Gram-positive pathogenic bacteria utilize cell-surface anchored proteins to bind and transport heme into the cell. These bacteria acquire iron from host proteins containing heme e.g., hemoglobin. Proteins like HmuT from Corynebacterium diphtheriae bind and help transport heme into the cell. Residues His136 and Tyr235 are utilized as the axial ligands, with a conserved Arg237 residue acting as the hydrogen bonding partner to the axial Tyr235. Similarly, Streptococcus pyogenes utilizes the cell anchored protein Shr to transfer heme into the cell. Shr-NEAT2 is hexacoordinated by two axial methionines and is prone to autoreduction where lysines are the most likely source of electrons. Lastly, PefR of Group A Streptococcus is a DNA transcription factor which regulates protein expression. Preliminary studies indicate a cysteine may coordinate the heme. A combination of UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies shows these proteins play a crucial role heme transport and regulation.

Bacillus thuringiensis (Bt) is a gram-positive common soil bacterium that produces crystals (Cry) containing proteins that are toxic to certain insects, in particular larvae of Lepidoptera and Diptera. The Bt toxin in the past has been widely used as a bioactive compound for the biological control of mainly lepidopteran pests. Most recently a variety of crops, including cotton and maize, have been genetically modified to express a Bt toxin to confer resistance to lepidopteran pests. However, the effect of abiotic environmental stress, such as drought and heat, which are typical for Africa, on Bt toxin expression in a genetically modified crop has so far not been fully evaluated. This study focuses on the expression and stability of the Cry1Ac insecticidal protein from Bacillus thuringiensis in genetically modified cotton plants under drought and heat stress. These include the physiological and biochemical characterization of the expressed Bt toxin gene under drought stress as well as the biological activity against first-instar larvae of the African cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae). Non-genetically modified cotton (Gossypium hirsutum cv. Opal), as well as genetically modified cotton (cv. Nuopal) expressing the Bt toxin Cry1Ac, were exposed to drought and heat stress. Drought stress was induced by withholding watering plants until the soil moisture content reached 25- 30 % of field capacity. Non-stressed control plants were watered and soil moisture content to 80-100 % of field capacity was maintained. For heat stress, plants were grown at 38 to 32 DC during the day and night, respectively, whereas control plants were grown in a growth cabinet at a 28/25 DC day/night cycle. For growth analysis plants were harvested every second week after planting. At each harvest, different parts of the plant were collected and their fresh and dry weight determined. For biochemical analysis and determining biological activity against first-instar larvae of H. armigera, two types of experiments were carried out, the first experiment four weeks after treatment induction and the second experiment eight weeks after treatment induction. Different plant material (leaves, flowers and immature green bolls) were used for Bt detection as well as for determining biological activity against first-instar larvae of H. armigera. Under drought stress conditions a reduction in leaf area and leaf dry weight were found in both Bt toxin expressing and non-expressing cotton plants, but no significant difference in physiological performance between Bt-expressing and non-expressing cotton plants was found. This study shows that the Bt toxin (Cry1Ac) level decreases in senescent plants and that drought stress did not affect the growth and development of genetically modified Bt plants when compared to non-Bt plants. Although the expression of Bt toxin (Cry1Ac) in Bt cotton plants decreased under drought stress no effect on the efficacy of the toxin against H. armigera was observed. In addition, no significant decrease of Bt toxin content was found in Bt cotton leaves after exposure to heat stress when compared to leaves from nonheat stressed plants. / Dissertation (MSc)--University of Pretoria, 2008. / Plant Science / unrestricted

Rapidly emerging strains of bacteria resistant to most advanced antibiotics have become issues of very important public health concern. Research currently directed towards marine actinomycetes presents a vast potential for new compounds that could be able to safely and effectively target resistant species. In this regard, ten putative Streptomyces strains isolated from the Nahoon beach were selected and assessed for antibiotic production and activity against a wide range of bacteria including reference strains, environmental strain and clinical isolates. The ethyl acetate extracts of the putative Streptomyces isolates showed activities against at least 6 and up to 26 of the 32 test bacteria. Inhibition zones were found to range between 9-32 mm diameters at a concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) of the crude extracts ranged from 0.039 - 10 mg/ml and the least minimum bactericidal concentration (MBC) demonstrated was 0.625 mg/ml against a reference strain Staphylococcus aureus ATCC 6538. Time kill kinetics of all extracts revealed bacteristatic and bactericidal activities. Average Log reductions in viable cell counts for all the extracts ranged from 0.86 Log10 and 3.99 Log10 cfu/ml after 3 h interaction and 0.01 Log10 and 4.86 Log10 after 6 h interaction at MIC, 2 × MIC, 3 × MIC and 4 × MIC concentrations. Most of the extracts were speedily bactericidal at 3 × MIC and 4 × MIC resulting in over 50 % elimination of most of the test bacteria within 3 h and 6 h interaction. The partial characterization of the crude extracts by IR spectral analysis revealed possibility of terpenoid, long chain fatty acids and secondary amine derivatives compounds in the extracts. It is therefore recommended that further investigation should address the relationship between the structure of the active component of the extracts and the broad spectrum activity, as well as a rapid method for large scale production and purification and whether this group of antibiotics has any application in managing human infectious disease.

<p><b>Part I: Design of a Photolabile Backbone Amide Linker for the Synthesis of C-terminally Modified Peptides</b></p> <p>A new photolabile backbone amide linker has been developed for the on-resin synthesis of cyclic and C-terminally modified peptides. The linker (Hcnb) is stable to strongly acidic conditions and instead releases the completed peptide through photolytic cleavage at 365 nm. Hcnb possesses four degrees of orthogonality and is amenable to the preparation of cyclic peptides, C-terminally modified peptides, and fully protected peptides due to its photolabile backbone amide linkage. The Hcnb precursor can be conveniently synthesized in 4 steps from commercially available 4-methyl-3,5-dinitrobenzoic acid. The C-terminal amino acid residue is loaded via reductive amination of the precursor followed by an O→N transacylation for the addition of the second residue in quantitative yields, even when employing sterically bulky residues. Standard Fmoc- or Boc-based synthesis can then be utilized to complete the desired peptide. Hcnb has been used to demonstrate the linear synthesis and subsequent on-resin cyclization of various cyclic peptides of interest, as well as synthesis of C-terminal thioesters on-resin. </p> <p><b>Part II: Development of II-HMG CoA Reductase Inhibitors for use as Gram-Positive Selective Antimicrobials. </b></p> <p>Bacterial resistance to antibiotic drugs is an issue that humans have faced since the first use of sulfa drugs in the 1930s. In recent years, the rate of production of new antimicrobial drugs has diminished, as they are no longer financially beneficial to pharmaceutical companies due to short term use and rapid resistance development. This places the burden of the development of new antimicrobial drug on the academic research field. In the work presented here, progress has been made toward the development of a novel class of antimicrobial compounds. These small molecule inhibitors target II-HMG CoA Reductase, a key enzyme involved in cell wall synthesis in gram-positive bacteria. Based on analysis of co-crystal structures obtained from first- and second- generation inhibitors, structural alterations were made to design a new generation of compounds. Efforts have also been made toward identification of a potential secondary target of these inhibitors. </p>

The work in the Brown laboratory has two long-range objectives. Both are derived from an interest in the replication of the genome of Gram-positive eubacteria. One objective is to gain a deeper understanding of the structure and function of DNA polymerase III, the unique species of DNA polymerase which is essential for chromosome replication. The second objective, the one from which this thesis is derived, is to determine whether a selective inhibitor of this DNA polymerase can serve as a basis for producing a new generation of clinically useful Gram-positive-selective antimicrobial agents. The polymerase III-specific inhibitor prototypes investigated in this work are members of a family of simple 6-substituted uracils. The following members of this family, TMAU and EMAU, were used as platforms for the manipulation of the N3 atom (arrow), the only ring component which could be substituted without severe reduction of inhibitory activity. The N3 position was substituted with a series of alkyl groups of increasing size. The resulting structure-activity relationships at the level of the polymerase was consistent with the presence of an N3-specific subdomain within the inhibitor binding site which could accommodate a wide variety of substituents. Although specific alkyl substituents at N3 also significantly enhanced the antibacterial potency of TMAU and EMAU, the respective compounds were found to have insufficient aqueous solubility for successful application in in vivo infection. To increase aqueous solubility, the N3 atom of the EMAU platform was substituted with selected hydroxy- and methoxyalkyl groups. The latter agents retained both anti-polymerase and antibacterial activity, and, as expected, they displayed a combination of lipid and aqueous solubility favorable to efficacy in in vivo infection. Two of the agents, N3-hydroxypropyl- and N3-methoxypropyl-EMAU were examined for their ability to protect mice from lethal staphylococcal infection. Both were found to be active in this model. In sum, the results of this work demonstrated, for the first time, that: (1) the eubacterial replication-specific DNA polymerase III is a valid target for antibiotic development, and (2) the N3-substituted 6-anilinouracil platform has strong potential as a basis for novel antibiotics useful against Gram-positive bacterial infection.

Thirteen bacterial cultures were isolated from the North arm of Great Salt Lake during the months of January and February of 1973. Eleven isolates were gram-negative pleomorphic rods which lysed in hypotonic solution. The remaining two were gram-positive cocci. All isolates and one known strain of Halobacterium salinarium were subjected to examination of morphological, cultural, physiological, and biochemical characteristics. A numerical taxonomic analysis was applied to the compiled characters to compute a coefficient of similarity for each individual isolate as compared to all other isolates. A comparative analysis was included in the similarity computation using characters assembled from those reported in the literature for six taxonomically accepted species of halophilic bacteria. The lake isolates proved to be extreme halophiles with relative high levels of similarity between each other and the known bacteria included in the numerical analysis.

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This investigation is concerned with the testing, in vitro, of oleoresins from one hundred higher plants common to North and South Central Texas. The plants used were selected in order to obtain a representative collection which might be of value in future research.