The usual strategy to prepare gold nanoparticles involves the reduction of a gold salt in solution by various reducing agents in the presence of a stabilizer. These particles are mostly spherical with poor monodispersity. An alternative means is to use biological material to mediate particle synthesis. Microorganisms such as fungi have demonstrated the ability to produce nanoparticles of different shapes and sizes extending beyond the scope of chemical means, and the microbial interaction with metals also supply eco-friendly methods for nanoparticle production. It has been hypothesized that the proteins involved in nanoparticle synthesis require a co-factor such as NADH / NADPH, as previous studies have indicated that NADH- and NADPH-dependent enzymes are important factors in the biosynthesis of metal nanoparticles. Thermus scotoductus SA-01, a thermophilic bacterium, isolated from an AngloGold Ashanti mine near Carletonville, Republic of South Africa, was used for purification of a gold(III) reducing and nanoparticle synthesizing protein. This bacterium has the ability to produce gold nanoparticles, and more than one pathway can be followed to produce these particles. A protein was purified to homogeneity by using a combination of several liquid chromatography resins. The N-terminal sequence was obtained by using automated Edman degradation. The protein purified is not a classical oxido-reductase and was identified as an ABC transporter peptide-binding protein (~70kDa). This discovery shows that gold nanoparticles can be produced by proteins other than oxidoreductases. The interaction of the protein extracted and purified from Thermus scotoductus SA-01, as well as the recombinant proteins, with liquid gold under varying physico-chemical conditions have been studied using TEM, EDS, and by measuring the plasmon resonance band, to illustrate the effect on particle morphology and to elucidate the protein mechanism. The size and the shape of particles could, to an extent, be manipulated by controlling the environmental parameters. The purified protein as well as the recombinant proteins was only able to produce nanoparticles in the presence of sodium dithionite and it is thus hypothesized that the donation of electrons via the disulphide bridge in the protein is involved in the reduction of the gold ions. Even though the recombinant proteins had the ability toproduce nanoparticles they were not as efficient as the native protein, but when the optimum parameters for the recombinant proteins are established they could be used in the upscale production of gold nanoparticles or gold nanosheets.

Avian pathogenic Escherichia coli (APEC), the causative agent of colibacillosis belong to the family enterobacteriaceae. The disease is manifested as localised or systemic infections which include peritonitis, airsacculitis, omphalitis, swollen head syndrome and colisepticaemia in poultry. Colibacillosis results in increased mortality, condemnations of carcasses at slaughter, reduced feed conversion and production due to morbidity and increased cost of treatment. The standard method for the control of colibacillosis has been the use of antibiotics either as a treatment or as prophylaxis through the addition of antibiotics into the animal feed at sub-minimal levels. The efficacy of antibiotics has drastically reduced over the years due to the emergence and increased prevalence of antibiotic resistance. The use of some antibiotics and antibiotic growth promoters such as fluoroquinolones has been banned and further bans are still impending. Therefore, colibacillosis can potentially cripple the poultry industry nationally and worldwide, if traditional treatment options are ineffective. There is a great demand for potential alternatives to the use of antibiotics and lytic bacteriophages offer attractive advantages over antibiotics in this regard. This study focused on the isolation and characterisation of lytic bacteriophages with the aim of determining their potential to control E. coli infections, in poultry. Nine bacteriophages were successfully isolated from sewage samples and five from chicken faecal matter. The lytic patterns of the phages against E. coli K12 and E. coli strains isolated from diseased poultry (presumed to be pathogenic) were determined. Among the phages isolated from sewage samples, 31-5, E13/2, E13/5 and BS4 lysed four or more of the E. coli strains. The widest host strain range was exhibited by E13/2 which infected 8 of the 11 strains. Thus, this isolate may hold the greatest potential for therapy. In contrast, isolates from chicken faecal matter had a very narrow host strain range, lysing a maximum of two E. coli strains including the original host. An interesting observation was made in that the laboratory strain; E. coli K12 was resistant to lysis by four out of the five isolates (from chicken faecal matter). This is an important factor to consider in any potential form of treatment where only the pathogenic strains are targeted while the normal microbiota should remain unaffected. The varied lytic patterns observed for most of the isolates further demonstrate the importance of employing phage cocktails rather than single preparations to treat or prevent colibacillosis. Additionally, some of the isolates may prove useful in phage typing due to their high discriminatory nature in lysis. Although much has been reported on APEC-specific coliphages isolated from sewage, this is one of a few studies where they have been successfully isolated from chicken faecal matter. Examination of morphological characteristics by transmission electron microscopy revealed the presence of a majority of tailed phages which are members of the Myoviridae and Siphoviridae families. For some viruses definite conclusions could not be made concerning their identity and they were therefore only tentatively placed into certain families. Molecular analyses included polymerase chain reaction (PCR) using primers specific for the g23 sequence of T4-type phages and the DNA polymerase of T7-like podophages. Although the identity of every phage isolate could not be inferred, the relatedness between isolates SK4 and SK5 was established using the PCR technique of random amplified polymorphic DNA (RAPD). Phylogenetic investigations revealed that 31-5 and SK5 were closely related followed by E13/5 and SK4. In conclusion, this study demonstrated that some of the isolated phages have the potential to prevent, eliminate or reduce APEC in poultry. Further research is however, necessary to fully characterise the phage isolates and perform efficacy tests in vivo.

Mycoplasma gallisepticum (MG) is an economically important pathogen of poultry worldwide, causing chronic respiratory disease in both chickens and turkeys. Little research has been done to characterize the MG field strains present in southern Africa. Field evidence however, suggested breaks in the vaccinations against MG, despite the proper cold chain and correct administration. Molecular methods were used to screen various poultry farms in South Africa and Zimbabwe for MG. Isolates were further characterized by gene-targeted sequencing (GTS). Portions of the cytadhesin pvpA, gapA, mgc2 genes and the uncharacterized surface lipoprotein gene designated MGA_0319 were sequenced and analysed. Three MG strains were identified in this study: a Ts-11-like strain; a strain showing closest percentage identity to the atypical MG RV-2 strain found in Israel, however with the lack of a large deletion within the pvpA region; and lastly an isolate from Zimbabwe, likely to be a novel MG strain. The latter contained a unique, large, in-frame nucleotide insertion in the mgc2 gene. Antigenically-significant variation (determined in silico) within the membrane surface proteins of this isolate was found compared to the MG Rlow strain used as an inactive vaccine on the poultry farm. It is thus postulated that by MG altering its antigenic profile, it allows effective avoidance of immune recognition and antibodies produced as a result of vaccination with inactivated and possibly live MG vaccines. Further research is however needed to substantiate this claim.

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.

The widespread and unrestricted use of antibiotics in animal production has led to a surge in antibiotic resistant bacterial strains. The poultry industry is steadily headed for a post antibiotic era, thus fuelling the search for alternative treatments for bacterial infections. One of these alternative treatments is the use of quaternary ammonium compound (QAC) based disinfectants. QACs are cationic surface active detergents widely used in the poultry industry because of their low relative toxicity and good antibacterial properties. Reports on QAC resistant bacteria have been on the increase in the food industry and thus studies on bacterial resistance to QACs are on the increase. In order to try and understand disinfectant resistance, it is important to gain a better understanding of the mode of action of QAC based disinfectants of bacterial cells, particularly in the light of a pending post antibiotic era. In order to do this, bacteria treated with DDAC were examined using Scanning electron microscopy (SEM) and Nano Scanning Auger Microscopy (NanoSAM). Staphylococcus aureus strain ATCC 2357 treated with DDAC revealed protuberances or âblebâ formations on their cell walls when observed with SEM. The DDAC treated cells were further investigated using NanoSAM. NanoSAM is the combination of Scanning Auger Microscopy (SAM) and etching with an Argon (Ar+) gun. SAM has the ability to perform semi-quantitative elemental analysis on extremely small volumes while visualizing the sample with SEM. Using NanoSAM technology we were able to visualize morphological changes caused by the disinfectant that SEM could not show. Clear evidence of a disruption of the cell membrane and the leaking out of cellular content was obtained. Resistance to QAC has been attributed to the presence of the qac resistance genes, smr, qacJ, qacG, qacH. During this study the presence of the qac resistance genes could be correlated to the degree of resistance QACs. The qac resistance genes were identified using conventional PCR in strains that displayed higher tolerance to the different QACs. No qac resistance genes where identified in the susceptible strain ATCC 25923 using conventional PCR even though this strain was resistant to one of the QACs, benzalkonium chloride. An increased resistance to the different QACs could not be attributed to the presence of one specific qac resistance gene.Real time PCR was introduced in this study since it is a technique known to be more sensitive than conventional PCR. Using real time PCR, it was revealed that all the bacterial strains contained more than one qac resistance gene. Interesting results were obtained with the susceptible strain ATCC 25923, where qac resistance genes were detected with real time PCR, while these genes were not detected using conventional PCR. Similar results were obtained with the Avian pathogenic Escherichia coli (APEC) strain isolated from poultry pens. After detecting the presence of the genes, the focus of the study changed to investigate the levels of expression of one of the qac resistance genes, smr. The expression study was performed using relative quantitative real time PCR. The hypothesis was that expression is increased when QACs are present in a culture medium. During the study it was revealed that there was no significant difference in the expression of the qac genes during cultivation in the presence of different QACs. There was, however a difference in the expression of the different strains tested where the smr was only expressed in the strain VB4-smr and not in the strains VB3-qacJ and ATCC 25923 during cultivation in the QAC didecyldimethylammonium chloride (DDAC). An additional hypothesis was subsequently formed. This hypothesis postulates that there is a difference in the expression of the smr gene over a time interval. During this study it was revealed that there was a significant difference in the expression of smr cultivated in different concentrations of DDAC, but there was no significant difference in the expression over a time interval. From this study, it has been established that qac resistance genes are present in various bacteria and that using the more sensitive real time PCR test, additional qac genes were found in most of the strains. From the expression studies, it can be concluded that the levels of resistance is not merely related to the presence or absence of a particular qac resistance gene. It was also established that resistance is also not always directly related to increased levels of expression of a particular qac resistance genes. From this study, it is evident that resistance to disinfectants is multi factorial and substantial additional research is required to fully understand resistance to disinfectants.

Opuntia ficus-indica, the prickly pear cactus, is well adapted for cultivation in arid and semi-arid regions, with a yield of 10 to 40 tonnes (dry wt) cladode biomass per ha. The cladodes (the âleavesâ, which in fact are the stems) might serve as lignocellulosic biomass feedstock for second generation bioethanol production, without competing for agricultural land or replacing significant natural vegetation. The main objective of this study was to investigate the feasibility of bioethanol production from an enzymatic hydrolysate of O. ficus-indica cladodes. The potential of a Kluyveromyces marxianus isolate UOFS Y-2791, a yeast capable of utilising a wider range of carbon substrates and of ethanol production at higher temperatures than Saccharomyces cerevisiae, was investigated for bioethanol production using an O. ficus-indica cladode enzymatic hydrolysate as feedstock. S. cerevisiae UOFS Y-0528, a wine yeast strain, was used as benchmark. Compositional analysis of the cladode biomass indicated that it had a low lignin content of 8% (dry wt). The content of readily fermentable carbohydrates in the cladode, which was 34.3 g per 100 g dry biomass of which 23 g was glucose, was comparable to other conventional biomass feedstocks such as sugar cane bagasse and corn stover, whereas it had a low xylose content. By applying a statistical design experimental approach where acid concentration and contact time were varied, optimum conditions for dilute acid pretreatment of the dried and milled cladode were determined to be 1.5% (w/w) sulphuric acid for 50 min at a temperature of 120oC and a dry biomass loading of 30% (w/v). Enzymatic hydrolysis experiments were performed with varied enzyme loadings of cellulase and β-glucosidase with or without the addition of pectinase, and the enzyme loadings chosen were 15 FPU cellulase, 15 IU β-glucosidase and 100 IU pectinase per gram of dry biomass. These parameters yielded an O. ficus-indica hydrolysate containing (per litre) 45.5 g glucose, 6.3 g xylose, 9.1 g galactose, 10.8 g arabinose and 9.6 g fructose. Using a chemically-defined medium with a sugar composition similar to the hydrolysate as benchmark, K. marxianus and S. cerevisiae were grown in the O. ficus-indica hydrolysate at 40oC and 35oC, respectively, under non-aerated conditions, whereas the performance of K. marxianus was also investigated under oxygen-limited conditions where the DOT was controlled at less than 1% saturation. The fermentation profiles of both yeasts were compared using separate hydrolysis and fermentation (SHF) and simultaneous hydrolysis and fermentation (SSF) process configurations, at a water-insoluble solids (WIS) content of 14%. Both yeasts achieved comparable ethanol yields in SHF and SSF under nonaerated conditions, although K. marxianus exhibited a lower volumetric ethanol productivity than S. cerevisiae. K. marxianus, cultivated under oxygen-limited conditions, achieved a lower ethanol yield than both yeasts cultivated without aeration. However, K. marxianus exhibited the highest volumetric ethanol productivity of 2.3 g l-1 h-1 and 1.57 g l-1 h-1 in SHF and SSF, respectively, although the ethanol produced was assimilated upon hexose depletion. K. marxianus utilised galactose poorly in the absence of aeration, but completely consumed the sugar under oxygen-limited conditions. The overall ethanol productivity of SSF was double that of SHF. An ethanol concentration of 20.6 g l-1; the highest concentration achieved in this study, was an improvement on the 14 g l-1 previously reported elsewhere. This study provided more information on the chemical composition of the O. ficus-indica cladode, particularly regarding its constituent carbohydrates, and also highlighted the feasibility of ethanol production from the cladodes, albeit at low concentrations from an industrial point of view. K. marxianus demonstrated its potential as an alternative to S. cerevisiae for bioethanol production from lignocellulosic biomass.

information indicate that CYP450s are prevalent in members of the bacterial phylum Deinococcus-Thermus as well as the archaeal family Halobacteriaceae that belong to the phyulm Euryarchaeota. A property shared by these phylogenetically distant extremophiles is the production of carotenoid pigments. It became the purpose of this study to use genome sequence information to clone and study new CYP450s from the genera Thermus and Halobacterium and to explore the role of these CYP450s in pigment production. The non-pigmented thermophilic bacterium Thermus scotoductus SA-01 was screened by PCR for the presence of a cytochrome P450 monooxygenase (CYP450). No CYP450 could be found and subsequent genome sequencing confirmed this finding. However, a CYP450 gene (CYP175A) was isolated from the closely related yellow pigmented strain Thermus sp. NMX2.A1 using oligonucleotides based on the DNA sequence of the β-carotene gene cluster from three Thermus strains. The genome sequence of T. scotoductus SA-01, revealed a ferredoxin (Fdx) and ferredoxin reductase (FNR) that were almost identical to those of Thermus thermophilus HB27. In T. thermophilus HB27 the Fdx and FNR are the native redox partners for CYP175A1, a β- carotene hydroxylase. After heterologous expression in Escherichia coli, we attempted to hydroxylate β-carotene with the CYP450 from Thermus sp. NMX2.A1 and the redox partners of T. scotoductus SA-01 using cell free extracts, but no products were detected. Thirty two CYP450s have been identified in the sequenced genomes of thirteen extremely halophilic archaea. Initial attempts to clone and heterologously express a CYP174A2- homologue from a Haloarcula LK-1 strain in E. coli and Pseudomonas fluorescens were unsuccessful. In order to study the physiological role of CYP450s in halophilic archaea and to create a strain that can be used for heterologous expression of CYP450s from halophiles CYP174A1 was deleted from H. salinarum R1. CYP174A1 is the only CYP450 in H. salinarum R1 and H. salinarum R1 is a genetically tractable strain. Upon culturing the wildtype and deletion strains, a difference in red pigmentation of stationary phase cultures was observed; implying that CYP174A1 might play a role in carotenoid synthesis. Microarray analyses revealed that the bop gene, which codes for bacterioopsin (BO) was severely repressed in stationary phase cultures of the deletion strain and sucrose gradient experiments showed a consequent loss of purple membrane (PM) in the deletion strain. The classical causes of bop repression e.g. insertion elements in the bop open reading frame as well as in the brz gene was ruled out by PCR screening. In addition to bop repression, the neighboring vng1459 and vng1468 genes (both part of the bopregulon) were also down regulated, but the genes normally involved in regulation of the bop gene were not affected. Currently the functions of vng1459 and vng1468 are unknown. Retinal, together with BO, is a key component of bacteriorhodopsin (BR) and essential for PM synthesis. Retinal is formed by the central cleavage of β-carotene which can be achieved by monooxygenases or dioxygenases.The Blh and Brp proteins in H. bacterium salinarum are very closely related to a confirmed bacterial 15,15â²-β-carotene dioxygenase and studies have shown that deletion of both brp and blh results in complete abolishment of retinal and BR. It is therefore unlikely that CYP174A1 plays a role in retinal biosynthesis. Another possible function for CYP174A1 might be the hydroxylation of β-carotene, since it is known that H. salinarum strains produce hydroxylated carotenoids such as transastaxanthin, but no genes encoding typical β-carotene hydroxylases or ketolases have been identified in the genomes of H. salinarum strains. This will imply that hydroxylated carotenoids play a role in the regulation of bop.

Candida albicans and C. dubliniensis are commensals of the gastrointestinal and genitourinary tract in healthy individuals. However, in diseased individuals they can cause superficial infections to deep seated mycoses. Both species form mycelial networks called biofilms, and formation of biofilms results in increased resistance towards antifungal compounds currently in use. Therefore, there is a need for alternative antifungal compounds such as fatty acids. Research has shown that supplementation of growth medium with polyunsaturated fatty acids (PUFAs), increased the unsaturation index and made cells susceptible to lipid peroxidation and cell death. During this study this phenomenon was evaluated on biofilms of C. albicans and C. dubliniensis using selected PUFAs. Due to differences in the carbon chain length and saturation of fatty acids, they interact differently with the cell membrane and will have different peroxidisability values. The results from the study showed C18:4 n-3 and C20:5 n-3 were taken in by the cell and resulted in increased unsaturation index. The results further indicated oxidative stress-induced apoptosis following supplementation with C18:4 n-3 and C20:5 n-3 in biofilms of both C. albicans and C. dubliniensis. The induction of apoptosis following supplementation by C18:4 n-3 and C20:5 n-3 was confirmed by mitochondrial membrane potential assay, Annexin V-FITC staining, TUNEL assay and DAPI staining. The use of C18:4 n-3 in synergism with amphotericin B resulted in decreased dosage of the antifungal compound needed to inhibit biofilms of C. albicans and C. dubliniensis.