Experiments were conducted to determine if phagocytosis-associated oxidative metabolic activity was responsible for the enhanced listericidal action of immunologically activated macrophages. Investigations into production of oxygen radicals by macrophages revealed that Listeria-immune antigen boosted macrophages produced significantly more superoxide, hydrogen peroxide, and chemiluminescence (CL) than resident, thioglycolate, or Listeria antigen elicited cells. Prolonged in vitro culture of immune boosted cells diminishes their long-term killing capacity with a parallel decrease in hydrogen peroxide production. Similarly, immune elicited macrophages cultured in excess of 29 h lose short-term listericidal activity with a concurrent diminution of CL. Scavengers of oxidative metabolites were found to be ineffective in significantly reducing the killing of Listeria in immune elicited cells. Resident murine peritoneal macrophages were rapidly rendered listericidal following exposure to lymphokine rich supernatants (LRS) derived from antigen-pulsed Listeria monocytogenes immune spleen cells. Subsequent examination of these cells showed that lymphokine activation failed to enhance the production of oxygen species. Additionally, quenchers of oxygen metabolites were unable to reduce lymphokine mediated listericidal action in resident macrophages. The production of superoxide and hydrogen peroxide by immune boosted macrophages in response to opsonized viable and heat-killed virulent Listeria monocytogenes A4413 was insignificant. In contrast, when immune elicited macrophages were exposed to Listeria A4413 a generous chemiluminescent response was noted. Similar results were obtained using avirulent Listeria monocytogenes 9037-7 and S. aureus 502A. The generation of oxidative metabolites by strains of Listeria and S. aureus 502A was also examined. Production of oxygen derived radicals was found to closely correlate with bacterial virulence. Virulent Listeria monocytogenes A4413 generated copious quantities of superoxide and hydrogen peroxide, while the avirulent Listeria 9037-7 and S. aureus 502A produced these species in greatly diminished amounts.

The involvement of tampons in development of toxic shock syndrome (TSS) was investigated in three stages. First, toxic shock syndrome toxin-1 (TSST-1) was isolated from a known TSST-1 producing strain of Staphylococcus aureus. Iodinated toxin was identified by reaction with specific anti-TSST-1 antibody and resolution of the antibody-adsorbed protein by SDS-PAGE and autoradiography. Additional toxin was purified by ion exchange chromatography on CM sephadex C25 or Rexyn 102 cation exchange resins followed by molecular exclusion chromatography. Each method yielded a purified toxin with a characteristic molecular weight of 22,000 daltons and a pI of 7.0. Purified TSST-1 was used to produce specific antiserum in a goat. Resulting immune IgG was used as a ligand for affinity purification of toxin. Secondly, production of TSST-1 in the presence of tampon fibers and magnesium (Mg$\sp{++}$) was studied in vitro. Polyester foam (PEF, Rely tampon) and polyacrylate rayon (PAR), two fibers more frequently associated with TSS stimulated TSST-1 production while cotton and rayon did not. The influence of Mg$\sp{++}$ appears to be different on PEF and PAR, PAR chelated Mg$\sp{++}$ from aqueous solutions and brain heart infusion broth (BHI) while PEF did not. Addition of Mg$\sp{++}$ had a direct effect on toxin production in the presence of PAR and had a modulating effect in PEF/BHI cultures. Lack of aeration decreased the amount of toxin produced in the presence of PEF but had no effect on toxin produced with PAR or cotton. Finally, intravaginal inoculation of guinea pigs with TSS associated strain MN-8 and PEF or cotton induced comparable levels of TSST-1 production in contrast to in vitro. Only the animals that had been implanted with PEF developed a serum antibody response to TSST-1 as measured by RIA and confirmed by immunoblot. This indicates a fiber specific enhancement of the humoral response and may have implications as to the effect of certain fibers on the development of toxic shock syndrome.

A gene encoding a B-xylosidase was cloned from a xylanolytic Clostridium via pBR322 into Escherichia coli. The gene was localized to a 3.1-kilobase EcoRI fragment. Either orientation of the fragment in pBR322 allowed for expression of B-xylosidase activity, indicating that the Clostridium promoter is utilized in E. coli. This activity was cell-associated and not inducible. B-Xylosidase was purified from cell extract of the recombinant strain E. coli DH5 (pIW1) by ammonium sulphate fractionation, hydrophobic interaction chromatography, ion exchange chromatography, and preparative isoelectric focusing. The molecular weight of B-xylosidase, as estimated by gel filtration, was 204,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified enzyme showed a single protein band with a molecular weight of 54,000. The isoelectric point of B-xylosidase was 4.8. B-Xylosidase activity was optimal at pH 6.5 and 55$\sp\circ$C. The enzyme was stable between pH 5.0 and 9.5 for 120 minutes at 20$\sp\circ$C and retained full activity at pH 6.0 after incubation at 50$\sp\circ$C for 30 minutes. Enzyme activity was totally inhibited by 1 mM Hg$\sp{2+}$, Cd$\sp{2+}$, Cu$\sp{2+}$, or Ni$\sp{2+}$. B-Xylosidase hydrolyzed p-nitrophenol-B-D-xylopyranoside (pNPX) and was slightly active on p-nitrophenol-B-D-galactopyranoside and p-nitrophenol-alpha-L-arabinopyranoside. With pNPX as substrate, the Km and Vmax values were 0.15 mM and 1.9 units/mg, respectively. Purified enzyme exhibited transferase activity with the substrate xylobiose. This activity was also detected with pNPX as substrate in the presence of methanol as a xylosyl acceptor.

Bibliography: pages 144-161. / A Tn7-like element was found in a region downstream of a cosmid (p818.1) isolated from a genomic library of Thiobacillus ferrooxidans ATCC 33020. A probe made from the Tn7-like element hybridized to restriction fragments of identical size from both cosmid p818.1 and T ferrooxidans chromosomal DNA. The same probe hybridized to restricted chromosomal DNA from two other T ferrooxidans strains (ATCC 23270 and 19859). There were no positive signals when an attempt was made to hybridize the probe to chromosomal DNA from two Thiobacillus thiooxidans strains (ATCC 19733 and DSM504) and a Leptospirillum ferrooxidans strain DSM 2705. A 3.5 kb BamHI-BamHI fragment was subcloned from p818.1 downstream the T ferrooxidans unc operon and sequenced in both directions. One partial open reading frame (ORFl) and two complete open reading frames (ORF2 and ORF3) were found. On the basis of high homology to previously published sequences, ORFI was found to be the C-terminus of the T ferrooxidans glmU gene encoding the enzyme GlcNAc I-P uridyltransferase (EC 1.7.7.23). The ORF2 was identified as the T ferrooxidans glmS gene encoding the amidotransferase, glucosamine synthetase (EC 2.6.1.16). The third open reading frame (ORF3) was found to have very good amino acid sequence homology to TnsA of transposon Tn7. Inverted repeats very similar to the imperfect inverted repeat sequences of Tn7 were found upstream of ORF3. The cloned T ferrooxidans glmS gene was successfully used to complement an E.coli glmS mutant CGSC 5392 when placed behind a vector promoter, but was otherwise not expressed in E.coli.

Aphid lethal paralysis virus (ALPV) could be detected by indirect immunofluorescent technique in dissected aphids. This technique was found to be more sensitive when compared to DAS-ELISA. The choice of a sensitive, low cost .detection method was of importance to test for low levels of virus in infected aphid body tissues where inapparent infection could cause detection problems. ALPV was visualized in ultrathin sections of diseased aphid body tissues by immunocytochemistry utilizing immunogold label. ALPV antigen was detected in the ovariole tissue, tracheocytes, symbionts of the mycetocytes, fat body cells, brain tissue, nerve tissue and stomach epithelial tissue. Virions were detected predominantly in the cytoplasm but were also found in the nucleus. ALPV antigen was not detected in muscle fibres or mitochondria. ALPV and Rhopalosiphum padi virus (RhPV) are transmitted transovarially. Different incidences of transmissions of ALPV were obtained for R. padi (2996) and Sitobion avenae (1696) and ALPV infections dramatically reduced the longevity and fecundity of these aphids. Infected apterous R. padi aphids were more fecund than alate aphids of the same clone. The percentage of viral infections in different aphid species (R. padi, S. avenae and Diuraphis noxia) was positively associated with temperature; higher temperatures dramatically increased the incidence of ALPV and RhPV and vice versa. The influence of ALPV on a natural R. padi aphid population was found to reduce the population size by 4996. This reduction coincided with a high death factor (70) of aphids per plant. A dramatic decline in R. padi aphid numbers and a high incidence of ALPV present in this aphid population was experienced. Parasitic fungal infections peaked at a later stage than ALPV, and a level of 21 parasitized aphids per plant was reached during this period. This appears to indicate that the presence of ALPV contributes to limit population development in R. padi aphids. Similar results were obtained with S. avenae aphids. Based on this data, ALPV could be considered as a major growth limiting factor in the development of small grain aphid populations in the western Cape. If the presence of the virus is taken into consideration, it could influence pest management strategies directly.

Includes bibliography. / This study investigated the feasibility of biolistic transformation for Ornithogalum. Two PDS 1000/He biolistic guns were used in independent studies, one in 1995 at the Floral and Nursery Plants Research Unit of the National Arboretum of the United States Department of Agriculture (USDA) and the other in 1996 at the Microbiology Department, University of Cape Town (UCT). In the study at the UCT, the Taxi system (Chen et al 1998) was also used in addition to conventional biolistics to determine which method was most effective in stable transformation studies with O. thyrsoides x O. dubium hybrid, designated A2.

Two component systems (TCSs) are one of the predominant methods for bacteria to detect and respond to environmental change. A typical TCS consists of a histidine kinase (HK), responsible for sensing inter- and intracellular changes, and a response regulator (RR) which as the name suggests responds to the detected stimuli, mostly by regulating DNA expression. In this project two predicted domains (Response_reg-like and HEF_like) were detected in 628 bacteria strains which share a high similarity to typical HKs and RRs with a 91.6% linkage observed across 644 genomic regions. The adjacent co-localisation between these genes carrying the novel predicted domains in addition to the conserved structural features of typical HKs and RRs, indicate that the Response_reg-like and HEF_HK-like domains could incorporate a new TCS in bacteria. With these HEF_HK-like and Response_reg-like domains, an overrepresented presence of restriction modification (RM) systems, especially type II RM was observed. The established presence of RM systems in the GHKL family domain HATPase_c_3 across most of the HEF_HK-containing genes could indicate an evolutionary relationship with the paraMORC family of ATPases. The aim of this project was to functionally characterize the putative HK and ultimately the entire novel TCS in Pectobacterium carotovorum subsp. brasiliense 1692 and optimise mutation analysis protocols for effective use in this enterobacteria species. / Dissertation (MSc (Microbiology))--University of Pretoria, 2020. / This research was funded by the National Research Foundation (NRF), South Africa through Competitive Funding for Rated Researchers (CFRR) 98993. W.J.S.P. studentship was supported by The Research Technology Fund (RTF) 98654. D.B-R. was supported by The University of Pretoria Post-Doctoral Fellowship. / Microbiology and Plant Pathology / MSc (Microbiology) / Restricted

abstract: Structural details about viruses and their components are important for understanding the many steps in a virus life-cycle, including entry into host cells, replication, assembly, and release of progeny virions. X-ray crystallography and electron microscopy, including cryo-EM, have been used extensively for virus structural studies. Recent advances with cryo-EM have significantly advanced the field with near-atomic resolution structures of viruses being achievable. X-ray free-electron lasers (XFELs) are a novel, developing method to solve structures for non-crystalline single particle targets like viruses. Diffraction patterns can be collected directly from particles at room temperature. High quality, homogeneous virus preparations are critical for both cryo-EM and XFEL studies. Thus, optimization of virus growth and sample preparation are important steps in virus structural studies. The work described in this thesis focused on optimization of protocols for growth and purification of bacteriophage PR772 for XFEL and complementary cryo-EM studies. PR772 is one of several model viruses used in the single particle initiative (SPI) experiments at the SLAC National Laboratory Accelerator Laboratory Linac Coherent Light Source (LCLS). SPI is a collaborative international effort that works towards identifying and solving challenges of high-resolution single particle imaging using XFELs. Single particle diffraction snapshots were collected from PR772 particles prepared with optimized protocols. PR772 preparations were also used for cryo-EM imaging, with the goal to obtain a high-resolution structure of the virus. The optimization and characterization employed to assure samples suitable for XFEL and cryo-EM are detailed, along with data collected with both approaches. / Dissertation/Thesis / Masters Thesis Microbiology 2019

Vpr, one of the four accessory proteins in HIV-1, is expressed in all primate lentiviruses, and has been shown to facilitate viral replication through degradation of various host factors in a DCAFCrl4 E3 ubiquitin ligase dependent manner and induce a G2/M cell cycle arrest. But the underlying mechanism of Vpr-dependent replication enhancement has remained unclear. Previous studies from our laboratory and in the literature have demonstrated the ability of HIV-1 Vpr to mediate enhanced gene expression from the viral long terminal repeat (LTR) in a DCAFCrl4-dependent manner. In recently published studies, Vpx, a homolog of Vpr, expressed by HIV-2/SIVmac/SIVsm lineage of lentiviruses has been shown to enhance proviral gene expression through degradation of components in Human Silencing Hub (HUSH) complex by hijacking the DCAF1 mediated ubiquitin proteasome pathway. The aim of this study is to determine if HIV-1 Vpr has the same role as Vpx in promoting viral expression through degradation of HUSH complex components. I specifically focused on the role of Vpr in mediating enhanced viral gene expression from unintegrated viral DNA. Unintegrated viral DNA can be present as 1-LTR or 2-LTR circles when viral cDNA integration is blocked by use of integrase inhibitors, such as Raltegravir, a commonly used drug in HIV treatment regimens. To assess Vpr’s role, I created HeLa cell lines in which expression of HUSH components, including the upstream SETDB1 protein, was diminished by use of targeted shRNAs. After assessing the knockdown efficiency using RT-qPCR, the cell lines are infected with luciferase expressing Vpr null (ΔVpr) or Wildtype (WT) HIV-1 that are normal or defective for viral DNA integration capability. Measurement of luciferase activity in infected cell lysates provided a quantitative measure of expression from viral DNA. Expression from unintegrated viral DNA was attenuated in the absence of Vpr compared to WT HIV-1. Furthermore, knockdown of HUSH complex subunits expression did not rescue luciferase reporter gene expression from unintegrated viral DNA in Vpr-null virus infections, suggesting that Vpr-mediated enhancement of viral gene expression from unintegrated viral DNA is not dependent on targeting HUSH complex functions. Future studies will be needed to fully understand the mechanism by which Vpr facilitates viral gene expression from unintegrated viral DNA.

Bibliography: pages 188-209. / Woodiness disease caused by virus infection is the most serious virus disease of passionfruit and affects economic production of this crop worldwide. A preliminary survey of diseased Passiflora material collected from various regions in South Africa revealed the presence of at least three different viruses. A diseased P. caerulea rootstock specimen from a woodiness diseased vineyard in Natal was selected as a source for isolation and further characterization of viruses. Two viruses that were present in a mixed infection were isolated and purified from this material: a spherical virus which appeared to be cucumber mosaic virus (CMV) and a filamentous virus which was initially presumed to be an isolate of passionfruit woodiness virus (PWV). The host range, transmission and prevalence of these viruses were studied by employing techniques such as electron microscopy (negative staining and immunosorbent), electroblot immunoassay, double antibody sandwich enzyme-linked imunoassay and nucleic acid hybridization. In transmission studies, the CMV-isolate and the potyvirus were found to be sap, aphid and graft transmissible. Separation of the two viruses was achieved by passage through a selective host range.