In the past few decades much research has been conducted on yeast alcohol dehydrogenases with emphasis on their role in S. cerevisiae. There are seven known Adh proteins that were identified in S. cerevisiae. Six of them are intensively studied on gene and translational level. After sequencing of S. cerevisiae chromosome II (Feldman et al. 1994), ADH5 was identified which is 76% and 77% identical to ADH1 and ADH2 respectively. Later studies on global localization on genes showed that ADH5 is localized in the cytoplasm (Hue et al. 2003). No further research has been performed on ADH5. In this study Adh5p was over-expressed, purified and characterized towards its primary substrate, ethanol, and preferred co-factor NAD+. Furthermore, indepth kinetic studies were performed using various alcohols, increasing in chain length and branching. Adh5p is capable of oxidizing alcohols from two carbons to ten carbons. However, the catalytic efficiency decreases with increasing chain length. Results showed that Adh5p functions in vitro the same as Adh1p and Adh2p, sharing a primary substrate (ethanol). Adh1p and Adh2p are capable of converting more substrate per unit enzyme per second Adh5p. The second part of this study was to determine if Adh5p could substitute the catalytic function of Adh1p in vivo. For this purpose, ADH5 expression needed to be similar to Adh1p levels in the cell. Thus an expression vector was used containing ADH5 gene flanked with the promoter and terminator regions of ADH1. S. cerevisiae TÎ123 was constructed with ADH4 and ADH5 still intact. S. cerevisiae TÎ123 was transformed with a pRS413 and pRS423 construct containing the ADH1 promoter and terminator fused to the ADH5 ORF. Growth was monitored in chemically defined media containing 7 g l-1 ethanol or 8 g l-1 glucose. Growth parameters were also compared to the S. cerevisiae W303-1A and the adh quadruple deletion strain (Q1) containing only ADH1. The S. cerevisiae TÎ123::ADH5_S and S. cerevisiae TÎ123::ADH5_M constructs were capable of limited growth on both glucose and ethanol as carbon sources. When comparing the biomass yield of S. cerevisiae TÎ123::ADH5_S and S. cerevisiae TÎ123::ADH5_M to the biomass yield for both S. cerevisiae W303- 1A and S. cerevisiae Q1 the constructs delivered a much lower biomass yield. To verify the expression of Adh5p, a Micro BCA⢠protein assay kit supplied by Pierce (Smith et al. 1985) was used. Protein concentrations were determined at various time intervals. Cell homogenization was standardized, S. cerevisiae Q1 cells were diluted to fit the OD600 obtained for both the S. cerevisiae TÎ123::ADH5_S and S. cerevisiae TÎ123::ADH5_M strains. To conclude, Adh5p is capable of oxidizing various alcohols, but ethanol is its primary substrate. Furthermore Adh5p is not capable of replacing Adh1p in cellular metabolic function. The low turnover number illustrated by Adh5p and the lack thereof to reduce acetaldehyde is the most prominent cause of cellular death. Unlike Adh1p, Adh5p is not capable of reducing acetaldehyde to ethanol and thus not capable of NAD+ - NADH regeneration.

Edible oils such as sunflower oil, soybean oil and palm oil are used today in the frying of food. During the frying process, various changes such as removal of antioxidants, hydrolysis, oxidation and polymerization occur in these oils. These reactions are responsible for a variety of physical and chemical changes observed in the oil during frying and may lead to the formation of breakdown products which include polar compounds (PCs) and polymerized triglycerides (PTGs). South African regulations state that oils that contain 16% and more PTGs and 25% and more PCs are harmful to human health. These oils may cause cancer and diarrhoea in humans and animals. However, little is known regarding the effect of oxidized oils on fungi. The oleaginous fungi Cryptococcus curvatus and Mucor circinelloides were used to determine the effect of palm oil breakdown products, measured as PTGs on lipid turnover, growth and morphology. In Mucor circinelloides we found, after seven days of growth, a decrease in biomass, lipid utilization and accumulation at increased PTG levels, at low and neutral pH. An increase in PTG concentration also influenced the morphology of M. circinelloides. Protrusions were observed on cell surfaces when grown on oil with 45% PTGs and not when the fungus was grown on fresh oil with 0.4% PTGs. In C. curvatus there was also a decrease in oil utilization and biomass production at increased PTG levels, at low and neutral pH. An increase in oil accumulation was observed at low pH while it remained constant at neutral pH for all PTG levels tested. Hairy and warty protuberances on cell surfaces were observed when C. curvatus was grown on oils with 15% and 45% PTGs, respectively. It is concluded that the changes observed in lipid turnover and morphology in both fungi are due to the presence of palm oil breakdown products. Oxidized oil breakdown products such as aldehydes are major sources of reactive oxygen species (ROS). Studies have shown that ROS has anti-mitochondrial action. It was also reported that acetylsalicylic acid (ASA), an anti-inflammatory and anti-mitochondrial drug, targets structure development and functions of yeasts, needing elevated levels of mitochondrial activity. Using antibody probes it was previously reported that sporangia of Mucor circinelloides also contain increased mitochondrial activity yielding high levels of 3-hydroxy (OH) oxylipins. This was however not found in Mortierella alpina (subgenus Mortierella). In this study, it is reported that oxidized palm oil breakdown products and ASA also targets sporangium development of M. circinelloides selectively while hyphae, with lower levels of mitochondrial activity, are more resistant. Similar results were obtained when the anti-inflammatory compounds benzoic acid, ibuprofen, indomethacin and salicylic acid were tested. Here, oxidized oils and antiinflammatory, anti-mitochondrial drugs exerted similar effects on this dimorphic fungus as found under oxygen limited conditions. Interestingly, sporangium development of M. alpina was found not to be selectively targeted by these compounds. Mortierella alpina, which could not expose dimorphic growth under oxygen limitation conditions, was also more sensitive to the anti-inflammatory drugs when compared to M. circinelloides. These results prompt further research to assess the applicability of these anti-mitochondrial antifungals to protect plants and animals against Mucor infections. It is concluded that indications exist that oxidized palm oil breakdown products target mitochondrial function. This may explain the inhibitory effect of these compounds on fungal growth, lipid turnover and altered cell wall morphology.

Cyanide is highly toxic to living organisms due to the potent inhibitory effect on the respiration system. This toxic compound can be deposited in the environment through various sources. Naturally occurring cyanide compounds can be synthesized (cyanogenesis) by various taxa including fungi, plants and bacteria. Cyanogenesis in bacteria is mostly linked to antagonistic activity against various microorganisms competing for the same nutrients in the same environment. Anthropogenic sources of cyanide include a wide variety of industries but the major contributor is the cyanidation process. This process extracts gold (silver can also be extracted) from ore and is responsible for the formation of metal-cyanide complexes in soil which can dissociate to form free cyanide under the correct conditions. Various microorganisms are capable to degrade free cyanide. The aims of this study were to identify microorganisms capable of utilizing cyanide as both a carbon and nitrogen source and to elucidate the mode of degradation. Samples were obtained from the Klipspruit Calcium Cyanide Factory site and were inoculated into minimal medium supplemented with NaCN. Eighteen isolates were identified from the samples and included organisms that could possibly be novel isolates based on the maximum identity percentage obtained when the 16S rRNA gene sequences (~1 500 bp) were used in a BLAST analysis against the NCBI database. The MIC was calculated for each of the 18 isolates and indicated that most of the organisms were capable of degrading cyanide at concentrations of above 2 M. This, in correlation with literature, is far above average. Gram stains were performed on the eighteen isolates. Five isolates were chosen for further studies based on 16S rRNA sequencing results, MIC determinations as well as information from literature that states that Bacillus and Pseudomonas species are often employed in bioremediation strategies. The five selected organisms included three gram positive (Bacillus sp.; Paenibacillus sp. and Leifsonia sp.) and two gram negative (Achromobacter sp. and Brevundimonas sp.) isolates. For comparative studies three control organisms (Bacillus pumilus, Pseudomonas fluorescens and Pseudomonas stutzeri) that are known and described in literature to be capable of cyanide degradation, were included in this study. The cyanide assay (100 mM NaCN) was performed on the five selected and three control organisms. The control organisms were unable to utilize the cyanide as the sole carbon and nitrogen source at this high concentration. In contrast, the selected organisms were capable of increasing their biomass over time indicating that these organisms can utilize the NaCN as the sole carbon and nitrogen source. To elucidate the mode of cyanide degradation primers were designed specific for the known genes involved in cyanide utilization in the three control organisms, and screening the five isolates with these primers for the presence of the these genes. The genes targeted were cyanide dihydratase (Bacillus pumilus), hydrogen cyanide synthase (Pseudomonas fluorescens) and cyanide degrading enzyme (Pseudomonas stutzeri). The specifically designed primers were used on the gDNA from the selected organisms and this led to various non-specific product formations and in many of the samples no product was obtained. With the failure to identify the presence of known cyanide degrading genes in the five selected organisms, one of these organisms, Bacillus sp. B4H3, was selected for pyrosequencing to elucidate the complete cyanide metabolism in this microorganism. The sequencing data was analyzed and it was observed that the 16S rRNA gene sequence obtained in Chapter 2, section 2.3.2.7 was not present in the genome of the isolate after pyrosequencing. The pyrosequencing data was analyzed and a total of twenty one enzymes involved in the cyanide metabolism of this isolate were identified. From the complete metabolic pathway it can be concluded that the single nitrogen can be utilized through the conversion of cyanide to formamide which in turn can be converted to ammonia. The utilization of the single carbon is based upon the hypothesis that the reaction between cyanide and glycine is reversible. This will lead to the carbon fixation metabolism which will prove that the isolate is capable of utilizing the single carbon as the sole carbon source.

Boerewors is a South-African fresh sausage, made from beef and pork and flavoured with salt, pepper and various spices, especially coriander. It may contain a maximum of 450 mg/kg SO2 as preservative. Until recently, the use of SO2 has had GRAS (Generally Regarded as Safe) status. Investigations have indicated certain asthmatic individuals were placed at risk by relatively small amounts of sulphites. The increased demand for minimally processed, extended shelf-life foods have renewed interest in exploitation of natural antimicrobials for food preservation uses. The aim of this study was to evaluate the efficacy of Citrox as a natural preservative in Boerewors. Citrox comprises of a range of phyto-alexins derived from the pith and rind of green Bergamont oranges and fruit acids. Seven Boerewors models were made following a typical commercial procedure: the control containing no preservative, current dosage of SO2 in Boerewors (450 mg/kg), two dosages of Citrox on its own at 1% and 2% addition, a reduced amount of SO2 (100 mg/kg) on its own and Citrox in combination with reduced amounts of SO2 (1% Citrox + 100 mg/kg SO2 and 2% Citrox + 100 mg/kg SO2). The models were stored at 4 ºC for 6 days under retail display conditions. The models were evaluated every 48 hours for microbiological growth as expressed by total aerobic plate count (TAPC), coliform count, Staph. aureus count, yeast and mould count. The models were also evaluated every 48 hours for colour changes as expressed by the colour a* value (redness), colour b* value (yellowness) and colour L* value (lightness). The models were evaluated after 6 days, and again after 100 days storage at -18 ºC, for lipid oxidation as expressed by TBARS (thiobarbituric acid reactive substance). The models were also evaluated organoleptically by a sensory panel. It was established that all the treatments had significantly lower counts than the control over the 6 day storage period on TAPC. There was no significant difference in the counts of 450 mg/kg SO2, 2% Citrox, 100 mg/kg SO2 + 1% Citrox and 100 mg/kg SO2 + 2% Citrox. The 450 mg/kg SO2 and 100 mg/kg SO2 + 2% Citrox had significantly lower coliform counts than the control. Against yeasts and moulds, 100 mg/kg SO2 + 2% Citrox had significantly lower counts than the control and 450 mg/kg SO2. Citrox had a negative effect, on its own and in combination with 100 mg/kg SO2 on the colour of the Boerewors. The colour a* values were significantly lower than those for the control and 450 mg/kg SO2. There was very little effect on the colour L* value, most values stayed relatively stable over the 6 days, the 2% Citrox and 100 mg/kg SO2 + 2% Citrox showed the highest values. For all the models, there were a slight decrease in the colour b* value. At the end of the 6 days, all models had similar values. Over the 6-day fresh display period there was no significant difference in the TBARS values for all the models. After 100 days storage at -18 ºC, 450 mg/kg SO2 had significantly lower values than the control and the rest of the models. The control had the same values as the rest of the models, which led to the conclusion that although the Citrox had no anti-oxidant capabilities, it also did not increase lipid oxidation. The sensory panel found that the models containing 2% Citrox had a slight sour flavour (can be explained by the fruit acids in Citrox in combination with the vinegar added to the Boerewors formulation), however the average scores for these models were still found to be âliked slightlyâ to âliked moderatelyâ.

Developments in the biosynthesis of nanoparticles have increased significantly during the last few years as a result of the growing interest in the unique properties displayed by nanoparticles. These particles are extremely small in size and have a large surface to volume ratio, giving them unique physical and chemical properties at this scale that differs considerably from when they are used in larger form. These exceptional properties are used in a wide variety of applications, ensuing nanotechnology to become a multidisciplinary field. Research into application structure types are extended daily and as a result, the next few years will be crucial as applications for nanomaterials in the industry are most likely to be increased. Gold nanoparticles is receiving more and more attention because of its wide variety of uses in optical, electronic, magnetic, catalytic, and biomedical applications, but even more due to them being the most stable of all the metal nanoparticles. Several methods are used to produce these metal nanoparticles, but are mostly making use of toxic chemicals in the synthesis protocol, which are harmful to the environment and human health. To overcome this problem, researchers are making use of more âgreenerâ alternatives through the use of biological systems and microorganisms in nanoscience and nanotechnology. These microorganisms have unique potential in producing nanoparticles that are environmentally friendly and display different shapes, sizes and distributions. Among the different microorganisms used, bacteria have received the most attention in the nanoparticle production process, but have not been as successful as chemical synthesis to produce monodisperse noble metal nanoparticles. In this study, successful gold reduction and nanoparticle formation with different shapes, sizes and distribution was obtained; however, these particles were not monodisperse. This was achieved with a thermostable protein of ± 70 kDa that was identified as an ABC transporter, peptide-binding protein and which was purified from Thermus scotoductus SA-01; an extremophile and thermophilic bacterium that was isolated from groundwater samples from Mponeng (a deep South African gold mine in the Witwatersrand Supergroup operated by AngloGold Ashanti) at a depth of 3.2 km with ambient rock temperature of 60ºC. The protein was expressed in Escherichia coli and Thermus thermophilus HB27, a mesophilic and a thermophilic expression host respectively. It was found that the expression host might have an influence on the way the protein is folded and therefore influence nanoparticle formation. Expression studies was also done on the protein that either included or excluded Histidine-Tags and a leader peptide, but it was found that neither the His-Tags nor the leader peptide had any influence on the nanoparticles produced. Gold reduction and nanoparticle formation was obtained through reduction of a surface exposed disulphide bond in the ABC transporter, peptide-binding protein, using sodium dithionite as electron donor and reducing agent. In general it was found that nanoparticle formation was dependant on environmental parameters but control of this process was not complete. Chemical reduction did influence the nanoparticle formation process in some instances, but overall it could be seen that the presence of the protein played a significant role in slowing down the reaction rate, yielding a level of control over the nanoparticles produced and ensuring a more environmentally friendly, biological process for the production of gold nanoparticles.

As the initiative for establishing a yeast culture collection began in the early 1980âs in the department of Microbial, Biochemical and Food Biotechnology, research pertaining to the identification of isolates became the main focus. Yeasts such as Candida shehatae and Pichia stipitis claimed potential applications in the fermentation industry and received the most research attention. This research area and others reflected the potential of the culture collection as one of the sources of microbial diversity. As a result, this granted this culture collection a Microbial Resources Centre by the United Nations Educational, Scientific and Cultural Organization (UNESCO) in 1996. Isolation, preservation, identification and determination of taxanomic affinities of yeasts are some of the important aspects of this culture collection in addition to others such as bioprospecting and elucidation of biotechnological applications of strains. As a general practice, these isolates must be identified reliably and rapidly using techniques such as polymerase chain reaction (PCR) and sequencing of the ribosomal DNA. Polymerase chain reaction (PCR) and sequenceing of the ribosomal DNA was mainly used in this study to identify isolates present in the UNESCO-MIRCEN Biotechnological Yeast Culture Collection. Ribosomal DNA was amplified and sequenced, followed by analysis of sequence data that was searched against available sequences on National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). In the case of new species, verification of novelty was performed by sequencing the internal transcribed spacers and doing phylogenetic analysis based on multigenic approach. New species were assigned to their phylogenetic groups and described using standardized traditional techniques. It was apparent from this study that using molecular identification methods, the number of identified ascomycetes and basidiomycetes as well as yeast-like taxa, with possible applications in the industrial, clinical, pharmaceutical, environmental and agricultural settings can be rapidly identified in great numbers. In this study, three basidiomycete strains representing new species in the genus Cryptococcus, with potential applications in cyanide bioremediation, were identified and described. These strains represent a new species which form part of a small group of yeasts (including Cryptococcus humicolus strain) with the ability to utilize inorganic cyanide as sole carbon and nitrogen source. Some of the applications and importance of strains representing known ascomycetes, basidiomycetes and yeast-like taxa are also highlighted here. Not only was the identification of isolates achieved in this study, but also full description of new yeasts present in the UNESCO-MIRCEN Biotechnological Yeast Culture Collection with possible applications in bioremediation. However, the applications of species such as Trichosporon africanensis sp. nov. will be elucidated elsewhere.

The involvement of histone acetylation in facilitating gene expression is well-established, particularly in the case of histones H3 and H4. It was previously shown in Saccharomyces cerevisiae that gene expression was significantly down-regulated and chromatin more condensed in stationary phase compared to exponential phase. We were therefore interested in establishing the acetylation state of histone H3 and H4 in stationary and in exponential phase, since the regulation of this modification could contribute to transcriptional shut-down and chromatin compaction during semi-quiescence. We made use of nano-spray tandem mass spectrometry to perform a precursor ion scan to detect an m/z 126 immonium ion, diagnostic of an N -acetylated lysine residue that allowed unambiguous identification of acetylated as opposed to tri-methylated lysine. The fragmentation spectra of peptides thus identified were searched with Mascot against the Swiss-Prot database, and the y-ion and bion fragmentation series subsequently analyzed for mass shifts compatible with acetylated lysine residues. We found that K9, K14 and K36 of histone H3 and K12 and K16 of histone H4 were acetylated in exponential phase (bulk histones), but could not detect these modifications in histones isolated from stationary phase cells. The corresponding unacetylated peptides were, however, observed. This result was confirmed by Western analysis (work not presented here). H4K16 acetylation was previously shown to disrupt formation of condensed chromatin in vitro.

Several aspects related to cereal fermentations were reviewed in the literature section of this study. These aspects included among others, the history of indigenous cereal fermentation, factors that affect cereal fermentation, potential microbiological hazards of cereal fermentation, recent advances in related industry, and the future of fermented foods. A wide variety of recipes exists across the African continent and the beers are known by many local names. In particular this study focussed on mahewu and umqombothi, both made from a combination of maize and sorghum, fermented beverages consumed in South Africa. Improved process technologies such as the use of starter cultures can lead to improved product characteristics and consequently better health attributes for the consumer of these indigenously fermented products. During the fermentation processes for umqombothi and mahewu, yeasts, lactic acid bacteria, moulds and enterobacteriaceae were present at the start of the process but as fermentation progressed yeasts and lactic acid bacteria were the dominant microorganisms. This was over a 48 h, and 3 day, fermentation period for the umqombothi and mahewu respectively. Home-, township-, and laboratorymade samples of umqobothi were compared while for mahewu, the comparison was between the home- and laboratory-made samples. Results from different production sites were not significantly different although there were instances that called for improved hygiene as contaminants were found in some samples. Yeasts isolated from umqombothi were identified and these included; Candida ethanolica, C. haemuloni, C. sorbophila, Dekkera anomala, Dekkera bruxellensis, Saccharomycopsis capsularis and Saccharomyces cerevisiae. The yeasts isolated from mahewu were identified and the predominant strains were Candida haemuloni, Candida sorbophila, Debaryomyces hansenii, Saccharomyces capsularis and Saccharomyces cerevisiae. Confirmation of the yeast identity was done using sequence analysis of the D1/D2 domain using primer pairs NL-1(5â-GCATATCAATAAGCGGAGGAAAAG) and NL-4(5â- GGTCCGTGTTTCAAGACGG). Scanning electron microscopy was performed on the interior surface of a pot used for indigenous cereal fermentation. The images obtained confirmed the presence of biofilm on the surface and the microorganisms present in this biofilm were predominantly yeasts and bacteria. The observed proximity of yeasts to the bacterial cells lent further support to the suggestion of a symbiotic relationship existing between these microorganisms in indigenous cereal fermentations. Biofilms are not homogenous in composition but are complex matrices composed of microcolonies interspersed with channels allowing the movement of fluids and nutrients. It therefore follows that a combination of different techniques that are available may be required to give the most accurate picture of the true biofilm structure and organisation. Indigenous cereal fermented products are an integral part of the diet of many African households. Several advantages do arise from the fermentation of cereals and improvement of this process will not only lead to better health attributes but also the production of fermented beverages with guaranteed and consistent quality. This augurs well for the producers of fermented products as more consumers become aware of the health properties of fermented products and this trend is only likely to improve in the future.

Cytochrome P450 monooxygenases are a superfamily of heme-containing enzymes that are found in all domains of life. P450s catalyse diverse reactions, many of which are difficult reactions to accomplish, even with the use of chemical catalysts. One such reaction is the terminal hydroxylation of alkanes, the first step in alkane degradation. The CYP153 family, found in alkane-utilising bacteria, is one of only two P450 families that can catalyse this reaction. One of the long-term goals of our groupâs research is the directed evolution of terminal alkane hydroxylases, using preferably a self-sufficient terminal alkane hydroxylase as the starting point. There are, however, no naturally self-sufficient CYP153s. Therefore, the first aim of this study was to create a self-sufficient CYP153 by fusing a CYP153 heme domain to the reductase (PFOR) domain of a self-sufficient P450. The gene encoding the heme domain of CYP153A6 from Mycobacterium sp. HXN-1500 was ligated to the DNA encoding the PFOR reductase domain of CYP116B3 from Rhodococcus ruber DSM 44319. The fusion gene was expressed in E. coli using a pET28a plasmid. The resulting protein was misfolded and expressed mainly in the insoluble fraction in the form of inclusion bodies. Factors possibly responsible for this were investigated including the expression conditions, the effect of an N-terminal His-tag on protein folding, the effect of the linker region sequence on protein folding, and the possibility of rapid expression resulting in protein misfolding, but with all of these experiments only low levels of P420s were observed in the soluble fraction and no P450 forms were detected. A whole-cell octane bioconversion experiment conducted using the expressed fusion revealed the presence of P450 forms of the protein, but no 1-octanol was produced, indicating that octane possibly facilitated the correct folding of the CYP153A6 heme domain but that the heme domain and the PFOR reductase domain were unable to form a functional complex. This theory does, however, require further research. In this study, CYP153A6 and its redox partners, ferredoxin reductase and ferredoxin were expressed in E. coli using the pET28b plasmid. Expression of CYP153A6 in E. coli using this plasmid has not previously been reported in literature. Whole-cell octane bioconversions conducted using the expressed CYP153A6 resulted in the production of 42 mM of 1-octanol after 24 hours, with the P450 concentration increasing during this time, a trend which was also observed with the fusion. The second aim of this study was to apply cassette PCR to the fusion to generate diverse selfsufficient terminal alkane hydroxylases, which would provide the genetic diversity required for directed evolution. Degenerate primers designed according to conserved N- and C-terminal regions of CYP153A amino acid sequences were used to amplify internal CYP153A gene fragments from environmental DNA extracted from enrichments of soil sampled at a dieselcontaminated site in the Eastern Cape. Three different sequences were identified, one of them being CYP153A6, which was excluded from the rest of the study. The two remaining sequences and two sequences originating from another project using environmental DNA from samples from the Beatrix Goldmine in the Free State were linked to the 5â- and 3â-ends of the CYP153A6 gene, generating full-length chimeric CYP153A genes. Because of the fact that the expression of the fusion was unsuccessful, the functionality of these chimeric genes was tested using the above-mentioned functional CYP153A6 operon. Expression was observed in the insoluble fraction in the form of inclusion bodies, with the proteins being misfolded. A whole-cell octane bioconversion did not result in P450 forms of the proteins and no 1-octanol was produced, indicating that these chimeras were non-functional.

Avibacterium paragallinarum is an avian pathogen and has the ability to cause vast economical losses. This bacterium forms part of the Pasteurellaceae family and factors contributing to pathogenicity, immunogenicity and serotyping are not clearly understood. One of the main questions that were addressed in this study was the identification of genetic tools are that is responsible for the NAD+- independence ability of this organism. NAD+ recycling genes were implicated for this bacterium and Av. paragallinarum seem to follow the recycling pathway as set out for the Pasteurellaceae family. Still the question regarding NAD+- independence remains unanswered as no complete pathway could be implicated for this trait. Furthermore, no plasmid(s) could be isolated that conferred this trait and no complete NAD+ synthesis pathway could be implicated. Only two genes were identified to form part of a NAD+-independent pathway which indicated that Av. paragallinarum may use genetic tools for NAD+-independence as set out for bacteria in general and not as the rest of the Pasteurellaceae family members. Plasmid isolation studies revealed only the plasmid p250 identical to the established plasmid p250 for Av. paragallinarum. Literature reports on two other native plasmids namely pYMH5 and pA14, however, these two plasmids could not be detected in any of the strains used in this study during plasmid screens. This study confirms and illustrates the integration of plasmid p250 within the genome of Av. paragallinarum by different serovars. In order to study this bacterium on a genomic level a whole genome sequencing project was launched. This project experienced vast amount of difficulties regarding the assembly of a complete genome for Av. paragallinarum. This bacterium contains numerous repeated regions within its genome which, with current sequencing technology, prevent the genome assembly into a single chromosome. A complete assembled genome can only be achieved when longer read lengths are available in high-throughput sequencing technologies and will thus enable clarification regarding the repeated regions. A pseudo-assembled molecule was sent to the JCVI for genome annotation. Annotated data revealed that Av. paragallinarum contains a high degree of complexity regarding its cell envelope which may shed light on pathogenicity and immunogenicity problems endured. This bacterium contains numerous mobile and extrachromosomal elements which contribute to the inability to close the genome of Av. paragallinarum. Nine site-specific integrases and 10 transposases were identified. These tools could allow for a high degree of genetic diversity within the Av. paragallinarum specie. Alongside these tools two putative prophages was identified for Av. paragallinarum. One prophage resembles the Mu-like prophages and was termed ФAvpmuC-2M, the other resembles the HP2-like prophages and was termed ФAvpC-2M-HP2.