Siderophores are low molecular weight, iron chelating compounds produced by many bacteria for uptake of iron in case of iron scarcity. Vicibactin is a trihydroxamate type siderophore produced by Rhizobium leguminosarum bv. trifolii ATCC 14479. This work focuses on identifying an outer membrane receptor involved in the transport of vicibactin. We have confirmed the presence of the putative fhuA gene in R. leguminosarum bv. trifolii ATCC 14479. This bacteria shows mutualistic symbiosis with the red clover plant Trifoliium prantense. Leghemoglobin, with its cofactor heme is present in the plant root nodules that surrounds the infecting organism present in the nodules. This work attempts to elucidate the ability of Rhizobium leguminosarum bv. trifolii ATCC 14479 to utilize heme-bound iron and genes involved in the transport. We have also elucidated the role of energy transducing proteins TonB- ExbB-ExbD on the heme-bound iron uptake system.

Mass spectrometry based proteomics has emerged as a powerful methodology for investigating protein expression. “Bottom up” techniques in which proteins are first digested, and resulting peptides separated via multi-dimensional chromatography then analyzed via mass spectrometry provide a wide depth of coverage of expressed proteomes. This technique has been successfully and extensively used to survey protein expression (expression proteomics) and also to investigate proteins and their associated interacting partners in order to ascertain function of unknown proteins (functional proteomics). Azospirillum brasilense is a free-living diazotrophic soil bacteria, with world-wide significance as a plant-growth promoting bacteria. Living within the rhizosphere of cereal grasses, its diverse metabolism is important for its survival in the competitive rhizospheric environment. The recently sequenced genome of strain Sp245 provided a basis for the proteome studies accomplished in this work. After initial mass spectrometer parameter optimization studies, the expressed proteomes of two strains of Azospirillum brasilense, Sp7 and Sp245, grown under both nitrogen fixing and optimal growth (non nitrogen fixing) conditions were analyzed using a bottom up proteomics methodology. Further proteome studies were conducted with A. brasilense strain Sp7 in order to ascertain the effect of one chemotaxis operon, termed Che1. In this study, proteomic surveys were conducted on two bacterial derivative strains, created earlier, which lacked either a forward signaling pathway or an adaptation pathway. The proteomic surveys conducted in this work provide a foundation for further biochemical investigations. In order to facilitate further investigation and a movement into functional proteomics, a set of destination vectors was created that contain a variety of tandem affinity tags. The addition of tandem affinity tags to a protein allow for generic purification schemes, and can facilitate future studies to investigate proteins of interest discovered in the first expression proteomic surveys of A. brasilense. Taken together, this dissertation provides a valuable data set for investigation into the physiology of A. brasilense and further provides biochemical tools for analysis of the functional protein interactions of A. brasilense cells.

Double strand breaks (DSB) are a common source of DNA damage in both prokaryotes and eukaryotes. If they are not repaired or are repaired incorrectly, they can lead to cell death (bacteria) or cancer (humans). In Escherichia coli, repair of DSB are typically accomplished via homologous recombination and mediated by RecA. This repair pathway, among others, is associated with activation of the SOS response. DNA adenine methyltransferase (dam) mutants have an increased number of DSB and, therefore, are notorious for being RecA-dependent for viability. Here, we show that the synthetic lethality of Δdam/ΔrecA is suppressed when clpP is removed, suggesting that there is a protein, normally degraded by ClpXP, which is preventing DSB from occurring.

Due to an increasing prevalence of bacterial resistance to antibiotic drugs and the overuse of commercial antibiotics, the need to discover novel antibacterial compounds is becoming more urgent. There is one promising avenue of novel drug discovery which has begun to be explored; the analysis of secondary metabolites. Rhodococcus is a genus of gram-positive bacterium known for their ability to catabolize a wide range of compounds, and more notably for its ability to produce bioactive secondary metabolites. Rhodococcus belongs to the class actinobacteria. A species of Rhodococcus, MTM3W5.2, has been discovered in Morristown, Tennessee and was found to produce a metabolite with inhibitory activity against closely related species. The aim of this study is to elucidate the structure of the inhibitory metabolite by first isolating and purifying it, and then characterizing it using spectroscopic techniques. The compound was isolated from MTM3W5.2 RM broth cultures using n-butanol extraction, which yielded an active crude extract. The crude extract was then subjected to fractionation using a Sephedex LH-20 column with a 100% methanol solvent. The inhibitory activity of the fractions was tested through disk diffusion assay using Rhodococcus erythropolis as an indicator of inhibitory activity. Further preparation was completed using preparative reverse-phase high-performance liquid chromatography. Advanced purification was conducted using multiple rounds of analytical reverse-phase HPLC and activity was tested at each subsequent step using disk diffusion assay. Throughout the study, the HPLC fractions were characterized and the stability was monitored using UV-Visible spectroscopy. Two pure samples at 58.63 and 72.72 minutes from HPLC (High-performance liquid chromatography) collections were selected for further structural identification and are currently being studied using spectroscopic techniques, most notably 2D NMR spectroscopy (two-dimensional nuclear magnetic resonance).

<p>Nonribosomal peptides (NRPs) are a major class of natural products known for their biological activities and are employed therapeutically as immunosupressants, anticancer agents, and antibiotics. Nonribosomal peptides are microbial products, biosynthesized by large assembly line-like enzymes, known as nonribosomal peptide synthetases (NRPSs) that can be found in large gene clusters within the genome. With the advent of genome sequencing, the gene clusters for known NRPs are easily identified within producing organisms, but more strikingly, this sequencing reveals that microbes often contain many gene clusters with no known products suggesting traditional methods of isolation are overlooking the majority of NRPs.</p> <p>Extensive studies of NRPS functions have revealed assembly line logic for the biosynthesis of NRPs and using this knowledge, the NRP products of NRPS gene clusters can be predicted. In this research, products from both a simple dimodular NRPS from <em>Staphylococcus aureus </em>and a complex 11 module NRPS from <em>Delftia acidovorans </em>were predicted and used to successfully identify and isolate two novel NRPs, aureusimine and delftibactin.<em> </em>Theses compounds fell outside traditional NRP activities, one being a virulence regulator and the other a gold-complexing metallophore. Subsequent biosynthetic studies of the aureusimine gene cluster within the heterologous host, <em>Escherichia coli</em>, provide insight into NRPS flexibility for the creation of NRP natural variants and highlighted the utility of <em>E. coli </em>for the heterologous production of NRPs.</p> <p>Realizing single NRP predictions are not always accurate, a strategy was devised to use a genomically predicted NRP fragment barcode databases with the LC-MS/MS dereplication algorithm, iSNAP, to chemoinformatically identify and physically locate genetically predicted NRPs within crude extracts. This final contribution eliminates the need for bioactivity guided approaches to discovery and provides a strategy to systematically discover all predicted NRPs from cryptic gene clusters. This thesis delivers strategies and technologies for the directed discovery of NRPs from microbial sources.</p> / Doctor of Philosophy (PhD)

The objective of this research was to investigate the effects of slow release urea on N metabolism in cattle. The ruminal behavior of Optigen®II and the effect of basal diet on the in situ degradability of urea and Optigen®II were evaluated. The effect of slow release urea and its interaction with degradable intake protein (DIP) level in the diet on N retention and excretion was evaluated utilizing 8 Holstein steers in a 4 x 4 Latin square experiment. In addition, the effect of slow release urea and DIP level on ruminal and systemic urea kinetics was evaluated using stable isotope techniques with 8 Holstein steers in a 4 x 4 Latin square experiment. Finally, slow release urea was evaluated under a practical beef production setting. The performance of slow release urea was compared to regular feed grade urea in a 42 day receiving study (288 Angus cross steers) as well as a 70 day growing study (240 Angus cross steers). High forage diets increased the ruminal degradation rate of both urea and slow release urea an increased the extent of degradation of slow release urea when compared to high concentrate diets. Lower DIP concentrations in the diet reduced systemic urea production, ruminal ammonia and plasma urea concentrations and urinary urea excretion under most circumstances but also led to a reduction in N retention, reduced diet digestibility, lower feed intake, lower growth rate and decreased feed efficiency. High DIP intakes increased N retention, growth rate, diet digestibility and improved feed efficiency but also lead to increased excretion on urea N in the urine. Slow release urea improved N retention and efficiency of N retention in high DIP diets when compared to urea and generally reduced plasma urea and ruminal ammonia concentrations. Compared to urea, slow release urea did not significantly improve the production of receiving cattle. However Optigen®II improved the feed efficiency when compared to urea on high concentrate diets but reduced feed efficiency on high forage diets.

In the gastrointestinal tract, probiotics have been shown to promote host immunity and to regulate immune signaling pathways. This study used Caco-2 cell line to examine the effects of a Lactobacillus rhamnosus isolate from “amabere amaruranu” a Kenyan traditional cultured milk, on the production inflammatory cytokines in enterocytes. Live Lactobacillus rhamnosus (MRS6AN), its cytoplasmic fraction (CF), filtered spent broth (FSB) or heat inactivated FSB (HIB) were used as treatments on differentiated Caco-2 cell monolayer in transwells. Cytokine content in the cell lysates, apical and basolateral supernatants were determined using ELISA. Caco-2 cell lysate treatments showed significantly increased anti-inflammatory TGF-β (ng/ml) levels on average about 100x more compared to the increase in pro-inflammatory IL-8 (pg/ml) levels. These levels were significantly reduced after inhibition of NF-κB. In conclusion, live Lactobacillus rhamnosus, its CF, FSB or HIB seemed to modulate the production of inflammatory cytokines in enterocytes partly via the NF-κB signaling pathway.

Bacteriophage MooMoo is a temperate phage that was isolated and propagated on Mycobacterium smegmatis (M. smeg). It typically produces turbid plaques, however spontaneous clear plaque mutants can be readily isolated. Both turbid (MooMoo-T) and clear plaque (MooMoo-C) formers can establish stable lysogens, but the parental turbid plaque forming phage has a higher lysogenic frequency. The phage repressor protein typically plays the central role in regulating the lysis/lysogeny decision. Therefore, we expected that the mutation responsible for the clear plaque phenotype would be located in the repressor gene. Remarkably, whole genome sequencing detected a single base pair mutation in the minor tail protein gene (gp19). The regulatory role of the repressor protein could not be excluded considering it was unclear how the mutation in gp19 was leading to the altered plaque phenotype. To locate the phage repressor, we used bioinformatics to identify several candidate genes with helix-turn-helix and DNA binding motifs (gp42, gp43 and gp44). We also cloned the parental and mutant gp19 genes. Each candidate gene was cloned into a shuttle vector. The clones of gp43, gp44 and both derivatives of gp19 did not prevent MooMoo growth, whereas the clones of gp42 inhibited phage growth. Based on these results, we concluded that gp42 is the phage repressor for MooMoo. To determine if the presence of gp19 alters lysogenic frequency, lysogeny assays of wild-type (WT) and mutant gp19 clones were evaluated. Compared to the MooMoo-C lysate, the cloned copy of the mutant gp19 showed a slight increase in lysogeny efficiency. The lysogeny frequencies on strains that carry cloned copies of gp19 (WT or mutant) were similar to those obtained on strains that lacked the plasmids. From these results, we concluded, the presence of either parental or mutant gp19 clones does not affect the lysogeny frequency. To determine if host cell physiology was affected by lysogeny, carbon, nitrogen, phosphorus and sulfur utilization resources were screened using high-throughput phenotypic microarrays. From these results, we concluded the presence of the WT or mutant prophage had no significant effect on the utilization of the resources tested.

Atribacteria (OP9), candidate phylum with no representatives in pure culture, is found in various anaerobic environments worldwide. “Caldatribacterium”, a lineage within Atribacteria that is predicted to be a strictly anaerobic sugar fermenter based on cultivation-independent genomic analyses, is currently being maintained in lab enrichment cultures with fucose as its sole growth substrate. Metagenomics and 16S rRNA gene tag sequencing indicated that the fucose culture was a co-culture of “Caldatribacterium” and an uncultivated member of the genus Thermodesulfobacterium. Due to failed attempts to isolate “Caldatribacterium” by dilution-to-extinction and plating, it was hypothesized that “Caldatribacterium” is dependent in some way on the Thermodesulfobacterium. To better understand the possible interaction, multiple isolates of the sulfate reducer were obtained under sulfate-reducing conditions with H2 as an electron donor, and one of the isolates was characterized. Whole genome and 16S rRNA gene sequence comparisons of the isolate and other related members of the genus Thermodesulfobacterium suggested the isolate represents a distinct species in this genus, for which the name T. auxiliatoris is proposed. T. auxiliatoris was capable of using H2, formate, and lactate as sole electron donors, but not fucose or other sugars, suggesting that its growth in the co-culture might be dependent on one or more fermentation substrates produced by “Caldatribacterium”. Addition of T. auxiliatoris to highly diluted samples of the co-culture that likely contained only “Caldatribacterium”, which did not exhibit growth on their own, demonstrated that T. auxiliatoris was sufficient to support growth of “Caldatribacterium” on fucose. When this dilution experiment was repeated with various other organisms and substrates, it was found that several other thermophilic sulfate reducers (T. commune, T. hveragerdense, or Thermodesulfovibrio yellowstonii) could also support growth, as well as supernatant from the T. auxiliatoris pure culture or yeast extract. This last finding allowed for isolation of “Caldatribacterium”, which could form colonies on solid media when yeast extract and casamino acids were present. Fluorescent in situ hybridization and nanometer-scale secondary ion mass spectrometry demonstrated that “Caldatribacterium” took up a variety of sugars and amino acids in mixed culture, and that addition of acetate or bicarbonate, substrates of T. auxiliatoris, stimulated sugar uptake in “Caldatribacterium”. These results support a model where T. auxiliatoris and “Caldateribacterium” are dependent on each other in co-culture on fucose, where “Caldatribacterium” provides growth substrates for T. auxiliatoris, which in turn provides “Caldatribacterium” with some sort of soluble, essential compound(s) that can be produced by other sulfate reducers and are present in yeast extract. Further characterization of the “Caldatribacterium” isolate, the first representative of the phylum Atribacteria, will allow for detailed study of its metabolic capabilities that can be extended to other members of this phylum. Further analysis of responses of T. auxiliatoris and “Caldatribacteirum” when grow in co-culture and the specific metabolite(s) that are exchanged between the two organisms could allow for testing whether these interactions occur in more complex, natural systems.

Invasive plant species are major threats to the biodiversity and ecosystem stability. The purpose of this study is to understand the impacts of invasive plants on soil nutrient cycling and ecological functions. Soil samples were collected from rhizosphere and non-rhizosphere of both native and exotic plants from three genera, Lantana, Ficus and Schinus, at Tree Tops Park in South Florida, USA. Experimental results showed that the cultivable bacterial population in the soil under Brazilian pepper (invasive Schinus) was approximately ten times greater than all other plants. Also, Brazilian pepper lived under conditions of significantly lower available phosphorus but higher phosphatase activities than other sampled sites. Moreover, the respiration rates and soil macronutrients in rhizosphere soils of exotic plants were significantly higher than those of the natives (Phosphorus, p=0.034; Total Nitrogen, p=0.0067; Total Carbon, p=0.0243). Overall, the soil biogeochemical status under invasive plants was different from those of the natives.