Role and regulation of glutamate dehydrogenase activity in Bacteroides fragilis bf1

Doctoral Thesis


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University of Cape Town

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.

Bibliography: leaves 101-115.