Master of Science / Department of Biological & Agricultural Engineering / Lisa R. Wilken / Microalgae has potential as a biofuel feedstock and as a source of valuable bioproducts for a variety of food, feed, nutraceutical, and pharmaceutical industries. However, several challenges are associated with bioproduct extraction from microalgae. The complexity of microalgae cell necessitates use of energy intensive disruption methods but current chemical or mechanical techniques can degrade economically valuable bioproducts. Aqueous enzymatic extraction (AEE), is a non-solvent and environmentally friendly bio-product recovery method that provides an opportunity to design an integrated process for protein and oil fractionation while reducing industrial costs. Based on the mechanistic understanding of biomolecule distribution and compartmentation, an aqueous enzymatic treatment for the release of internally stored proteins and lipid bodies in wild type Chlamydomonas reinhardtii was developed. In this study, we optimized harvesting times that maximized lipid and protein yields in nitrogen depleted cultures of the microalgae Chlamydomonas reinhardtii. Furthermore, an aqueous enzymatic extraction (AEE) treatment was developed. First, four lytic enzymes were tested for their ability to permeate C. reinhardtii cell walls. After cells were permeable, another set of enzymes were tested for their ability to release internally stored bioproducts. Protein recovery and lipid characterization after enzymatic treatment indicated a 54% release of total soluble protein and a localization of lipids to the chloroplast. Additionally, the development of secondary enzyme treatment for chloroplast disruption achieved about 70% total lipids released into the supernatant. Taken together, results indicate the application of an enzymatic treatment scheme for protein and oil recovery as a promising alternative to traditional extraction processes.

Conversion of <i>Campylobacter upsaliensis</i> to the nonculturable but viable coccoid form was characterized. Chloramphenicol did not prevent the conversion. Severe decreases in isocitrate dehydrogenase activity and oxygen uptake and extensive degradation of ribosomal RNA suggest that the coccoid form is a degenerative form rather than part of a life cycle. The autolysins of spiral and coccoid forms of <i>C. upsaliensis</i> were also studied. Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of <i>C. upsaliensis</i> could not be demonstrated by native (nondenaturing) PAGE. Autolysins were detected, however, by using denaturing SDS-PAGE gels containing either purified <i>E. coli </i> peptidoglycan or whole cells of <i>Micrococcus luteus</i> as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained <i>E. coli</i> peptidoglycan, 14 autolytic bands were detected ranging from 200 kDa to 12 kDa. In similar gels containing whole cells of <i>M. luteus</i> , only a single band appeared having a molecular weight of 34 kDa. This band corresponded to one of the bands present in the gels containing <i>E. coli </i> peptidoglycan. This common autolysin was isolated by adsorbing it from <i>C. upsaliensis</i> lysates onto <i>M. luteus</i> cells and then subjecting these cells to renaturing SDS-PAGE in gels containing <i>E. coli</i> peptidoglycan. The 34 kDa autolysin differed from a single 51 kDa autolysin unique to the <i>M. luteus cells</i>. The 34 kDa autolysin was isolated from an SDS-PAGE gel and was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34 kDa autolysin to have 67% identity with a part of antigenic protein PEB4 of <i>Campylobacter jejuni</i>. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular weight of 34 kDa, and thus it seems unlikely that the 34 kDa autolysin was derived from any of the other autolysins that were detected. / Ph. D.

As the spread of antibiotic resistance threatens our ability to treat infections, avoiding the return of a pre-antibiotic era urgently requires the discovery of novel antibiotics. Actinomycetes, a family of bacteria commonly isolated from soil, are a proven source of clinically useful antibiotics. However, easily identifiable metabolites have been exhausted and the rediscovery of common antibiotics thwarts searches for rarer molecules. Sequencing of actinomycete genomes reveals that they contain far more biosynthetic gene clusters with the potential to encode antibiotics than whose products can be readily observed in the laboratory. The work presented in this thesis revolves around developing approaches to mine these previously inaccessible metabolites as a source of new antibiotics. First, I describe how inactivation of biosynthetic gene clusters for common antibiotics can uncover rare antibiotics otherwise masked in these strains. By applying CRISPR-Cas9 to knockout genes encoding nuisance antibiotics, I develop a simple strategy to reveal the hidden biosynthetic potential of actinomycete strains that can be used to discover rare or novel antibiotics. Second, I describe the use of the evolutionary history of biosynthetic gene clusters to prioritize divergent members of an antibiotic family, the glycopeptide antibiotics, that are likely to possess new biological activities. Using these predictions, I uncover a novel functional class of glycopeptide antibiotics that blocks the action of autolysins, essential peptidoglycan hydrolases required for remodelling the cell wall during growth. Finally, I apply target-directed genome mining, which makes use of target duplication as a predicted resistance mechanism within an antibiotic’s biosynthetic gene cluster. Using this approach, I discover the association of a family of gene clusters with the housekeeping protease ClpP and characterize the produced metabolite’s effect on ClpP function. These three research projects mine previously inaccessible chemical matter from a proven source of antibiotics, actinomycetes. The techniques and antibiotics described are required now more than ever to develop life-saving antibiotics capable of combatting multidrug-resistant pathogens. / Dissertation / Doctor of Philosophy (PhD) / Antibiotics are essential for treating life-threatening infections, but the rise of antibiotic resistance renders them ineffective. To treat these drug-resistant infections, new antibiotics that work in new ways are required. A family of bacteria commonly isolated from soil called Actinomycetes produce most antibiotics we use today, but it has become increasingly difficult to find new antibiotics from this source. My work describes three techniques that can be applied to actinomycetes to help overcome the challenges associated with antibiotic discovery. Specifically, these techniques guide discovery efforts by making use of regions in actinomycete genomes called biosynthetic gene clusters that often encode antibiotics. In doing so, I describe ways to uncover rare antibiotics from actinomycete strains that produce common and uninteresting antibiotics, use antibiotic family trees to discover antibiotics that work in new ways, and apply antibiotic resistance to identify biosynthetic gene clusters likely to act on a certain bacterial target.

Staphylococcus aureus (SA) is a major human pathogen that colonizes the anterior nares in 30% of the human population. Though nasal carriage of SA is a known risk factor for the subsequent spread of SA infections, the dynamics of SA nasal colonization is poorly understood. Our research focuses on understanding the host and bacterial factors that might contribute to the human nasal colonization by SA. In an attempt to elucidate the host response to SA, we performed an autologous human in vivo nasal colonization study, which showed decreased survival rates of SA in hosts who elicited a robust immune response. We also identified a significant correlation between SA nasal colonization and the expression of host proinflammatory, chemotactic and growth factors. Additionally, we functionally disrupted a major autolysin, atl a surface expressed bacterial protein that plays multiple roles in cell separation, adhesion and biofilm formation of SA. Microscopic analysis of the ∆atl strains showed phenotypic differences, including cell clumping and cluster formation due to defective cell separation, which confirmed the functional loss of atl. Subsequent analysis of the ∆atl and wild-type strains revealed that there was no significant difference in their ability to adhere to human nasal epithelial cells (hNEC) in an ex vivo hNEC model. Similarly, our competitive in vivo human nasal colonization study, in which equal colony-forming units of each wild-type and ∆atl SA strain were inoculated in the anterior nares of donors, showed similar survival rates between wild-type and ∆atl. These results suggest that Atl might not be directly involved in the adherence and colonization of SA to the anterior nares. Furthermore, our study suggests that host factors might play a predominant role in determining SA colonization to human anterior nares.

Biofilms formed on medical devices pose significant challenges, compromising device efficiency and serving as sources of infection. Staphylococcus epidermidis, an opportunistic pathogen, relies on the autolysin protein, notably its R2ab and amidase domains, to attach to polystyrene surfaces and initiate biofilm formation. Despite their pivotal role, the structural intricacies of these proteins’ interactions with surfaces remain elusive. In this dissertation, the multifaceted aspects of protein interactions with polystyrene surfaces and the implications of these interactions for biofilm control are studied. Over the course of this study, it is found how the R2ab and amidase domains influence biofilm formation on polystyrene surfaces. Pretreatment of polystyrene plates with these domains effectively inhibits biofilm growth, underscoring their strong affinity for polystyrene surfaces. Furthermore, these domains demonstrate a remarkable propensity for interactions with polystyrene nanoparticles (PSNPs). The insights gained from this study offer promising avenues for the development of novel biofilm eradication strategies, with the potential to enhance the longevity and effectiveness of medical devices. Shifting to a broader context of nanotechnology, the influence of nanoparticle size on protein adsorption and unfolding stabilities is studied using two distinct proteins, R2ab and GB3. Isothermal titration calorimetry reveals tighter binding to smaller PSNPs for both proteins, with enthalpy as the driving force. Structural changes in the adsorbed proteins are detected through fluorescence spectroscopy and circular dichroism, indicating a propensity for protein unfolding upon adsorption. Importantly, this unfolding effect is less pronounced with larger PSNPs, which has implications for protein binding on macroscopic surfaces. The significance of side-on interactions between neighboring proteins is underscored in this work, since they appear to stabilize proteins bound to surfaces with low curvature, an observation with critical implications for the protein corona formed around nanoparticles and its potential to preserve the structure of surface-adsorbed proteins in vivo. This dissertation also investigates the molecular-level interaction between R2ab and PSNPs of varying sizes. By utilizing lysine methylation in mass spectrometry and hydrogen-deuterium exchange (HDX) NMR spectroscopy, this work investigates how changes in methylation patterns and hydrogen-deuterium exchange rates in specific regions of R2ab reflect conformational changes upon binding to PSNPs. In conclusion, this dissertation comprehensively explores protein-surface interactions and reveals several important and surprising features of the proteins that drive biofilm formation.