Development of PCR-based detection assays for Legionella pneumophila in water

Please read the abstract on p 79-80 of this documentDissertation (MSc Agric (Microbiology))--University of Pretoria, 2005.Microbiology and Plant Pathologyunrestricte

Identification and characterization of glycoside hydrolase family 32 enzymes from Aspergillus niger

This thesis focuses on the identification and characterization of sucrose and fructan modifying enzymes present in the genome of the industrially important filamentous fungus, Aspergillus niger. In addition to three known activities, encoded by the genes suc1 (invertase activity; designated sucA), inuE (exo-inulinase activity) and inuA/inuB (endo-inulinase activity), two new putative intracellular invertase-like proteins were identified (designated sucB and sucC; Chapter 2, Yuan et al., 2006; Pel et al., 2007). These enzymes were similarly regulated and all strongly induced on sucrose and inulin. sucB, but not sucC, is expressed at a low level, and up-regulated in sucrose- or inulin-containing media. Biochemical analysis of heterologously expressed SucB indicated that it is an invertase with high substrate affinity for sucrose (Chapter 3, Goosen et al., 2007). Disruption of the sucB gene in A.niger indicated that it was not essential for sucrose or fructan metabolism; however, it might be (in)directly responsible for the observed early onset of sporulation. The presence of SucB homologues in other fungal species indicates that these proteins may all play an important, but not essential role in the intracellular metabolism of sucrose (Chapters 2 and 3; Yuan et al., 2006; Goosen et al., 2007). Heterologous expression, purification and characterization of the extracellular exo-inulinase AngInuE (InuE) showed that his monomeric enzyme completely hydrolyses inulin, levan and sucrose to free fructose and glucose. AngInuE also produces small amounts of inulin-, levan and neo-series inulo-oligosaccharides from sucrose, 1-kestose and nystose (Chapter 4, Goosen et al., submitted for publication). Finally, site-directed mutagenesis in the putative inulin-binding domain (Ser469, domain G) of AngInuE indicated that this domain was essential for activity of this enzyme (Chapter 4, Goosen et al., submitted for publication)

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis

Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger

As a soil fungus, Aspergillus niger can metabolize a wide variety of carbon sources, employing sets of enzymes able to degrade plant-derived polysaccharides. In this study the genome sequence of A. niger strain CBS 513.88 was surveyed, to analyse the gene/enzyme network involved in utilization of the plant storage polymer inulin, and of sucrose, the substrate for inulin synthesis in plants. In addition to three known activities, encoded by the genes suc1 (invertase activity; designated sucA), inuE (exo-inulinase activity) and inuA/inuB (endo-inulinase activity), two new putative invertase-like proteins were identified. These two putative proteins lack N-terminal signal sequences and therefore are expected to be intracellular enzymes. One of these two genes, designated sucB, is expressed at a low level, and its expression is up-regulated when A. niger is grown on sucrose- or inulin-containing media. Transcriptional analysis of the genes encoding the sucrose- (sucA) and inulin-hydrolysing enzymes (inuA and inuE) indicated that they are similarly regulated and all strongly induced on sucrose and inulin. Analysis of a ΔcreA mutant strain of A. niger revealed that expression of the extracellular inulinolytic enzymes is under control of the catabolite repressor CreA. Expression of the inulinolytic enzymes was not induced by fructose, not even in the ΔcreA background, indicating that fructose did not act as an inducer. Evidence is provided that sucrose, or a sucrose-derived intermediate, but not fructose, acts as an inducer for the expression of inulinolytic genes in A. niger.

Molecular and Biochemical Characterization of a Novel Intracellular Invertase from Aspergillus niger with Transfructosylating Activity

A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger. The sucB gene was expressed in Escherichia coli and an invertase-negative strain of Saccharomyces cerevisiae. Enzyme purified from E. coli lysate displayed a molecular mass of 75 kDa, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Its optimum pH and temperature for sucrose hydrolysis were determined to be 5.0 and 37 to 40°C, respectively. In addition to sucrose, the enzyme hydrolyzed 1-kestose, nystose, and raffinose but not inulin and levan. SucB produced 1-kestose and nystose from sucrose and 1-kestose, respectively. With nystose as a substrate, products up to a degree of polymerization of 4 were observed. SucB displayed typical Michaelis-Menten kinetics with substrate inhibition on sucrose (apparent K(m), K(i), and V(max) of 2.0 ± 0.2 mM, 268.1 ± 18.1 mM, and 6.6 ± 0.2 μmol min(−1) mg(−1) of protein [total activity], respectively). At sucrose concentrations up to 400 mM, transfructosylation (FTF) activity contributed approximately 20 to 30% to total activity. At higher sucrose concentrations, FTF activity increased to up to 50% of total activity. Disruption of sucB in A. niger resulted in an earlier onset of sporulation on solid medium containing various carbon sources, whereas no alteration of growth in liquid culture medium was observed. SucB thus does not play an essential role in inulin or sucrose catabolism in A. niger but may be needed for the intracellular conversion of sucrose to fructose, glucose, and small oligosaccharides

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis