Raman Spectroscopy Based Toolkit for Mapping Bacterial Social Interactions Relevant to Human and Plant Health

Bacteria interact and co-exist with other microbes and with higher organisms like plants and humans, playing a major role in their health and well being. These ubiquitous single celled organisms are so successful, because they can form organized communities, called biofilms, that protect them from environmental stressors and enable communication and cooperation among members of the community. The work described in this thesis develops a toolkit of analytical techniques centered around Raman microspectroscopy and imaging representing a powerful approach to non-invasively investigate bacterial communities, yielding molecular information at the sub-micrometer length scale.

Bacterial cellular components of non-pigmented and pigmented rhizosphere strains are characterized, and regiospecific SERS is used for cases where resonantly enhanced background signals obscure the spectra. Silver nanoparticle colloids were synthesized in situ, in the presence of the cells to form a proximal coating and principal component analysis (PCA) revealed features attributed to flavins. SERS enabled in situ acquisition of Raman specra and chemical images in highly autofluorescent P.aeruginosa biofilms. In combination with PCA, this allowed for non-invasive spatial mapping of bacterial communities and revealed differences between strains and nutrients in the secretion of virulence factor pyocyanin. The rich potential of using Raman microspectroscopy to study plant-microbe interactions is demonstrated. Effect of exposure to oxidative stress, on both the wild type Pantoea sp. YR343 and carotenoid mutant ΔcrtB, was assessed by following the intensity of the 1520 cm^-1 and 1126 cm^-1 Raman bands, respectively, after treatment with various concentrations of H₂O₂. Significant changes were observed in these marker bands even at concentrations (1 mM) below the point at which the traditional plate-based viability assay shows an effect (5-10 mM), thus establishing the value of Raman microspectroscopy as a tool for high sensitivity studies of bacterial environmental stressors. The use of PCA in Raman imaging can also discriminate between spectral contributions from plant and bacterial cells. Finally, spectroscopy compatible microfluidic corral platforms are fabricated and a simple microfluidic technique is demonstrated for capturing bacterial cells. This opens up the possibility of studying bacterial communication in settings where it is possible to control population size and microenvironment.