Defining atomic-level composition in mammalian and bacterial whole cells and new discoveries in biofilm assembly, structure, and function

Bacteria inhabit and thrive in an astonishingly diverse range of environmental habitats across the Earth and in association with many living organisms including plants, fish and humans. In nature, bacteria are most commonly found in multicellular communities termed biofilms. Biofilms are found everywhere and promote beneficial functions as well as undesirable consequences. Bacteria found in symbiotic association within the root nodules of plants fix nitrogen from air to provide essential nitrogen-containing compounds for plants. Yet, bacterial biofilms are also associated with chronic and difficult-to-treat bacterial infections in human hosts. Bacteria produce and secrete molecular components to assemble extracellular matrix (ECM) architectures that serve to envelop individual cells and enmesh bacterial communities. Depending on the organism and ECM, both chemical and physical properties of the ECM can afford protection from environmental threats such as desiccation, host defenses, and antibiotics. The ECM of most bacteria contain a heterogeneous entanglement of biopolymers such as polysaccharides and proteins. The ECM poses a challenge to chemical analysis approaches in terms of quantitative composition and structure due to its heterogeneity, coupled to the presence of typically non-crystalline insoluble polymeric. Traditional methods rely on genetic approaches and biochemical detection to define crucial parts lists. However, these methods are usually not well suited to estimate or compare abundance of ECM components across samples, particularly with polysaccharide and insoluble macromolecular protein assemblies. Solid-state nuclear magnetic resonance (NMR) has emerged as an exciting tool for ECM analysis because the ECM material can be studied as an intact, native biocomposite. Recently, a previously unidentified chemically modified cellulose (pEtN cellulose) was discovered using solid-state NMR in the ECM material from Escherichia coli and also is produced by Salmonella enterica serovar Typhimurium. This modification escaped detection for decades, likely due to cellulose hydrolysis methods used to liberate glucose for solution-based detection. This solid-state NMR discovery thus provided the first experimental determination of a chemically modified cellulose produced in nature. Additionally, solid-state NMR has been used to define the ECM composition and quantify the ratio of curli (functional amyloid fibers) and pEtN cellulose in a pathogenic bacterial biofilm. Moving beyond compositional analyses of curli and pEtN cellulose producing bacteria, outstanding questions remain unanswered regarding the way curli and pEtN cellulose come together and mix to form intricate macromolecular structures. Here I present interbacterial complementation studies to recapitulate biofilm formation in mixes of E. coli strains that alone are unable to produce the hallmark biofilm morphology. Using a host of compositional analytical methods including 13C CPMAS experiments and a newly introduced Congo red fluorescence assay, we discovered that mixed cultures of bacteria individually able to produce just curli or pEtN cellulose could recapitulate the biofilm phenotypes of that from single bacteria producing both polymers. Towards exciting implications for mixed species biofilms that are commonly found in host niches, such as in the human GI tract, we expanded this exploration and discovered that mixed cultures of E. coli producing only curli and Salmonella Typhimurium producing only pEtN cellulose formed biofilms that were compositionally and functionally similar to biofilms ascribed to single organisms producing both polymers. Through these studies we provide new data for this model in which curli and pEtN cellulose biofilm architecture, assembly, and function are attributed to atomic level interactions between these two components, weaving curli and pEtN cellulose into basket-like structures surrounding cells.

Keywords: Chemistry.
Advisor: Cegelski Lynette
Boxer Steven G. (Steven George)
Du Bois Justin