Theses and Dissertations at Montana State University (MSU)
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Item The biofilm matrix in sulfate-reducing bacterial biofilms: potential roles for electron mediators and large proteins(Montana State University - Bozeman, College of Letters & Science, 2019) Krantz, Gregory Peter; Chairperson, Graduate Committee: Matthew Fields; Kilean Lucas, Erica L.-Wunderlich, Linh T. Hoang, Recep Avci, Gary Siuzdak and Matthew W. Fields were co-authors of the article, 'Bulk phase resource ratio alters carbon steel corrosion rates and endogenously produced extracellular electron transfer mediators in a sulfate-reducing biofilm' in the journal 'Biofouling' which is contained within this dissertation.; Peter J. Walian, Marty Boyl-Davis, Kara De Leon, Judy D. Wall and Matthew W. Fields were co-authors of the article, 'Large extracellular proteins sense hydrodynamic force and drive biofilm formation in Desulfovibrio vulgaris' which is contained within this dissertation.; Marty Boyl-Davis, Kara De Leon, Judy D. Wall and Matthew W. Fields were co-authors of the article, 'Characterization of extracellular biofilm mutants cultivated on 1018 carbon steel in Desulfovibrio vulgaris Hildenborough' which is contained within this dissertation.Sulfate-reducing bacteria grow and form biofilms in soil and benthic environments across much of the Earth's surface. Formation of these prevalent biofilms requires the secretion of an extracellular polymeric substance (EPS) to allow the cells to stick together, as well as adhere to a surface. The specific interactions that occur between EPS components of an SRB biofilm are poorly understood. The data presented in this dissertation suggest the presence of two extracellular mechanisms utilized in these communities. The first mechanism was observed in a study altering the lactate (electron donor) and sulfate (electron acceptor) ratios to create limiting nutrient conditions in Desulfovibrio alaskensis G20 (G20) biofilms. G20 was grown under two conditions: electron donor limited (EDL) and electron acceptor limited (EAL) conditions. When grown on a 1018 carbon steel substrate, the G20 consumes all of the available lactate, and once limited, it turns to the high energy electrons in the Fe 0 for growth. Corrosion rates in the steel increased two fold compared to the EAL condition. Global metabolomic analysis revealed increased lumichrome levels under the EDL condition, which suggested higher flux through the riboflavin/FAD biosynthetic pathway. Previous research showed that synthetically adding riboflavin and FAD increases the corrosion rate of a SRB biofilm on 1018 carbon steel, and paired with these results, suggest G20 produces a flavin-based extracellular electron transfer molecule endogenously, and uses it to harvest high energy electrons from Fe 0 when limited for electron donor. The second mechanism was observed in Desulfovibrio vulgaris Hildenborough (DvH) biofilms grown on glass. Two proteins, DVU1012 and DVU1545 were found to be the most abundant extracellular peptides in a DvH biofilm. Single deletion strains for these proteins grew biofilm similar to the wild type strain, but a double deletion strain had decreased ability to form biofilm, demonstrating that at least one of the peptides must be present in order to form a biofilm. Exposure to increased shear force caused an large increase in wild-type biofilm biomass, yet eliminated the double mutant biofilm. These proteins are required for a DvH biofilm to respond to shear force.Item Metabolic interactions and activity partitioning in a methanogenic, interdomain biofilm(Montana State University - Bozeman, College of Letters & Science, 2019) Camilleri, Laura Beth; Chairperson, Graduate Committee: Matthew Fields; Kristopher A. Hunt, Aurelien Mazurie, Jennifer Kuehl, Alex Michaud, James Connolly, Egan Lohman, Zack Miller, Adam M. Deutschbauer and Matthew W. Fields were co-authors of the article, 'Differential gene expression of a bacterial-archaeal interdomain biofilm producing methane' submitted to the journal 'Biofilms' which is contained within this dissertation.; B.P. Bowen, C.J. Petzold, T.R. Northen and M.W. Fields were co-authors of the article, 'Activity partitioning in an archaeal-bacterial biofilm' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methanococcus maripaludis factor causes slowed growth in Desulfovibrio vulgaris Hildenborough' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Growth effects of sulfopyruvate and sulfoacetate on the sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, and the methanogenic archaeon Methanococcus maripaludis S2' submitted to the journal 'Scientific reports' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methane production in Pelosinus fermentans JBW45' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.Biofilms are an ancient survival strategy in which communities of organisms can grow as a cohesive unit, generally attached to a surface and/or at interfaces. Despite the paradigm that 99% of microorganisms grow as a biofilm in the environment, current research methods are largely limited to monoculture planktonic studies. Although more investigations are trying to improve culture complexity by evaluating interactions between two or more populations, experiments are still more readily performed with microorganisms in the planktonic growth mode. The research presented here aims to elucidate the complexity of interactions between two microorganisms from different domains of life that results in enhanced metabolism due to localization of cells in close proximity within an anaerobic biofilm. Desulfovibrio vulgaris Hildenborough (DvH) and Methanococcus maripaludis S2 (Mmp) form a syntrophic mutualism when grown in sulfate-limited media that requires electron flux from DvH to Mmp through what is commonly assumed to be interspecies hydrogen transfer, thereby establishing cross-feeding. The biofilm has been shown to promote a stable and more even carrying capacity for both populations that is likely linked to improved hydrogen transfer (and/or other potential carbon and electron co-metabolites) as compared to planktonic populations. Transcriptomic and proteomic analyses, utilizing RNA-seq and deuterated water respectively, were used to elucidate genes and proteins that contribute to the biofilm growth mode that results in a more efficient metabolism for the syntrophic co-culture (defined by biomass per substrate flux). The results demonstrate the expression of many genes with unknown functions, and others that contribute to cell-cell interactions as well as active proteins in electron processing (e.g., lactate oxidation) in DvH and CO2 reduction (e.g., methanogenesis) in Mmp. A metabolic model of the coculture provided reinforcement for transcriptomic assumptions and aided in the identification of a sulfonate and other amino acids as important syntrophic metabolites. Assessment of biofilm co-culture activity utilizing a new method, Biorthogonal Noncanonical Amino Acid Tagging (BONCAT), showed Mmp was less active in the uptake of a methionine analog as compared to DvH. Alternate assessments confirmed that Mmp was in fact active (based upon methane generation) although translational activity was below the detection limit. Further investigation of the system under sulfate stress showed that the metabolic pairing is more stable than previously thought and could indicate survival strategies that drive the seemingly 'mutualistic' relationship as a forced cooperation. The sulfate stress response coincided with observed lags in DvH growth when grown in Mmp spent medium that was associated with a decoupling of lactate-oxidation and sulfate-reduction. Together the results demonstrate metabolic interactions and activity partitioning within a methanogenic archaeal-bacterial biofilm. The dogma of mutualism being synonymous with equal reciprocity is challenged as it pertains to this model biofilm system. Moreover, this unique bacterial-archaeal biofilm represents interdomain interactions that could represent systems that contributed shared metabolic processes that lead to the development of eukaryotic life.Item The roles of hibernation promoting factor in resuscitation of Pseudomonas aeruginosa from dormancy(Montana State University - Bozeman, College of Letters & Science, 2018) Akiyama, Tatsuya; Chairperson, Graduate Committee: Michael Franklin; Kerry S. Williamson, Robert Schaefer, Shawna Pratt, Connie B. Chang and Michael J. Franklin were co-authors of the article, 'Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation' in the journal 'Proceedings of the National Academy of Sciences of the United States of America' which is contained within this thesis.; Kerry S. Williamson and Michael J. Franklin were co-authors of the article, 'Expression and regulation of the Pseudomonas aeruginosa hibernation promoting factor' submitted to the journal 'Molecular microbiology' which is contained within this thesis.Microbial biofilms are surface-attached communities of microorganisms. Biofilms are often associated with chronic infections due to antibiotic resistance. Pseudomonas aeruginosa causes chronic pulmonary infections in cystic fibrosis patients and chronic wound infections in diabetic ulcers. One mechanism for biofilm-associated resistance is a formation of non-dividing, metabolically dormant cells resisting antibiotics. The goals of this research were to understand the molecular mechanisms involved in formation, maintenance, and resuscitation of dormant cells, with the ultimate goal of developing enhanced treatment strategies for chronic biofilm-associated infections. While dormant, bacteria must maintain cellular and macromolecular integrity required for resuscitation. Previous study found the high abundance of messenger RNAs for ribosome accessory proteins, hibernation promoting factor (HPF) and ribosome modulation factor (RMF), in the dormant subpopulation of P. aeruginosa biofilms. In this research, we characterized the activity and expression of the ribosome hibernation factor. By exposing the hpf and rmf deletion mutant strains to nutrient starvation, we found that HPF, but not RMF, is essential for cell viability maintenance during starvation-induced dormancy. Viability loss in the hpf mutant strain corresponded to loss of ribosomal RNA, and by inference, loss of cellular ribosome content during dormancy. Single-cell level studies using fluorescence in situ hybridization showed the heterogeneous ribosomal RNA levels for both the hpf and wild-type cells. Single-cell level studies using drop-based microfluidics also showed heterogeneity in resuscitation from dormancy. While the majority lost ability to resuscitate from dormancy, a fraction of hpf mutant cells recovered but with an extended lag time. We also determined the regulation of HPF expression using a transposon-based yellow fluorescent protein (YFP) reporter fused to HPF. The results showed that hpf is expressed from at least two different promoters. HPF expression is also controlled by mRNA folding, and an autofeedback mechanism. The complex regulatory mechanism at transcriptional and post-transcriptional levels may allow the bacteria to respond to nutrient limitation and enter a dormant state. Our results show the importance of HPF on ribosome preservation during starvation, as well as how this hibernation factor is regulated. The results provide new information of this novel target for treatment of dormant infectious bacteria.Item Nutrient limitation alters metabolism, CR(VI) response, and biofilm matrix composition in desulfovibrio vulgaris Hildenborough(Montana State University - Bozeman, College of Letters & Science, 2017) Franco, Lauren Christine; Chairperson, Graduate Committee: Matthew Fields; Grant Zane, Sadie Steinbeisser, Judy Wall and Matthew W. Fields were co-authors of the article, 'Cr(VI) reduction and physiological toxicity is impacted by resource ratio in Desulfovibrio vulgaris Hildenborough' submitted to the journal 'Applied microbiology and microbiology' which is contained within this thesis.; Julijana Ivanisevic, Gary Siuzdak and Matthew W. Fields were co-authors of the article, 'Nutrient limitation causes decline in metabolites important for cell cycle progression in bacterial biofilm' submitted to the journal 'Applied microbiology and microbiology' which is contained within this thesis.; Siva Wu, Michael Joo, Joel Mancuso, Jonathan Remis, Amita Gorur, Ambrose Leung, Danielle M. Jorgens, Joaquin Correa, Manfred Auer and Matthew W. Fields were co-authors of the article, 'Extracellular membrane structures in Desulfovibrio vulgaris Hildenborough biofilms' which is contained within this thesis.; Chris Petzold and Matthew W. Fields were co-authors of the article, 'Outer membrane vesicles and associated proteins produced by Desulfovibrio vulgaris Hildenborough biofilms' submitted to the journal 'Applied microbiology and microbiology' which is contained within this thesis.Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that live in anoxic environments and play critical roles in biogechemical cycling, namely linkages between the carbon and sulfur cycles. Desulfovibrio vulgaris Hildenborough (DvH) is a model organism for SRB that has been studied for its ability to reduce toxic heavy metals to insoluble forms and its involvement in microbially induced corrosion in oil pipelines and other industrial settings. The described work investigated how the availability of electron donor/carbon sources and electron acceptors affected Cr(VI) reduction, metabolism, and biofilm growth and composition in DvH. DvH was grown planktonically at 20°C and 30°C in batch mode or as a biofilm under continuous flow at 20°C. In the second chapter of this dissertation, it is established that electron acceptorlimitation (EAL) predisposes cells to Cr(VI) toxicity compared to a balanced electron donor to electron acceptor (BAL) condition and electron donor-limited (EDL) condition. The effect of nutrient limitation on DvH biofilms is investigated, and microscopy revealed unique extracellular membranous structures that have not previously been observed. The extracellular structures were heterogeneously distributed, connected to cells, co-localized with metal precipitates, and more prevalent under EAL compared to BAL condition. Differential staining indicated that the structures were composed of lipid, consistent with the observation that these structures are membrane derived. Metabolomic analysis revealed an up-regulation of fatty acids under the EAL condition, which was confirmed and quantified via GC-MS. Down-regulated metabolites for biofilm grown under the EAL condition included those involved in DNA turnover, N-cycling, and peptidoglycan turnover, indicating that EAL may induce a switch from growth to fatty acid production that may coordinate with alternative electron transfer mechanisms. Outer membrane vesicles (OMVs) were purified from DvH biofilm and proteins detected in OMVs included porins, lipoproteins, hydrogenases, and oxidative stress response proteins. The results presented here show that nutrient limitation and resource ratio affect DvH physiology in both biofilm and planktonic growth modes. The analysis of the DvH biofilm matrix highlights the importance of investigating extracellular capabilities that are unique to the biofilm growth mode and has implications for activities and physiological states in the environment.Item Understanding the physiology of Pseudomonas aeruginosa biofilms in an in vitro chronic wound model(Montana State University - Bozeman, College of Letters & Science, 2016) White, Benjamin Michael; Chairperson, Graduate Committee: Michael FranklinPseudomonas aeruginosa is a common colonizer of cutaneous abrasions and burns. These Gram-negative, aerobic bacteria are problematic due to their natural resilience to antibiotics and their metabolic versatility. P. aeruginosa can produce a prodigious extracellular matrix. Within this matrix P. aeruginosa can divide and form a multicellular community called a biofilm. Biofilms have become a health concern worldwide, as these communities are highly resistant to antibiotics. This thesis reports the effort to model the wound environment. A chronic wound exudate medium was designed and P. aeruginosa was grown at 33°C under low flow in a drip-flow biofilm reactor. Bacterial cells were grown planktonically and in biofilms. Biofilms were treated with the fluoroquinone, ciprofloxacin for 24 hours and transcriptomic and metabolomic data were collected from treated and untreated biofilms and planktonic cells. Cells growing in biofilms demonstrated a shift in in the regulation of their tricarboxylic acid cycle, amino acid degradation, and siderophore biosynthesis genes as compared to planktonic cells. Ciprofloxacin treatment altered the transcriptomic landscape within the biofilm. Changes were observed in the transcription of DNA repair, prophage, and phenazine biosynthesis genes. An important virulence factor, the type VI secretion system, was also differently regulated in these samples and is likely important for the persistent infection of wounds. From the information collected, target genes have been identified for future gene-knockout and ciprofloxacin susceptibility assays. A reduction in fitness may indicate genes that are relevant drug targets to enhance antibiotic treatment of these resilient communities.Item Bacterial colonization and modification of grain boundaries on 316L stainless steel(Montana State University - Bozeman, College of Agriculture, 1993) Gillis, Richard JohnItem Rapid in situ physiological assessment of disinfection in bacterial biofilms(Montana State University - Bozeman, College of Agriculture, 1994) Yu, Feipeng PhilipItem Pathogens in model distribution system biofilms(Montana State University - Bozeman, College of Agriculture, 1996) Warnecke, Malcolm RobertItem Biomass distributions, activity, growth, and carbon utilization in heterotrophic bacterial communities(Montana State University - Bozeman, College of Agriculture, 1999) Ellis, B. D.Item Interaction between human neutrophils and Pseudomonas aeruginosa biofilm : morphological and biochemical characterization(Montana State University - Bozeman, College of Agriculture, 2003) Papke, Maiko Sasaki; Chairperson, Graduate Committee: Michael Franklin
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