Includes bibliographical references (leaves 135-154). / The objective of this study was to identify a potential probiotic bacterium that increased the growth and decreased the susceptibility of farmed abalone to pathogenic bacterial infection. The mechanism by which the probiotic is able to increase growth rates and reduced susceptibility to pathogen infection was investigated. A number of bacterial strains were isolated from the digestive tract of Haliotis midae that are capable of degrading a wide variety of different polysaccharides (Erasmus, 1996). Strain C4 was selected for further investigation as a result of its ability to degrade alginate since H. midae is predominantly fed a kelp diet of which the major component is alginate.

Includes bibliographical references. / Angiotensin converting enzyme is an ectoprotein prone to regulated proteolytic solubilisation by an as yet unknown protease or sheddase. Proteolytic cleavage of membrane proteins is an essential cellular process that controls their expression and function, and modulates cellular and physiological processes. Testis ACE (tACE) is shed at a higher rate than somatic ACE and it has been proposed that regions in its ectodomain direct its shedding. Discrete secondary structures on the surface of the distal ectodomain of tACE were replaced with their N-domain counterparts to determine their role in the ectodomain shedding of ACE. None of the regions investigated proved to be an absolute requirement for shedding, but the mutant ACE proteins were subject to variations in shedding compared to wild-type tACE. To investigate the role of the proximal ectodomain in shedding the residues H610-L614 were mutated to alanines, causing a decrease in shedding. An extension of this mutation on the N-terminal side to seven alanines resulted in a reduction in ACE activity and, more importantly, it affected the processing of the protein to the membrane, resulting in expression of an underglycosylated form of ACE. When E608-H614 was mutated to the homologous region of the N-domain, processing was normal and shedding only marginally reduced. These data suggest that this region is more crucial for the processing of ACE than is for regulating shedding. Construction of a P628L mutation in tACE showed an increase in shedding. Furthermore, MALDI analysis of a tryptic digest established that the putative glycosylation site N620WT became glycosylated. Further mutagenesis of the P628L mutant to remove the newly formed glycosylation site, resulted in an even greater increase in shedding. Soluble fluorogenic peptides mimicking the ACE stalk were used in a cell-based assay to characterise the contribution of the stalk to ACE shedding. Hydrolysis of the wild-type peptide Abz-NSARSEGPQ-EDDnp was not responsive to phorbol ester or the hydroxamate inhibitor (TAPI), however, it was inhibited by EDTA. The aminopeptidase inhibitor bestatin did not inhibit cleavage or alter the cleavage site. Therefore the protease involved in the cleavage of the ACE stalk peptides is likely different to the sheddase responsible for ACE shedding. Substitution of the P1 and P1' sites of the peptides did not significantly influence the rate of cleavage. All the peptides were cleaved at the E-G bond, which is C-terminal to the physiological R-S cleavage site. Removal of the fluorogenic capping groups resulted in no cleavage of the peptides and lengthening of the peptide did not result in cleavage. This confirms the need for the ACE sheddase and its substrate to be anchored in the membrane and suggests the use of soluble peptide substrates in a cell assay has limited application for investigating the ectodomain shedding of ACE.

To investigate the structural determinants of NitN specificity on short aliphatic amide substrates by analyzing binding and interactions of these molecules with the NitN binding pocket. To probe the catalytic role of the two active site glutamate residues (Glu61 and Glu139) using NitN as a model enzyme. To monitor the activity, interactions and reactivity of the WT NitN and the Glu61 and Glu139 NitN mutants with ACR.

Includes abstract. Includes bibliographical references (leaves 170-198).

Bibliography: leaves 101-115. / Bacteroides fragilis is a gram-negative obligate anaerobe that exists normally in the human large intestine. It is, however, capable of causing a variety of infections once outside of this environment. Previous studies have suggested that the regulation of the ammonia assimilatory pathways in B. fragilis occurs in a manner distinct from that found in other gram-negative bacteria, with glutamate dehydrogenase (GDH) activity serving as the primary route of ammonia incorporation in both ammonia-limited and ammonia-excess environments. While these studies showed that B. fragilis produces two distinct GDH enzymes, one NAD(P)H-dependent (GdhA) and the other NADH-dependent (GdhB), their specific roles in nitrogen assimilation were not clearly resolved. In this dissertation, the physiological and molecular factors affecting the regulation of GDH activity in B. fragilis were examined, with a view to extending our understanding of nitrogen assimilation in this organism. Physiological analysis revealed that activities of the two B. fragilis GDH enzymes were differently regulated in response to both the nitrogen source (ammonia or peptides) and availability in the growth medium. GdhA activity was present in B. fragilis cells during growth in both ammonia-limited and ammonia-excess environments. Its activity was, however, found to be greatest in ammonia-limited cultures, and was down-regulated when peptides replaced ammonia as the sole nitrogen source for growth. GdhB activity, by contrast, was up-regulated following growth with peptides, but not ammonia, as the sole nitrogen source. These findings, taken together, suggest that the two GDH enzymes fulfil distinct functions in nitrogen nutrition in B. fragilis. The gdhB structural gene was cloned via heterologous complementation of an E. coli glutamate auxotroph. The gene was found to encode a deduced polypeptide of 445 amino acid residues with homology to previously studied GDH enzymes. Northern blot analysis showed that the gdhB gene is transcribed as a monocistronic 1.5 kb mRNA, and that the regulation of GdhB activity occurs at the level of transcription. The transcriptional regulation of the gdhB gene was further examined by primer extension analysis, and transcriptional fusions using a xylosidase reporter gene. The results demonstrated that gdhB gene expression occurs from an inducible promoter located upstream of the gdhB gene, with transcription being induced by the increased availability of peptides in the growth medium. Deletion analysis of the gdhB promoter region resulted in the identification of several remote sequence-elements that may be involved in the transcriptional activation of this gene. The gdhB gene was expressed in E. coli, and the recombinant enzyme (rGdhB) purified. Immunoblot analysis, using antiserum raised against the rGdhB enzyme, identified a single immunoreactive protein in cell free extracts obtained from B. fragilis grown with high concentrations of organic nitrogen. Experiments designed to establish whether the GdhB enzyme was regulated at the post-translational level, showed that the activity of the pre-existing enzyme was modulated by rapid inactivation in response to a sudden decrease in peptide availability in the growth ""medium. Analysis of fractionated B. fragilis cells demonstrated that GdhB activity was predominantly associated with the membrane fraction of the cell. The presence of GdhB at the B. fragilis cell-surface was further confirmed by immunogold labelling of B. fragilis cells, followed by electron microscopy. The cell-surface localisation of this enzyme may facilitate the utilisation of glutamate, derived from peptides incorporated from the external milieu.

Studies on resurrection plants and other anhydrobiotic organisms show expression of Late Embryogenesis Abundant (LEA) proteins associated with desiccation tolerance. However, the precise role of these proteins has not been described. This study was undertaken to investigate expression, structure and function of XhLEA1-4S1 and XhLEA1-1S2, Group 1 LEA proteins from Xerophyta humilis, in order to shed light on their role in desiccation tolerance. Complementary DNA (cDNA) of these XhLEAs were cloned into bacterial expression vectors and the recombinant proteins expressed in E. coli. Antibodies were generated and used in determination of expression conditions and immunolocalization studies.

Includes abstract. / Includes bibliographical references. / Rhodococcus rhodochrous ATCC BAA-870 (BAA-870) had previously been isolated on selective media for enrichment of nitrile hydrolysing bacteria. The organism was found to have a wide substrate range, with activity against aliphatics, aromatics, and aryl aliphatics, and enantioselectivity towards beta substituted nitriles and beta amino nitriles, compounds that have potential applications in the pharmaceutical industry. This makes R. rhodochrous ATCC BAA-870 potentially a versatile biocatalyst for the synthesis of a broad range of compounds with amide and carboxylic acid groups that can be derived from structurally related nitrile precursors. The selectivity of biocatalysts allows for high product yields and better atom economy than nonselective chemical methods of performing this reaction, such as acid or base hydrolysis. In order to apply BAA-870 as a nitrile biocatalyst and to mine the organism for biotechnological uses, the genome was sequenced using Solexa technology and an Illumina Genome Analyzer. The Solexa sequencing output data was analysed using the Solexa Data Analysis Pipeline and a total of 5,643,967 reads, 36-bp in length, were obtained providing 4,273,289 unique sequences. The genome sequence data was assembled using the software Edena, Velvet, and Staden. The best assembly data set was then annotated automatically using dCAS and BASys. Further matepaired sequencing, contracted to the company BaseClear® BV in Leiden, the Netherlands, was performed in order to improve the completeness of the data. The scaffolded Illumina and mate-paired sequences were further assembled and annotated using BASys. BAA-870 has a GC content of 65% and contains 6997 predicted protein-coding sequences (CDS). Of this, 54% encodes previously identified proteins of unknown function. The completed 5.83 Mb genome (with a sequencing coverage of 135 X) was submitted to the NCBI Genome data bank with accession number PRJNA78009. The genome sequence of R. rhodochrous ATCC BAA-870 is the seventh rhodococcal genome to be submitted to the NCBI and the first R. rhodochrous subtype to be sequenced. An analysis of the genome for nitrile

Includes abstract. / Includes bibliographical references. / This thesis reports the first assessment of OR variation in Bathyergidae, and therefore, the first for a family of subterranean mammals. Using a PCR-sequencing approach, 178 unique OR sequences, corresponding to 119 unique OR genes are characterised from 14 mole-rat species. Bathyergidae OR genes are classified using sequence similarity and phylogenetic comparison with more than 50 mammalian OR subgenomes.

Includes bibliographical references.

Includes bibliographical references. / Progression of the HIV disease is characterised by a massive depletion of CD4+ T cells and it has been shown that patients living with a more advanced HIV infection have a higher risk of developing diarrhoea due to the disruption of the gastrointestinal microbiota caused by either the HIV-infection or the use of antibiotics and drugs such as highly active antiretroviral therapy (HAART). An imbalance in the microbial composition, attributable to a disturbed mucosal barrier, as well as increased permeability and inflammation caused by HIV, can influence the metabolic (carbohydrate fermentation) and protective functions provided by the microbiota. The effect of HIV on the intestinal microbiota has not been widely examined and those studies that have focused on HIV and the gastrointestinal tract, have investigated it mainly from a virological perspective. Consequently, the aim of the study was to ascertain whether the diversity and/or abundance of the endogenous intestinal microbiota of South African HIV-positive patients was disrupted on account of HIV within the gastrointestinal tract. An additional aim was to determine whether the administration of HAART affected the microbiota during a 6 month longitudinal study. The diversity of the intestinal microbial composition was characterised with respect to the total bacteria, Bifidobacterium and Lactobacillus species using PCR-DGGE. qPCR was used to determine the abundance of total bacteria, Bifidobacterium, Lactobacillus, Escherichia coli, the Bacteroides/Prevotella, Clostridium coccoides and Clostridium leptum groups. ... In addition, three potential intestinal pathogens (Clostridium difficile, Campylobacter jejuni and Salmonella enterica) were monitored by qPCR during this period, to determine their prevalence in the HIV-positive patients.