Browsing by Subject "Bacillus"
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Item Characterization of Extremophilic Bacteria for Potential in the Biofuel and Bioprocess Industries(2014-04-18) Haynes, Abria RIndustrial bioprocesses are constrained by the availability of microbes that are optimized for harsh bioprocess conditions. Over 500 soil and sediment samples collected from 77 saline and/or thermal sites in the continental United States, Hawaii, and Puerto Rico were used to inoculate fermentations in an effort to optimize the microbial communities for a biofuel process developed at Texas A&M (MixAlco?). A diverse bacterial isolate library (N= 1,866 isolates) was constructed by employing a variety of culturing techniques across thirty-four of the fermentation communities selected based on superior performance in the biofuel platform. Phylogenetic analysis of partial 16S rDNA sequences placed 1,200 of these isolates in the genus Bacillus. Other genera include, but are not limited to: Ureibacillus, Bacillus, Geobacillus, and Caldalkalibacillus. The central hypothesis of this dissertation was that selection due to the original site conditions of the natural inocula, and also, survival during fermentation likely favored isolates in the library that possess biofuel/bioenergy relevant traits (e.g. hydrolysis of lignocellulosic biomass, utilization of hydrolysis products, and tolerance to inhibitory compounds released during hydrolysis). The phylogeny for this library was used to identify and prioritize a diverse subsample of the library (n=207) for high-throughput screens of extracellular cellulase activity, n-butanol tolerance, vanillin utilization, and lignin degradation, as indicated by decolorization of the surrogate Congo red. Many isolates exhibited the capacities screened, including several isolates that were positive for more than one of these traits. Subsequently, a subset of the 207 screened isolates were studied further for tolerance and/or utilization of bioprocess byproducts (e.g. lignocellulosic hydrolysate, bio-oil) that are known to harbor both compounds with inhibitory effects on growth and pentose sugars released after hydrolysis. Polyhydroxyalkanoates (PHA) are bio-plastics produced by some bacteria. There is interest in industry in the identification of bacteria that can utilize wastes from some bioprocesses while creating bio-plastics, thus, a subset of the library (n=43 isolates) was screened for PHA accumulation associated with growth with the bio-diesel byproduct glycerol as the carbon source. Several isolates from the library are PHA producers with glycerol based on a fluorescence screen conducted. Microfluidic microbial fuel cells (MFCs) take advantage of microbial metabolism to convert organic matter to electricity. Microbial communities collected directly from soils were screened in a MFC array developed at Texas A&M. The screening of natural microbial communities identified electricigens with enhanced power generation abilities. The variation identified in these industrially relevant traits across isolates provides a proof of concept for both the existence of this variation in nature and the efficacy of employing fermentation and culturing approaches to enrich for these phenotypes. Also, it became clear this library could serve as a resource for bioprocess isolates for industry, either directly or as a starting point to advance the bioprocess optimization prior to some form of genetic engineering targeting a particular function.Item Effect of Phytate-Degrading Probiotics on Broiler Performance(2013-10-30) Askelson, Tyler EProbiotics have been demonstrated to promote growth, stimulate immune responses, and improve the microbial food safety of poultry. While widely used, their effectiveness is mixed and the mechanisms through which they contribute to poultry production are not well understood. Phytases isolated from microorganisms are increasingly supplemented in feed to improve digestibility and reduce anti-nutritive effects of phytate. The microbial origin of these enzymes suggests a potentially important mechanism of probiotic functionality. Our objective was to investigate phytate degradation as a novel probiotic mechanism using recombinant Lactobacillus cultures expressing Bacillus subtilis phytase. B. subtilis phyA was codon optimized for expression in Lactobacillus and cloned into the expression vector, pTRK882. The resulting plasmid, pTD003, was transformed into Lactobacillus acidophilus, Lactobacillus gallinarum, and Lactobacillus gasseri. SDS-PAGE revealed an approximately 44 kDa protein in the culture supernatants of Lactobacillus pTD003 transformants corresponding to the predicted molecular weight of B. subtilis phytase. The phytate degrading ability of these cultures was evaluated by determining the amount of inorganic phosphate released from sodium phytate. Expression of B. subtilis phytase increased phytate degradation of L. acidophilus, L. gasseri, and L. gallinarum approximately 4-, 10-, and 18-fold over the background activity of empty vector transformants. The effect of administration of recombinant phytase-expressing L. gallinarum and L. gasseri was evaluated in broiler chicks fed a phosphorous deficient diet (0.25% aP). Phytase-expressing L. gasseri improved weight gain (P < 0.05) of broiler chickens to a level comparable to chickens fed a phosphorous adequate control diet (0.40% aP) demonstrating proof of-principle that administration of phytate-degrading probiotic cultures can improve performance of livestock animals. Additionally, this will inform future studies investigating whether probiotic cultures are able to combine the performance benefits of feed enzymes with the animal health and food safety benefits traditionally associated with probiotics.Item On the structure and assembly of staphylococcal leukocidin: a study of the molecular architecture of beta-barrel pore-forming toxins(Texas A&M University, 2006-08-16) Miles, Jr., George EmmettStaphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other β-PFTs form homooligomeric pores. For example, the staphylococcal α- hemolysin is a homoheptamer. Limited and controversial data exist on the assembly and molecular architecture of the leukocidin pore. In this work, biochemical and biophysical methods were used to characterize the leukocidin pore produced by the LukF (HlgB) and LukS (HlgC) components encoded by Staphylococcus aureus. I demonstrate that LukF and LukS assemble to form an SDS-stable pore on rabbit erythrocyte membranes. In addition, the pore-forming properties of recombinant leukocidin were investigated with planar lipid bilayers. Although leukocidins and staphylococcal α-hemolysin share partial sequence identity and related folds, LukF and LukS produce a pore with a unitary conductance of 2.5 nS (1 M KCl, 5 mM HEPES, pH 7.4), which is over three times greater than that of α-hemolysin measured under the same conditions. The subunit composition and stoichiometry of a leukocidin pore were determined by two independent methods, gel shift electrophoresis and sitespecific chemical modification during single channel recording. Four LukF and four LukS subunits were shown to co-assemble into an octameric transmembrane structure. The existence of an additional subunit in part explains properties of the leukocidin pore, such as its high conductance. Additionally, this is the first time that either technique has been applied successfully to assess the composition of a heteromeric membrane protein. It is also relevant to understanding the mechanism of assembly of β-PFT pores, and suggests new possibilities for engineering these proteins. In additional studies, the HlyII pore encoded by Bacillus cereus was found to form a homoheptameric transmembrane pore with properties conforming in general with those of other members of the class of β-PFTs. HlyII possesses additional properties which make it an attractive candidate for applications in biotechnology, such as an oligomer with a high thermal stability in the presence of SDS and the ability of the pore to remain open at high transmembrane potentials.