roots.

For a deeper and comprehensive understanding of the composition and function of rhizosphere microbiomes, we need to focus at the scale of individual roots in standardized growth containers. Root exudation patterns are known to vary along distinct parts of the root even in juvenile plants giving rise to spatially distinct microbial niches. To address this, we analyzed the microbial community from two spatially distinct zones of the developing primary root (tip and base) in young Brachypodium distachyon grown in natural soil using standardized fabricated ecosystems known as EcoFABs as well as in more conventional pot and tubes. 16S rRNA based community analysis showed a strong rhizosphere effect resulting in significant enrichment of several OTUs belonging to Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. However, microbial community composition did not differ between root tips and root base or across different growth containers. Functional analysis of bulk metagenomics revealed significant differences between root tips and bulk soil. The genes associated with different metabolic pathways and root colonization were enriched in root tips. On the other hand, genes associated with nutrient-limitation and environmental stress were prominent in the bulk soil compared to root tips, implying the absence of easily available, labile carbon and nutrients in bulk soil relative to roots. Such insights into the relationships between developing root and microbial communities are critical for judicious understanding of plant-microbe interactions in early developmental stages of plants.

Spatiotemporal Dynamics of Molecular Messaging in Bacterial Co-Cultures Studied by Multimodal Chemical Imaging.

Microbial community behavior is coupled to a set of genetically-regulated chemical signals that correlate with cell density - the quorum sensing (QS) system - and there is growing appreciation that the QS-regulated behavior of bacteria is chemically, spatially, and temporally complex. In addition, while it has been known for some time that different species use different QS networks, we are beginning to appreciate that different strains of the same bacterial species also differ in their QS networks. Here we combine mass spectrometric imaging (MSI) and confocal Raman microscopy (CRM) approaches to investigate co-cultures involving different strains (FRD1 and PAO1C) of the same species (Pseudomonas aeruginosa) as well as those involving different species (P. aeruginosa and E. coli). Combining MSI and CRM makes it possible to supersede the limits imposed by individual imaging approaches and enables the spatial mapping of individual bacterial species and their microbial products within a mixed bacterial community growing in situ on surfaces. MSI is used to delineate the secretion of a specific rhamnolipid surfactant as well as alkyl quinolone (AQ) messengers between FRD1 and PAO1C strains of P. aeruginosa, showing that the spatial distribution and production rate of AQ messengers in PAO1C far outstrips that of FRD1. In the case of multiple species, CRM is used to show that the prolific secretion of AQs by the PAO1C strain of P. aeruginosa is used to mediate its interaction with co-cultured E. coli.

Surface-Growing Communities of Pseudomonas aeruginosa Exhibit Distinct Alkyl Quinolone Signatures.

A cascade of events leads to the development of microbial biofilm communities that are thought to be responsible for over 80% of infections in humans. However, not all surface-growing bacteria reside in a stationary biofilm state. Here, we have employed confocal Raman microscopy to analyze and compare variations in the alkyl quinolone (AQ) family of molecules during the transition between surface-attached motile-swarming and stationary biofilm communities. The AQs have been established previously as important to Pseudomonas aeruginosa biofilms, interspecies competition, and virulence. The AQ Pseudomonas quinolone signal (PQS) is also a known quorum-sensing signal. We detail spatial identification of AQ, PQS, and 2-alkyl-4-hydroxyquinoline N-oxide (AQNO) metabolites in both swarm and biofilm communities. We find that AQNO metabolites are abundant signatures in active swarming communities.

Quantitative SIMS Imaging of Agar-Based Microbial Communities.

After several decades of widespread use for mapping elemental ions and small molecular fragments in surface science, secondary ion mass spectrometry (SIMS) has emerged as a powerful analytical tool for molecular imaging in biology. Biomolecular SIMS imaging has primarily been used as a qualitative technique; although the distribution of a single analyte can be accurately determined, it is difficult to map the absolute quantity of a compound or even to compare the relative abundance of one molecular species to that of another. We describe a method for quantitative SIMS imaging of small molecules in agar-based microbial communities. The microbes are cultivated on a thin film of agar, dried under nitrogen, and imaged directly with SIMS. By use of optical microscopy, we show that the area of the agar is reduced by 26 ± 2% (standard deviation) during dehydration, but the overall biofilm morphology and analyte distribution are largely retained. We detail a quantitative imaging methodology, in which the ion intensity of each analyte is (1) normalized to an external quadratic regression curve, (2) corrected for isomeric interference, and (3) filtered for sample-specific noise and lower and upper limits of quantitation. The end result is a two-dimensional surface density image for each analyte. The sample preparation and quantitation methods are validated by quantitatively imaging four alkyl-quinolone and alkyl-quinoline N-oxide signaling molecules (including Pseudomonas quinolone signal) in Pseudomonas aeruginosa colony biofilms. We show that the relative surface densities of the target biomolecules are substantially different from values inferred through direct intensity comparison and that the developed methodologies can be used to quantitatively compare as many ions as there are available standards.

Spatially dependent alkyl quinolone signaling responses to antibiotics in Pseudomonas aeruginosa swarms.

There is a general lack of understanding about how communities of bacteria respond to exogenous toxins such as antibiotics. Most of our understanding of community-level stress responses comes from the study of stationary biofilm communities. Although several community behaviors and production of specific biomolecules affecting biofilm development and associated behavior have been described for Pseudomonas aeruginosa and other bacteria, we have little appreciation for the production and dispersal of secreted metabolites within the 2D and 3D spaces they occupy as they colonize, spread, and grow on surfaces. Here we specifically studied the phenotypic responses and spatial variability of alkyl quinolones, including the Pseudomonas quinolone signal (PQS) and members of the alkyl hydroxyquinoline (AQNO) subclass, in P. aeruginosa plate-assay swarming communities. We found that PQS production was not a universal signaling response to antibiotics, as tobramycin elicited an alkyl quinolone response, whereas carbenicillin did not. We also found that PQS and AQNO profiles in response to tobramycin were markedly distinct and influenced these swarms on different spatial scales. At some tobramycin exposures, P. aeruginosa swarms produced alkyl quinolones in the range of 150 µm PQS and 400 µm AQNO that accumulated as aggregates. Our collective findings show that the distribution of alkyl quinolones can vary by several orders of magnitude within the same swarming community. More notably, our results suggest that multiple intercellular signals acting on different spatial scales can be triggered by one common cue.

In Situ Probing of Laser Annealing of Plasmonic Substrates with Surface-Enhanced Raman Spectroscopy.

In this work, we in situ monitor the laser annealing of template-fabricated silver substrates using surface-enhanced Raman scattering (SERS) and 4-mercaptobenzoic acid (4-MBA) as a molecular probe. The annealing process, which exhibits a strong dependence on the laser power, yields a large (>50×) increase in the SERS of the immobilized 4-MBA. This increased SERS response is correlated with the changing substrate morphology using optical and scanning electron microscope images. We attribute the large enhancement to the formation of nanogaps facilitated by binding of the 4-MBA through both thiol and COO- groups in a sandwich structure, resulting in both electromagnetic and chemical enhancement. This annealing effect, associated with the continuous increase of SERS intensity, was not limited to the AgNP arrays but included Ag films deposited on a variety of nanoporous templates. This study provides a simple strategy for in situ optimization of plasmonic SERS substrates.

Spatial Mapping of Pyocyanin in Pseudomonas Aeruginosa Bacterial Communities Using Surface Enhanced Raman Scattering.

Surface enhanced Raman spectroscopy (SERS) imaging was used in conjunction with principal component analysis (PCA) for the in situ spatiotemporal mapping of the virulence factor pyocyanin in communities of the pathogenic bacterium Pseudomonas aeruginosa. The combination of SERS imaging and PCA analysis provides a robust method for the characterization of heterogeneous biological systems while circumventing issues associated with interference from sample autofluorescence and low reproducibility of SERS signals. The production of pyocyanin is found to depend both on the growth carbon source and on the specific strain of P. aeruginosa studied. A cystic fibrosis lung isolate strain of P. aeruginosa synthesizes and secretes pyocyanin when grown with glucose and glutamate, while the laboratory strain exhibits detectable production of pyocyanin only when grown with glutamate as the source of carbon. Pyocyanin production in the laboratory strain grown with glucose was below the limit of detection of SERS. In addition, the combination of SERS imaging and PCA can elucidate subtle differences in the molecular composition of biofilms. PCA loading plots from the clinical isolate exhibit features corresponding to vibrational bands of carbohydrates, which represent the mucoid biofilm matrix specific to that isolate, features that are not seen in the PCA loading plots of the laboratory strain.

Metal-assisted polyatomic SIMS and laser desorption/ionization for enhanced small molecule imaging of bacterial biofilms.

Mass spectrometry imaging (MSI) has become an important analytical tool for many sectors of science and medicine. As the application of MSI expands into new areas of inquiry, existing methodologies must be adapted and improved to meet emerging challenges. Particularly salient is the need for small molecule imaging methods that are compatible with complex multicomponent systems, a challenge that is amplified by the effects of analyte migration and matrix interference. With a focus on microbial biofilms from the opportunistic pathogen Pseudomonas aeruginosa, the relative advantages of two established microprobe-based MSI techniques-polyatomic secondary ion mass spectrometry (SIMS) and laser desorption/ionization-are compared, with emphasis on exploring the effect of surface metallization on small molecule imaging. A combination of qualitative image comparison and multivariate statistical analysis demonstrates that sputtering microbial biofilms with a 2.5 nm layer of gold selectively enhances C60-SIMS ionization for several molecular classes including rhamnolipids and 2-alkyl-quinolones. Metallization also leads to the reduction of in-source fragmentation and subsequent ionization of media-specific background polymers, which improves spectral purity and image quality. These findings show that the influence of metallization upon ionization is strongly dependent on both the surface architecture and the analyte class, and further demonstrate that metal-assisted C60-SIMS is a viable method for small molecule imaging of intact molecular ions in complex biological systems.

Label-free molecular imaging of bacterial communities of the opportunistic pathogen Pseudomonas aeruginosa.

Biofilms, such as those formed by the opportunistic human pathogen Pseudomonas aeruginosa are complex, matrix enclosed, and surface-associated communities of cells. Bacteria that are part of a biofilm community are much more resistant to antibiotics and the host immune response than their free-floating counterparts. P. aeruginosa biofilms are associated with persistent and chronic infections in diseases such as cystic fibrosis and HIV-AIDS. P. aeruginosa synthesizes and secretes signaling molecules such as the Pseudomonas quinolone signal (PQS) which are implicated in quorum sensing (QS), where bacteria regulate gene expression based on population density. Processes such as biofilms formation and virulence are regulated by QS. This manuscript describes the powerful molecular imaging capabilities of confocal Raman microscopy (CRM) and surface enhanced Raman spectroscopy (SERS) in conjunction with multivariate statistical tools such as principal component analysis (PCA) for studying the spatiotemporal distribution of signaling molecules, secondary metabolites and virulence factors in biofilm communities of P. aeruginosa. Our observations reveal that the laboratory strain PAO1C synthesizes and secretes 2-alkyl-4-hydroxyquinoline N-oxides and 2-alkyl-4-hydroxyquinolones in high abundance, while the isogenic acyl homoserine lactone QS-deficient mutant (ΔlasIΔrhlI) strain produces predominantly 2-alkyl-quinolones during biofilm formation. This study underscores the use of CRM, along with traditional biological tools such as genetics, for studying the behavior of microbial communities at the molecular level.

Multimodal chemical imaging of molecular messengers in emerging Pseudomonas aeruginosa bacterial communities.

Two label-free molecular imaging techniques, confocal Raman microscopy (CRM) and secondary ion mass spectrometry (SIMS), are combined for in situ characterization of the spatiotemporal distributions of quinolone metabolites and signaling molecules in communities of the pathogenic bacterium Pseudomonas aeruginosa. Dramatic molecular differences are observed between planktonic and biofilm modes of growth for these bacteria. We observe patterned aggregation and a high abundance of N-oxide quinolines in early biofilms and swarm zones of P. aeruginosa, while the concentrations of these secreted components in planktonic cells and agar plate colonies are below CRM and SIMS detection limits. CRM, in conjunction with principal component analysis (PCA) is used to distinguish between the two co-localized isomeric analyte pairs 4-hydroxy-2-heptylquinoline-N-oxide (HQNO)/2-heptyl-3-hydroxyquinolone (PQS) and 4-hydroxy-2-nonylquinoline-N-oxide (NQNO)/2-nonyl-hydroxyquinolone (C9-PQS) based on differences in their vibrational fingerprints, illustrating how the technique can be used to guide tandem-MS and tandem-MS imaging analysis. Because N-oxide quinolines are ubiquitous and expressed early in biofilms, these analytes may be fundamentally important for early biofilm formation and the growth and organization of P. aeruginosa microbial communities. This study underscores the advantages of using multimodal molecular imaging to study complex biological systems.

Using Raman Spectroscopy and SERS for in-situ studies of rhizosphere bacteria.

Bacteria colonize plant roots to form a symbiotic relationship with the plant and can play in important role in promoting plant growth. Raman spectroscopy is a useful technique to study these bacterial systems and the chemical signals they utilize to interact with the plant. We present a Raman study of Pantoea YR343 that was isolated from the rhizosphere of Populus deltoides (Eastern Cottonwood). Pantoea sp. YR343 produce yellowish carotenoid pigment that play a role in protection against UV radiation, in the anti-oxidative pathways and in membrane fluidity. Raman spectroscopy is used to non-invasively characterize the membrane bound carotenoids. The spectra collected from a mutant strain created by knocking out the crtB gene that encodes a phytoene synthase responsible for early stage of carotenoid biosynthesis, lack the carotenoid peaks. Surface Enhanced Raman Spectroscopy is being employed to detect the plant phytoharmone indoleacetic acid that is synthesized by the bacteria. This work describes our recent progress towards utilizing Raman spectroscopy as a label free, non-destructive method of studying plant-bacteria interactions in the rhizosphere.