No description available.

No description available.

A major objective of biodiversity-ecosystem functioning (BDEF) research is to determine the consequences of species loss, caused both naturally and anthropogenically, on the functioning of ecosystems. The impact of plant species loss on the soil microbial community has not received much attention even though soil microbes influence many important ecosystem functions such as decomposition and nutrient cycling. The objective of this research was to investigate how the functional group composition of the aboveground plant community influenced the belowground microbial community. Plant functional groups (graminoids, legumes and non-leguminous forbs) were removed from a northern grassland system in the Yukon Territory, Canada. One metre square plots had one of the three functional groups removed or left intact as a control and this was crossed with a fertilizer treatment and a fungicide treatment that targeted mycorrhizal fungi. After five seasons (2003-07) of implementing treatments the soil microbial community was analyzed using substrate-induced respiration (SIR, a measure of metabolic diversity) and phospholipid fatty acid analysis (PLFA, a measure of community composition). Plant functional group removal had almost no effect on the soil microbial community. The only response detected was an increase in stress (indicated by the PLFA stress ratio of cy19:0 to 18:1ω7c) which occurred when legumes were removed and fertilizer was not added, indicating that legumes had a positive effect on the nutrient status of microbes. Likewise, soil properties (total carbon, pH, moisture and nutrients) showed limited response to plant removals. Fertilization decreased the metabolic diversity of the soil microbial community. We detected no soil microbial or plant biomass response to the fungicide indicating that mycorrhizae had little influence in this system. Based on the low-productivity of the grassland, and the lack of response in both the soil properties and the microbial community, we hypothesize that the main determinants of the microbial community may be litter input. When litter decomposition rates are slow, such as in this northern system, five growing seasons may not be sufficient to detect the impact of a changing plant community on the soil microbes.

No description available.

No description available.

The interesterification yield (IY) and changes in fatty acid positional distribution of selected butter fat triacylglycerols were investigated, using a wide range of commercial microbial lipases and organic solvent media. The interesterification of butter fat by lipase from Mucor miehei was carried out in hexane, hexane-chloroform, and hexane-ethyl acetate; the results showed that the addition of 30% of either chloroform or ethyl acetate to the hexane resulted in a 23% increase in the IY. The interesterification of butter fat in a microemulsion co-surfactant system, containing Brij 35 as surfactant and 1-heptanol as co-surfactant, resulted in an increase in the triacylglycerols that contain C18:0 at sn-2 position, located originally at sn-1,3 positions, with a concomitant interchange with C14:0 and C18:1 at the same position. The interesterification of butter fat by lipase from Rhizopus niveus, in a phosphatidylcholine reverse micellar system, showed an increase in C16:0 at the sn-2 position, with a concomitant decrease in the proportion of small chain fatty acids (C4-C10:0); however, the interesterification of butter fat in co-surfactant free microemulsion systems, containing hexane and ionic (phosphatidylcholine) and non-ionic (sorbitol monostearate and polyoxyethylene sorbitan monostearate) surfactants, showed that the interesterified selected triacylglycerols were enriched with C18:0 and C18:1, originally located on sn-1,3 position, at sn-2 position with concomitant interchange with C12:0, C14:0 and C16:0, originally located at the same position. The interesterification of butter fats, in co-surfactant free microemulsion system, by four microbial lipases showed that those catalyzed by lipase from R. niveus demonstrated a 46% increase in the proportion of C18:1 at sn-2 position whereas those catalyzed by enzymes from M. javanicus, R. delemar and M. miehei were enriched with C16:0 at the same position, by 21%, 35% and 41%, respectively. In addition, lipase from

Microbial and chemical contamination of inland and coastal waters in Southern Africa is a major challenge facing the water industry and regulatory authorities. Increased stresses on these surface water resources through human and environmental influences have resulted in deteriorating water quality that has severely encumbered the country’s capability to provide sufficient water to meet its needs and to ensure environmental sustainability. In addition, indiscriminate use of antibiotics has resulted in widespread contamination of surface waters, leading to accelerated development of antibiotic resistance and proliferation of resistant water-borne diarrhoeal-related pathogens, such as Escherichia coli and Vibrio cholerae. Despite the high level of contamination of South African surface waters, the microbiological quality of rivers and beaches in Durban, South Africa, have not been adequately investigated. Therefore, the current study assessed the seasonal fluctuations of the microbial and physicochemical quality of two rivers (Umgeni River and Umdloti River) and six beaches (Virginia Aerodome, Beachwood, Umgeni South, Battery, Sunkist, Addington) in Durban, using several bacterial indicator organisms and physico-chemical parameters as indices. The antibiotic resistance profiles (ARPs) of E. coli and V. cholerae strains, recovered from the water samples, were determined and changes in the microbial community of the water samples were monitored over a seasonal cycle, using denaturing gradient gel electrophoresis (DGGE). Spatial and seasonal fluctuations of the physico-chemical parameters differed significantly (p < 0.05) among the water samples with high heavy metal concentrations detected across the seasonal cycle. Temperature profiles ranged from 13°C to 26.5°C for the Umgeni River, 13°C to 27°C for the beaches and 12°C to 26°C for the Umdloti River while pH ranged from 6.30 to 8.45 (Umgeni River), 6.37 to 8.30 (beaches) and 5.96 to 7.94 (Umdloti River). Turbidity ranged from 0.53 NTU to 15.6 NTU (Umgeni River); 0.57 NTU to 2.37 NTU (beaches) and 2.23 NTU to 18.8 NTU (Umdloti River). During spring and summer, all river and beach water samples had < 500 μg/L phosphate concentrations; however, these concentrations increased significantly (p < 0.05) during autumn and winter in both rivers. Majority of the samples had low concentrations of ammonia and nitrates. Sulphate concentrations for the beach samples ranged from 2355 mg/L (B5 – summer) to 2899 mg/L (B2 – winter) as compared to the Umgeni and Umdloti Rivers which ranged from 3.90 mg/L (A4 – autumn) to 2762 mg/L (A1 – summer) and 4.47 mg/L (C4 – winter) to 168 mg/L (C1 – winter), respectively. According to the South African Target Quality Range guidelines for the heavy metals (in surface waters), all river and beach water samples exceeded the set limits for lead (Pb2+), mercury (Hg2+) and cadmium (Cd2+) across all seasons. During spring and summer all water samples complied with the aluminium guideline of 0 – 0.15 mg/L. Bacterial population profiles indicated that all sampling points failed to comply with the set guidelines (domestic use) for presumptive total coliform (TC), faecal coliform (FC) and total heterotrophic bacterial (THB) counts during all four seasons. Estimated TC, FC and THB populations as high as 8.6 x 101, 3.7 x 101 and 2.15 x 105 cfu/100ml, respectively, were obtained for some of the samples with peak indicator levels and generally a higher microbial load observed during the summer season. High prevalence of resistance to ampicillin [67.82% (Umgeni River)] was encountered among the E. coli isolates from the water samples followed by amikacin [53.33% (Umdloti River)], augmentin [49.6% (Umdloti River)], tetracycline [42% (Umgeni River)], streptomycin [37.1% (beaches)] and cotrimoxazole [33% (Umgeni River)]. The most frequently encountered form of resistance among the V. cholerae isolates was against cotrimoxazole [93.34% (Umgeni River)], streptomycin [84% (beaches)], erythromycin [78.7% (Umgeni River)], trimethoprim [77.7% (Umdloti River)], rifampicin [70% (Umgeni River)] and cefoxitin [45% (Umdloti River)]. Multidrug resistance among the E. coli isolates was indicated by twenty nine (Umgeni River), twenty six (beaches) and fourteen (Umdloti River) different resistance patterns, while the V. cholerae isolates produced eighteen (Umgeni River), thirty five (beaches) and twenty nine (Umdloti River) different resistance patterns. In addition, proportional resistances of the E. coli and V. cholerae strains to the different classes of antibiotics ranged from six to eleven and four to eleven different antibiotic classes, respectively. The present study suggests that the bacterial communities detected in the water samples collected from the rivers and beaches in Durban, followed seasonal dynamics and could possibly be the consequence of fluctuations in certain environmental factors. A total of 87 different DGGE bands were detected among the Umgeni River water samples, 127 different DGGE bands among the six beach water samples and 107 bands in the Umdloti River samples, over the four seasons. Twenty one dominant bands were found among all sampling sites, indicating widespread phylotypes, whereas 14 bands were exclusively detected at only one sampling site (C1) potentially indicating unique phylotypes. Some bands appeared all year-round, whereas some other bands were specific to a particular season. Overall, the present study successfully demonstrated the poor microbiological quality of the investigated river and beach water resources which raise concerns over the management of these water resources and the subsequent deleterious effects these waters could have on the end users. This emphasizes the need for implementation of improved management strategies of these river catchments and beaches for continued sustainability. Furthermore, the high level of multi-antibiotic resistance demonstrated by the E. coli and V. cholerae strains, recovered from the water samples, reiterates the need to continuously monitor the changing trends in antimicrobial resistance patterns of these diarrhoeal-related bacterial pathogens. Therefore, continued surveillance of these surface waters used for recreational or domestic purposes and development of adequate prevention strategies are needed for public health reasons. Lastly, combining the use of conventional faecal indicators with molecular-based techniques, such as DGGE, can provide more information on the microbial load and diversity of surface waters. In addition, information regarding the effects of seasonal variations on microbial diversity as observed in this study is important for the sustainable management of surface water resources. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2010.

Chryseobacterium species have been reported to cause spoilage in food products such as fish, meat, poultry and dairy products. Some of these Chryseobacterium species can grow at temperatures below 7 °C which gives them the ability to grow and cause spoilage in milk kept at refrigerated temperatures. Protease and lipase enzymes produced by psychrotolerant species in milk are responsible for spoilage by the production of off-odours and âflavours. The aim of this study was to determine the growth and spoilage characteristics of Chryseobacterium species in milk and compare the results to those of Pseudomonas fluorescens which is regarded as the major organisms causing spoilage in milk. The specific growth rates and cardinal temperatures for C. joostei, C. bovis and Ps. fluorescens were determined by optical density measurements. Chryseobacterium joostei had the highest maximum specific growth rate followed by Ps. fluorescens and C. bovis. All three organisms were able to grow at 4 °C, but Ps. fluorescens showed the highest growth rate temperatures below 7 °C. All three organisms can thus be classified as psychrotolerant mesophiles due to the fact that growth was observed at 4 °C with optimum temperatures ranging between 25 and 30 °C. Arrhenius plots for the three species showed that C. joostei had the lowest activation energy followed by Ps. fluorescens and C. bovis. This means that C. joostei was the least sensitive to temperature changes that may take place while C. bovis was the most sensitive of the three tested organisms. The protease activity per mg protein for the three organisms was determined with the azocasein method. Chryseobacterium joostei showed the highest activity followed by C. bovis while Ps. fluorescens showed significantly lower activity than the Chryseobacterium species. All three species showed protease activity at 4 °C. Determination of the lipolytic activity of C. joostei and Ps. fluorescens were done by measuring the amount of FFAs present in fat-free and full cream milk incubated either at 4 or 25 °C. High levels of lipolysis were observed for all the inoculated samples. Although differences between the different sample treatments were observed, these differences were not significant enough to differentiate between the spoilage potential of C. joostei and Ps. fluorescens in the different milk samples. Secondary lipid oxidation of the two organisms was determined with the TBA method. Although inoculated milk samples demonstrated higher levels of oxidation compared to non-inoculated samples, there were no signigicant differences between any of the inoculated milk samples. Sensory analysis was done on the samples and C. joostei produced stronger odours than Ps. fluorescens. All the samples that were inoculated with C. joostei scored significantly higher spoilage scores than the control samples as well as the Ps. fluorescens samples. The odours described for the two organisms also showed distinct differences. In addition to the âsmelly feetâ, âblue cheeseâ and âagarâ odours described for all the inoculated samples, C. joostei also produced âputridâ and âsourâ odours while Ps. fluorescens produced odours described as âfruityâ, ânuttyâ and âbitterâ. The volatile compounds produced by C. joostei and Ps. fluorescens were identified with headspace SPME-GC/MS analysis. Ketones, alcohols and fatty acids were the main compounds produced. Ketones are responsible for the production of fruity and floral odours. Fatty acids produce sour odours, with isovaleric acid being responsible for the smelly feat odour. Alcohols do not usually contribute to the odour production in milk. The results could not be used to differentiate between the two organisms in the different milk samples.

Molecular chaperones are proteins which enable other protein molecules to fold to their native conformation and this property has been widely used to improve the solubility of proteins expressed in Escherichia coli. The effect of co-expressing of heterologous, thermophilic DnaK chaperones alongside with Thermus thermophilus DNA polymerase in E. coli was investigated in this study. A novel approach of co-expressing these proteins was also attempted. To construct the plasmid vectors, high-copy expression vectors pET22 and pET28 commercial plasmid were used to provide the backbone for the new vectors. The KJEA operon, encoding DnaK chaperone, DnaJ co-chaperone, GrpE nucleotide exchange factor and the DnaK/DnaJ assembly factor, DafA, was amplified from Thermus scotoductus SA-01 and T. thermophilus HB8 by PCR. Similarly, the arabinose-inducible promoter PBAD and its regulator protein-encoding gene, AraC, were amplified from the pBAD commercial vector. All fragments were subcloned into pGEM®-T easy before cloning them into the pET vectors. PBAD was first ligated to either TsKJEA or TtKJEA to make a promoter-DnaK fusion gene that was then subcloned into pGEM®-T easy and later cloned into pET22 and pET28, along with AraC to yield four recombinant vectors, p22TsK, p22TtK, p28TsK and p28TtK. Induction of PBAD in these vectors with 5 mg ml-1 of L-arabinose resulted in expression of DnaK chaperone proteins from only p22TsK and p28tsK, which express T. scotoductus DnaK proteins. The problem in p22TtK and p28TtK has been attributed to non-expression of AraC protein due to the long distance between AraC and its promoter region which lies in the PBAD region, and is inverted in these vectors; however, this has yet to be investigated. The T. thermophilus DNA polymerase, TthPolI was cloned into the MCS of the chaperone expression vectors. The same polymerase was also fused to a superfolder green fluorescent protein, sGFP, and cloned into the chaperone vectors. Only the pET22-series of chaperone vectors were used as cloning into the pET28 line was unsuccessful. Expression of these two proteins was initiated by induction of the T7/lac promoter with 1mM IPTG. A commercial plasmid, pKJE7, encoding E. coli DnaK, DnaJ and GrpE was also used to co-express both proteins, for comparison. Expression was achieved in all DnaK-expressing vectors as well as non-expressing negative controls. Purification of uncoupled TthPolI by affinity chromatography was possible from cell expressing DnaK and the negative controls; however, only TthPol-sGFP protein expressed from p22TsK was purified successfully, demonstrating the need for molecular chaperones when folding large proteins and the superiority, in folding activity, of the thermophilic DnaK chaperone system, in comparison to the E. coli system. Activity assays were carried out to test the processivity, thermostability and fidelity of Tth polymerases purified from these vectors. Results show that Tth Polymerase amplifies short fragments, such as 771 bp sGFP, with high fidelity and is comparable to commercial Taq polymerase. However, Tth polymerase purified from strains co-expressing the commercial DnaK proteins had a poorer activity and yielded lower product than the other polymerases. It was able to amplify 3518bp-TtKJEA operon but the yield of product was lower than that obtained from the two commercial Taq polymerases. It was also unable to amplify a 6.6 kb plasmid, p22Cyp153A6, although even the commercial Taq polymerases only produced a mixture of DNA fragments, none of which were the correct size. This problem has been linked to the thermostability of Tth polymerase, which has a half-life of 20 min while that of Taq polymerase is 40 min. This means that while, Tth might be able to amplify large fragments, as a result of its low processivity, it is soon denatured from the high temperature cycles in long-distance PCR and substantial amplicons are not generated in time but Taq polymerase, though stable, has poor affinity for longer templates and dissociates before it can complete elongation of the template. Extended incubation of Tth polymerase at 95°C inactivates it and it is unable to amplify even the relatively short sGFP template. According to literature, the poor thermostability is a property of Tth polymerase and cannot be altered or improved by molecular chaperones.

Most of what is known about the biology and function of oxylipins, oxygenated polyunsaturated fatty acids and metabolites, including the eicosanoids such as prostaglandins, comes from the study of mammalian biology. These compounds are ubiquitous in nature and found in all eukaryotic organisms, including the fungal domain. It is also in this group of organisms that the least is known about the metabolic pathways leading to the production of oxylipins, including those derived from arachidonic acid (AA) (n-6 fatty acid), and the functions of these compounds in the biology of fungi and yeasts. Candida species has the ability to produce proinflammatory eicosanoids, such as prostaglandin E2 (PGE2), from host derived AA. Candida albicans is an important opportunistic pathogen in humans causing systemic infections. An important virulence factor in C. albicans is the ability to produce pro-inflammatory PGE2, which enhances biofilm formation and influences host immune responses. Biofilms increase damage in host cells and are more resistant to antifungal drugs than planktonic yeast cells. This is an important area of research which may aid in the understanding of the complex interactions between host and pathogen, leading to the identification of novel antifungals or drug targets. This study evaluated the production of the prostaglandins, PGE2 and PGF2α, from exogenous AA, by biofilms of C. albicans and the closely related C. dubliniensis as well as the effect of different AA metabolism inhibitors on PGE2 production. Candida albicans and C. dubliniensis biofilms were both capable of producing PGE2 and PGF2α, from exogenous AA. The use of different inhibitors suggested that cytochrome P450s and multicopper oxidases are involved in PGE2 production by these Candida biofilms. It is known that mammalian cells cannot produce PGE2 from non-methylene interrupted fatty acids (NMIFAs), such as sciadonic acid (SA) (n-6 fatty acid). This property provides these fatty acids with potential anti-inflammatory activities. This study indicated the incorporation of SA into the lipids of epithelial cells, which reduced PGE2 production and influenced cytokine profiles in SA supplemented epithelial cells infected with C. albicans or C. dubliniensis. This suggest that the incorporation of n-6 NMIFAs, such as SA, might lead to a reduction in pro-inflammatory prostaglandins, especially PGE2, which could benefit the host during a Candida infection. Interestingly, both C. albicans and C. dubliniensis biofilms were unable to produce PGE2 from exogenous SA. Further genomic hybridization studies were used to evaluate the regulation of C. albicans biofilm genes during incubation in the presence of exogenous AA and SA. Transcriptional analysis indicated that the genes differentially expressed in the presence of AA had diverse functions not normally required for cell growth. Genes encoding for oxidoreductase and hydrolase activity were regulated, but were not clearly involved in PGE2 synthesis. Interestingly, genes that encode for ABC transporters, as well as genes associated with filamentous and hyphal growth, carbohydrate metabolic processes and oxidative stress response were differentially expressed by the presence of AA. Further studies of these differentially expressed genes are needed to evaluate how they may be involved in AA metabolism and PGE2 production.