Shamoo, Yousif2023-08-092023-052023-04-18May 2023Song, Xinhao. "Developing Microdroplet Emulsions as a System for the Study of Microbial Social Interactions." (2023) Diss., Rice University. <a href="https://hdl.handle.net/1911/115086">https://hdl.handle.net/1911/115086</a>.https://hdl.handle.net/1911/115086The evolution and maintenance of social behaviors, especially cooperative interactions, is an intensively-studied field in ecology. Social microbes that interact through the secretion of diffusible chemical signals are ideal model organisms for both fundamental and translational studies of this fascinating process. In this thesis, I investigate the social interactions among soil-dwelling Gram-positive bacteria Streptomyces spp. Streptomyces species have contributed to more than two-thirds of current antibiotics and have a remarkable capability for the synthesis of secondary metabolites. Moreover, in their natural soil habitats, neighboring Streptomyces species can form complex social interaction networks that are mediated by diffusible secondary metabolites, further adding to their qualities as model organisms for the study of microbial social interactions. One of the key factors for the evolution and maintenance of social interactions among microbes is spatially structured environments. Such environments limit the diffusion of “public good” molecules such as beneficial metabolites and chemical signals and may favor social traits with indirect fitness benefits. In this thesis, I used microdroplet emulsions to construct artificial and configurable spatially segregated environments for the investigation of nascent social interactions. Two comprehensive case studies of inhibition and cooperative Streptomyces pairs were conducted. First, a collection of Streptomyces environmental isolates was obtained and characterized both phenotypically and genomically. From this collection, isolates T4-11 and AMS-5 were identified as inhibitive and cooperative partner strains for the model strain S. venezuelae. Next, using both plasmid-based and genome integration approaches, various RFP and GFP fluorescence reporter strains were constructed to facilitate the investigation of social interactions. Subsequently, interactions between T4-11/S. venezuelae and AMS-5/S. venezuelae pairs were thoroughly investigated using conditioned medium and co-culture assays in both liquid suspension and microdroplet environments. The inhibition of T4-11 on S. venezuelae was potent and reproducible across various conditions, whereas the interactions between AMS-5 and S. venezuelae varied significantly in different environments. To expand further the set of tools available to study social interactions, a volatile gas-based S. venezuelae reporter strain was constructed and characterized. It is well known that fluorescence reporters suffer from limitations when used in microdroplets, such as inaccurate readings due to strong background or limited sensitivity during early growth stages. To overcome such limitations, I showed that volatile gas produced by S. venezuelae gas reporters could be quantified by GC-MS in situ and could serve as an alternative non-optical detection approach for microbes encapsulated microdroplets. In addition, a 100-1000 fold improvement in sensitivity was observed for the gas reporter when compared to an RFP fluorescence reporter.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.microfluidicsStreptomycesmicrobial social interactionmethyl halide transferaseThesis2023-08-09