Department of Microbiology

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    50 years of Emmonsia disease in humans : the dramatic emergence of a cluster of novel fungal pathogens
    (Public Library of Science, 2015) Schwartz, Ilan S.; Kenyon, Chris; Feng, Peiying; Govender, Nelesh P.; Dukik, Karolina; Sigler, Lynne; Jiang, Yanping; Stielow, J. Benjamin; Munoz, Jose F.; Cuomo, Christina A.; Botha, Alfred; Stchigel, Alberto M.; De Hoog, G. Sybren
    New species of Emmonsia-like fungi, with phylogenetic and clinical similarities to Blastomyces and Histoplasma, have emerged as causes of systemic human mycoses worldwide. They differ from classical Emmonsia species by producing a thermally-dependent, yeast-like phase rather than adiaspores, and by causing disseminated infections, predominantly in immunocompromised patients and often with high case-fatality rates. Such differences will be important for clinicians to consider in diagnosis and patient management, and for microbiologists who may encounter these fungi with increasing frequency.
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    The ability of antimicrobial peptides to migrate across the gastrointestinal epithelial and vascular endothelial barriers
    (Stellenbosch : Stellenbosch University, 2018-03) Dreyer, Leane; Smith, Carine; Dicks, Leon Milner Theodore; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Antibiotic resistance has become a major threat to humankind, necessitating research and development of alternative antimicrobial compounds. Many bacteria, including lactic acid bacteria, produce small antimicrobial peptides, referred to as bacteriocins. These peptides are generally not toxic, are active at low concentrations and have a narrow spectrum of antimicrobial activity. However, they usually have low in vivo stability due to degradation by proteolytic enzymes. Drug delivery systems are thus required to transport bacteriocins to the site of infection. Probiotic bacteria are an attractive delivery system, since these bacteria normally colonise the gastrointestinal tract and produce bacteriocins. Numerous studies have been done on the probiotic Entiro™ which consists of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA. The bacteriocins produced by these strains, plantaricin 423 and bacST4SA, respectively, are active against a variety of pathogens and are potential alternatives to antibiotics. However, it is unknown whether these bacteriocins are able to migrate across the gastrointestinal epithelium and vascular endothelium in order to enter the bloodstream. The aim of this study was to evaluate the stability, cytotoxicity and permeability of plantaricin 423 and bacST4SA in vitro and evaluate the potential use of these peptides as an alternative to antibiotics. The well-known lantibiotic, Nisin A, produced by Lactococcus lactis subsp lactis, was used as control and Listeria monocytogenes EGDe as target (sensitive) organism. Migration of the lantibiotic, nisin A and class IIa bacteriocins, plantaricin 423 and bacST4SA across simulated models of the vascular endothelial and gastrointestinal epithelial barriers was studied by growing human umbilical vein endothelial cells (HUVEC)- and human colonic adenocarcinoma (Caco-2) cells on transmigration inserts and adding fluorescently labelled nisin A, plantaricin 423 and bacST4SA to the inserts. All three peptides diffused across HUVECs and Caco2 cells. Only 21% nisin A, 11% plantaricin 423 and 12% bacST4SA remained attached to Caco-2 cells and only 6% nisin A and 3% bacST4SA attached to the HUVECs, and plantaricin 423 did not attach. The viability of both cell types remained unchanged when exposed to 50 μM nisin A, 50 μM plantaricin 423 and 50 μM bacST4SA, respectively. Furthermore, little extracellular lactate dehydrogenase (LDH) activity was recorded when cells were exposed to 100 μM of each peptide, suggesting that the peptides are not cytotoxic. The three peptides retained 60% of their antimicrobial activity when 25 μM of each were exposed to 80% human plasma for 24 h. However, at higher concentrations (50 μM) 68% of the original antimicrobial activity was recorded and at 100 μM the peptides retained 79% of their activity. This is the first report of nisin A, plantaricin 423 and bacST4SA migrating across simulated gastrointestinal- and vascular barriers. In vivo studies are required to confirm these findings and determine the effect these peptides may have in the treatment of systemic infections.
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    The abundance and diversity of Acidobacteria in fynbos soil: a closer look at culturability and function
    (Stellenbosch : Stellenbosch University, 2021-04) Conradie, Tersia Andrea; Jacobs, Karin; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH SUMMARY: The Acidobacteria are considered to be one of the most widespread and highly abundant soil bacterial phyla. This phylum was first described in 1997 with only three cultured representatives. Currently, the Acidobacteria is divided into 15 class-level subdivisions, of which only 5 subdivisions contain the 62 successfully cultured and fully described species in the Acidobacteria. The reason for their low representation in culture collections, is partly due to their unculturable, or difficult to culture nature. The application of 16S rRNA gene techniques has revealed that this phylum can represent almost 50% of the soil bacterial community, averaging around 10-20% of the global soil bacteria. Their proliferation in soils suggests that the Acidobacteria play an important role in biogeochemical processes. Microorganisms are an essential part of the terrestrial environment and are important in maintaining ecological functions. These functions are especially important in biomes where nutrient availability is low, and plants depend on their symbiotic relationships with the soil microbiome. One example of such an environment include the fynbos biome in the Cape Floristic Region (CFR) of South Africa. Despite the dominant presence of the Acidobacteria in several habitats, little is still known about their diversity and distribution in the fynbos biome. The aim of this study, therefore, was to explore the Acidobacterial communities in fynbos soils with the use of 16S rRNA gene sequencing, and how they respond to environmental change, as well as agricultural practices. Further, we aimed to isolate as many species as possible from the fynbos biome, and taxonomically characterise novel species. In Chapter 2, we explored the distribution patterns of the Acidobacteria in different fynbos soils from native conservation areas, and in Chapter 3 how the Acidobacteria responds to seasonal changes and the cultivation of Aspalathus linearis (rooibos) and Cyclopia spp. (honeybush), two indigenous plants used in commercial agriculture. A total of 27 soil samples were collected at three nature reserves, namely Jonkershoek, Hottentots Holland, and Kogelberg. In addition, data from two previous studies from our research group, with GenBank accession numbers DRA003953 for Cyclopia spp. (honeybush) and DRA004000 for Aspalathus linearis (rooibos), were included in our analysis. A total of 33 acidobacterial operational taxonomic units (OTUs) were identified in the nature reserve samples, and a total of 32 and 31 OTUs were identified for honeybush and rooibos, respectively. The majority of OTUs in all samples were classified as representatives of subdivisions 1, 2, and 3. Significant differences were observed in the distribution and composition of these OTUs between nature reserves, between seasons for both honeybush and rooibos, as well as between the agricultural practices in some cases. Several OTUs and subdivisions correlated significantly with soil pH, potassium, phosphorus, and in some instances carbon and calcium. In Chapter 4, we successfully isolated two novel species, both within subdivision 1. We proposed the classification of strain HDX4T as the type strain of Edaphobacter sabuleum nom. prov., and strain ADX1T as the type strain of Terriglobus capensi nom. prov. Based on the genome sequences, both strains had the genomic potential for several complete carbohydrate metabolic pathways, organic nitrogen metabolism, as well as several survival mechanisms that contributes to their survival in the soil environment. In short, this study has contributed greatly to our knowledge of the Acidobacteria and their distribution and diversity in the fynbos biome. The successful isolation of two novel species were added to the list of cultivated Acidobacteria from around the globe.
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    Anaerobic digestion application in the treatment of gelatin-manufacturing effluent
    (Stellenbosch : Stellenbosch University, 2000-12) Lloyd, Magaretha Hester; Van der Merwe-Botha, M.; Britz, T. J.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: A severely polluted industrial effluent is generated by the local gelatinmanufacturing industry. Due to increasingly stringent restrictions on discharge qualities enforced by the National Water Act of 1998 and National Environmental Management Act of 1998, as well as increasing trade-effluent charges implemented via the Local Municipal Bylaws, the industry is compelled to consider a system to pre-treat the polluted effluent. A study was undertaken to examine the viability of anaerobic treatment of the gelatin-manufacturing effluent, since the anaerobic digestion technology is well recognised for the high success rate in the treatment of high-strength, complex wastewaters. Various laboratory and pilot-scale studies were done, using different hybrid Upflow Anaerobic Sludge Blanket (UASB) and contact designs. Two mesophilic laboratory-scale hybrid UASB digester designs, fitted with polyethylene (AD-1) and polyurethane (AD-2), performed well at a hydraulic retention time (HRT) of 1.0 d. Chemical oxygen demand (COD) removal efficiencies of up to 90% (avg. 53%) for AD-1 and 83% (avg. 60%) for AD-2 at organic loading rates (OLR) of 9.56 and 4.62 kg COD.m-3.d-1, respectively, were obtained. High sulphate (S04) removal efficiencies of up to 96% (avg. 86%) for AD-1 and 98% (avg. 82%) for AD-2 were also achieved, respectively. A maximum total solid (TS) removal of 65% (avg. 25%) for AD-1 and 62% (avg. 28%) for AD-2 was reported. An average methane content of 80% (AD-1) and 79% (AD-2) with average methane yields per COD removed of 2.19 and 1.86 m3. kg CODremoved.df-o1r AD-1 and AD-2 were found, respectively. When the same digesters (AD-1 and AD-2) were combined in a muItiphase series configuration, a total COD removal efficiency of up to 97% (avg. 80%) at an OLR of 8.32 kg COD.m-3.d-1,was achieved. Excellent total S04 removals of 96% (avg. 69%) were accomplished. Up to 82% TS (avg. 29%) was also removed during this study and the biogas consisted of 89% methane (avg. 79%). For this multi-phase combination up to 92% volatile fatty acids (VFA) (avg. 48%) were removed, indicating possible selective phase separation of the respective fatty acid producing/utilising bacterial populations. The use of a laboratory-scale UASB bioreactor with recirculation, resulted in COD removal efficiencies of up to 96% (avg. 51%) at an HRT of 3.0 d, and 95% (avg. 54%) at a HRT of 1.0 d. Low performances were generally found, with average S04 and TS removals of 59% (max. 97%) and 26% (max. 67%), respectively at an HRT of 1.0 d. The biogas production was very low throughout the study (0.05 - 0.63 I,d-1 ). A pilot-scale UASB reactor (300 I) was constructed and performed satisfactory with a 58% average COD removal and maximum of 96%. S04 and TS removals up to 96% (avg. 44%) and 93% (avg. 63%), respectively, were obtained. The methane content of the biogas was 85%. The pilot-scale studies were conducted under actual field conditions, where various shock and organic loads had to be absorbed by the system. The pilot-scale contact configuration (300 I) did not perform satisfactory as a result of continuous blockages experienced in the feed and recirculation lines. Maximum COD, S04, VFA and TS removal efficiencies of 41% (avg. 27%), 62% (avg. 41%), 64% (avg. 27%) and 39% (avg. 21%), respectively, were obtained. The results of all the studies indicated acceptable COD removals with increasing OLR's. Indications of the presence of active methanogenic and sulphate-reducing bacterial populations were apparent throughout the studies. One possibility for the successful start-up and commissioning of the anaerobic reactors was the use of a well-adjusted biomass, which consisted of highly selected and adapted microbial consortium for the specific gelatinmanufacturing effluent. It was clear from this study that gelatin-manufacturing effluent can be treated successfully, especially with the use of the UASB design. A welldefined data base was constructed which could be of great value for further upscaling to a full-scale digester.
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    Analysis of an 18kb accessory region of plasmid pTcM1 from Acidithiobacillus caldus MNG
    (Stellenbosch : University of Stellenbosch, 2009-03) Louw, Lilly-Ann; Rawlings, Douglas E.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
    Biomining organisms are generally found in metal-rich, inorganic environments such as iron and sulfur containing ores; where they play a vital role in mineralization and decomposition of minerals. They are typically obligatory acidophilic, mesophilic or thermophilic, autotrophic, usually aerobic, iron-or sulfur oxidizing chemolithotrophic bacteria. The most prominent biomining organisms used in bioleaching of metal sulfides are Acidithiobacillus ferrooxidans, At. thiooxidans, At. caldus, Sulfobacillus spp. and Leptospirillum spp. Biomining enables us to utilize low grade ores that would not have been utilized by conventional methods of mining. Research has focused on the backbone features of plasmids isolated from bacteria of biomining environments. The aim of this study is to sequence and analyze an 18 kb region of the 66 kb plasmid pTcM1 isolated from At. caldus MNG, focusing on accessory genes carried by this plasmid. Fifteen putative genes / open reading frames were identified with functions relating to metabolism and transport systems. The genes are located in two divergently located operons. The first operon carries features related to general metabolism activities and consists of a transcriptional regulator (ORF 2), a succinate / fumarate dehydrogenase-like subunit (ORF 3), two ferredoxin genes (ORF 4 and ORF 7), a putative HEAT-like repeat (ORF 6) which is interrupted by an insertion sequence (ORF 5) and a GOGAT-like subunit (ORF 8). The second operon contains an ABC-type nitrate / sulfonate bicarbonate-like gene (ORF 9), a binding protein-dependent inner membrane component-like gene, another ABC sulfonate / nitrate-like gene (ORF 12i and 12ii) which is interrupted by an insertion sequence (ORF 13) and two hypothetical proteins with unknown functions (ORF 14 and ORF 15). Southern hybridization analysis have shown that most of the genes from the two operons are found in other At caldus strains #6, “f”, C-SH12 and BC13 from different geographical locations. Expression of the GOGAT-like subunit and the succinate / fumarate-like subunit was demonstrated in At. caldus MNG showing that these genes are functional and actively transcribed. The transcriptional regulator (ORF 2) has been shown to repress the downstream genes of putative operon 1. The persistence of these genes on plasmids together with the fact that they are being expressed, represents a potential metabolic burden, which begs the question why they have been maintained on the plasmid from geographically separated strains (and perhaps also growing under very different nutrient availability conditions) and therefore what possible role they may play.
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    Analysis of arsenic resistance in the biomining bacterium, Acidithiobacillus caldus
    (Stellenbosch : University of Stellenbosch, 2007-03) Kotze, Andries Albertus; Rawlings, Douglas E.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: In this study the chromosomal arsenic resistance (ars) genes shown to be present in all Acidithiobacillus. caldus isolates were cloned and sequenced from At. caldus #6. Ten open reading frames (ORFs) were identified on a clone conferring arsenic resistance, with three homologs to arsenic genes, arsC (arsenate reductase), arsR (regulator) and arsB (arsenite export). This ars operon is divergent, with the arsRC and arsB genes transcribed in opposite directions. Analysis of the putative amino acid sequences of these arsRC and arsB genes revealed that they are the most closely related to the ars genes of Acidithiobacillus ferrooxidans. These ars genes were functional when transformed into an Escherichia coli ars deletion mutant ACSH50Iq, and conferred increased levels of resistance to arsenate and arsenite. ArsC was required for resistance to arsenate, but not for resistance to arsenite. None of the other ORFs enhanced arsenic resistance in E. coli. A transposon located arsenic resistance system (TnAtcArs) has been described for highly arsenic resistant strains of the moderately thermophilic, sulfur-oxidizing, biomining bacterium At .caldus #6. In the latter study it was shown that TnAtcArs confers higher levels of resistance to arsenate and arsenite than the chromosomal operon. TnAtcArs was conjugated into a weakly ars resistant At. caldus strain (C-SH12) and resulted in greatly increased arsenite resistance. RT-PCR analysis revealed that arsR and arsC are co-transcribed. Despite ORF1 (cadmium inducible-like protein) and ORF5 (putative integrase for prophage CP-933R) not being involved in resistance to arsenic, ORF1 was co-transcribed with arsRC and ORF5 with arsB. Using arsR-lacZ and arsB-lacZ fusions it was shown that the chromosomal ArsR-like regulator of At. caldus acts as a repressor of the arsR and arsB promoter expression. Induction of gene expression took place when either arsenate or arsenite was added. The chromosomal located ArsR was also able to repress TnAtcArs, but the transposon-located ArsR was unable to regulate the chromosomal system.
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    An analysis of glycerol synthesis by Saccharomyces cerevisiae
    (Stellenbosch : Stellenbosch University, 2002-12) Cronwright, Garth Rupert, 1974-; Prior, B. A.; Rohwer, J. M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Glycerol metabolism is paramount to the physiological adaptation by Saccharomyces cerevisiae to hyper-osmotic stress conditions. Glycerol metabolism also -plays a fundamental role in maintaining a redox state favourable for growth under fermentative conditions. All aspects of the relationship between redox balancing and glycerol metabolism are not yet fully defined and attempts to manipulate this relationship, i.e., to increase or decrease glycerol yields from fermentation, result in a redox disturbance that is often detrimental to other aspects of metabolism. Another aspect of glycerol metabolism that is not thoroughly understood, is how the various parameters of the glycerol synthesis pathway, each independently and in conjunction with each other, control the rate at which glycerol is synthesized. Addressing these questions has been the topic of this thesis. In this regard, the theory of metabolic control analysis (MeA) was adopted and calculations were performed with the aid of an experimentally validated kinetic model. To ascertain the in vivo substrate, product, coenzyme and known modifier concentrations of the glycerol synthesis pathway, reliable techniques to halt metabolism, extract and measure these metabolites had to be established. The metabolite concentrations constitute a portion of the parameters of the pathway and are necessary to construct a detailed kinetic model. Measuring the concentration of an intracellular metabolite enzymatically requires the cell extract to have an adequate quantity of the metabolite in question. This may be achieved by concentrating the cells, before extracting the metabolite, by means of rapid filtration. Then by freezing the cells with liquid nitrogen, metabolism can be halted instantly. It was found that when metabolites were measured, yields were largely dependent on the method of extraction, since different metabolites are sensitive to different pH and temperature conditions. Methods of extraction found to be reliable for the metabolites of interest in this study are presented in Chapter 3. Metabolic control coefficients calculated by the model helped identify the parameters that control flux through the glycerol synthesis pathway most rigidly. The first reaction of the pathway, catalyzed by NAO+-dependent glycerol 3- phosphate dehydrogenase, had a flux control coefficient ( c; )of 0.83 to 0.87 and exercises the majority of control of flux through the pathway, while the subsequent reaction, catalyzed by glycerol 3-phosphatase, had far less control (C:2 = 0.13 to 0.17). The response coefficients (RJ ) of various parameter metabolites indicate [x] that flux through the pathway is most responsive to the concentration of the substrate, DHAP (RJ = 0.48 to 0.69), followed by the concentration of the [DHAP] inhibitor, ATP (RJ =-0.21 to -0.5). Interestingly, the model also predicts that [ATP] the pathway responds far more severely to the ATP/ADP ratio than to the NADH/NAD ratio, because of the weak response coefficient attributed to NADH (RJ = 0.03 to 0.08). Thus, the model suggests that the targets most strategic [NADH] for altering glycerol synthesis would be the Vmax of the glycerol 3-phosphate dehydrogenase reaction and the concentrations of DHAP and ATP. Ideally, the approach would entail manipulating each of these parameters to their optimal levels in conjunction with each other, with the least detrimental physiological effect possible.
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    An analysis of population structure using microsatellite DNA in twelve Southern African populations of the Mozambique tilapia, Oreochromis mossambicus (Peters)
    (Stellenbosch : Stellenbosch University, 2001-12) Hall, Edward G.; Lewis, Rupert; Brink, Danie; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: DNA micro satellite loci express extensive allelic variation making them convenient markers for research in many fields employing population genetic tools, including aquaculture and conservation genetics. Twelve Oreochromis mossambicus populations from wild, captive and introduced sources in Southern Africa were screened for genetic variation at ten CA repeat micro satellite loci. Three of the loci - UNHI04, UNHlll, and UNH123 - were sufficiently well resolved to screen extensively and were interpreted according to a model of Mendelian inheritance. Data was analyzed in terms of genetic structure and levels of genetic variation, the effect of management regime in captivity through successive generations on genetic diversity, and the nature of phylogenetic relationships present between populations. Exact tests, carried out using Monte Carlo type multiple resampling techniques, and F-Statistics were used to detect and quantify genetic structure among the twelve populations. The Exact test X2 (P < 0.001), a FST of 0.27 (P < 0.001), eST of 0.26, RsT of 0.28, and a ST of 0.17 all indicated significant structuring among the populations. The evident genetic structuring endorsed the practice of maintaining the populations as separate genetic stocks, in separate tanks, in order to preserve unique genetic material for aquaculture strain development. Populations also exhibited some significant deviations from Hardy Weinberg equilibrium characterised by an overall reduced heterozygosity across the loci. In microsatellite studies, null alleles are often suggested as major contributors to heterozygote deficits. To test for null alleles, two controlled crosses of 0. mossambicus were made. The progeny from each cross were examined for expected parental allelic ratios at the UNHI04, UNHlll and UNH123 loci. All three loci presented evidence of possible null alleles. Accelerated inbreeding and genetic drift through successive generations in captivity can reduce heterozygosity and gene diversity. To investigate loss of diversity a sample taken from the Bushmans population in 1999 (N = 25) was compared with a Bushmans 2000 sample (N = 36). The comparison highlighted altered allele frequencies, a significant increase in average observed heterozygosity and a non-significant change in average expected heterozygosity using the UNHI04 and UNH123 loci. Calculation of genetic distances and phylogenetic comparisons between the populations provided insight into the degree of management required in conserving genetic diversity in natural populations of Mozambique tilapia. UPGMA and Neighbour-Joining techniques were used to construct phylogenetic trees using Dm and ({)~)2 distance matrices. Clustering of populations appeared to reflect geographic locality of the source populations, however certain populations were not congruent with geography. Mantel tests were used to expose a possible association between genetic distance matrices generated from each individual locus. An association would support a geographic background to population genetic structure. The Mantel tests did not provide conclusive evidence. Mantel tests for association between the combined locus Dm and (81l)2 genetic distance matrices and a geographic distance matrix were similarly non-significant. Multi-dimensional scaling (MDS) plots of Euclidean distance values for Dm and (81l)2 matrices presented a two-dimensional view of the genetic distance data. The degree of similarity with the UPGMA and Neighbour-Joining tree-clustering pattern was higher for the (81l)2 than for the Dm MDS plots. Scatter plots indicated a reliable non-linear correlation between Euclidean distance and genetic distance for the two-dimensional MDS. The micro satellite markers employed in this research provided molecular information needed for complimenting a co-study on quantitative genetic evaluation of the twelve populations. The quantitative co-study provided measures of average length and weight gain indices for the populations based on progeny growth trials. No significant correlation of average heterozygosity (gene diversity) with either average weight or length gain was found. The significant genetic diversity and structure present between the twelve populations provided rationale for implementing strategies to conserve natural 0. mossambicus populations as genetic resources, and manage captive populations for long term maintenance of genetic diversity.
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    Analysis of the mobilization region of the broad host-range IncQ-like plasmid, pTC-F14, and its ability to interact with a related plasmid, pTF-FC2
    (Stellenbosch : Stellenbosch University, 2003-12) Van Zyl, Leonardo Joaquim; Rawlings, D. E.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: The 14.2 kb plasmid pTC-FI4 was isolated from the moderately thermophilic (45°- 50°C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic bacterium Acidithiobacil/us caldus and has a replicon that is closely related to the promiscuous, broad host-range, IncQ-family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins and an origin of transfer, which are related to the DNA processing (Tral) region of IncPI plasmids, rather than to the three Mob protein systems of the IncQ-l-group plasmids (e.g. plasmids RSFIOIO or R1162). Plasmid pTC-F14 is the third example of an IncQ family plasmid that has five mob genes, with the others being pTF-FC2 and pRAS3.1. The minimal region that was essential for mobilization included the mobA, mobB and the mobC genes as well as the oriT. The mobD and mobE genes were non-essential, but together enhanced the mobilization frequency by approximately 300-fold. The repB gene increased the mobilization frequency but was not essential for mobilization. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3500-fold less efficient than the mobilization ofpTF-FC2. When both plasmids were co-resident in the same E. coli host, pTC-FI4 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-FI4 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. pTF-FC2 could mobilize the oriT of pTC-FI4 whereas pTC-F14 could only mobilize the pTFFC2 oriT if provided with some of the mobilization genes from the pTC-FC2 mobilization region. Unexpectedly either the mobEDC genes or the mobAB genes would allow the mobilization of the pTF-FC2 oriT by pTC-F14 even though there was no common gene between the two subsets. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.
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    Antibiotic resistance in surface waters and biofilm-response to environmental contaminants
    (Stellenbosch : Stellenbosch University, 2021-12) Tucker, Keira; Wolfaardt, Gideon M.; Botes, Marelize; Feil, Edward; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Ensuring water security for the future has become important due to rapid urbanisation and diminishing freshwater resources. South Africa’s water resources are scarce and as a result, reclamation of alternative freshwater resources such as treated wastewater is being investigated. There is growing evidence that drinking and wastewater treatment is either non- compliant to quality standards or lacking in certain communities. In areas with no infrastructure for wastewater removal, open sewers create a health risk for humans, animals, and the environment. Poor antimicrobial stewardship, over-use and incorrect disposal has led to increased resistance to antibiotics, rendering some bacterial infections untreatable. There is a concern that sub- inhibitory concentrations of antibiotics create a selection pressure that promotes horizontal gene transfer and emergence of bacterial communities that are resistant to antibiotics. Antibiotics, antibiotic resistant bacteria (ARB), as well as other contaminants that have been shown to promote antimicrobial resistance (AMR) such as heavy metals, enter surface waters and wastewater treatment works (WWTW) in trace concentrations via multiple pathways. As a result, WWTW are deemed hotspots for the emergence and dissemination of AMR. In addition, environmental waters are home to various matrices, including biofilms that are especially problematic in a clinical setting due to their antibiotic resistant and persistent nature. The research presented in this dissertation aimed to contribute to the knowledge surrounding the abundance of ARB in WWTW and surface waters in a South African context. Although ARB and antibiotic resistance genes (ARG) were detected in WWTW effluent, the abundance of both were reduced compared to the influent, suggesting that WWTW played a role in reducing AMR in receiving waters, while exposure to sub-inhibitory concentrations of antibiotics did not result in a significant change in the number of target ARG in isolates selected as representatives of a cultured population. This was emphasised in an expanded study that monitored various regions over a year. In addition, it was shown that surface waters, biofilms and sediments influenced by anthropogenic activities from residential and industrial sectors had higher prevalence of ARB compared to samples influenced by agricultural activity. Metagenomic analysis revealed that ARG relating to efflux pumps were the most common compared to those specific for target antibiotics. Due to heavy-metals and antibiotics being present in the environment in trace concentrations, exposure of mixed-community biofilms to sub-inhibitory concentrations of these contaminants was investigated. AMR in the biofilms did not increase, but it was suggested that the sub-inhibitory exposure promoted the development of persistent mixed community biofilms. Treatment interventions are crucial for removing pollutants and AMR already present in the environment. However, with due recognition of the complexity involved when considering humans, animals, the environment and a diverse pool of contaminants, this dissertation argues the need to expand the approach for mitigation of emergence or dissemination of AMR in the environment by incorporating greater emphasis on antibiotic stewardship, policies around antibiotic usage in all sectors, and overall public awareness.
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    Antimicrobial susceptibility and population dynamics of a defined biofilm community under different nutrient conditions
    (Stellenbosch : Stellenbosch University, 2004-03) Garny, Kerstin; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Little is known about the impact of nutrient conditions on antimicrobial resistance in biofilms grown under continuous flow conditions. Furthermore, community-level response of biofilms to antimicrobial substances and different nutrient regimes are poorly described. A better understanding of the influence of environmental conditions on biofilm behavior and antimicrobial susceptibility may contribute to the efforts, addressing the problems associated with increased antimicrobial resistance. Therefore, the aim of this study was to evaluate the survival and population dynamics in a defined mixed-species biofilm community grown under different nutrient conditions and when subjected to biocide treatment. Epi-fluorescence microscopy in conjunction with the LIVE/DEAD® BacLight™ viability kit, a conventional cultivation technique (plate counts), and culture-independent techniques (terminal restriction fragment length polymorphism and fluorescent in situ hybridization) were applied to observe biofilm and planktonic antimicrobial susceptibility, as well as population dynamics. A defined mixed-species community, consisting of four bacterial strains, was cultivated and monitored in a flow cell system. Two nutrient types were used: 1) a complex growth medium [tryptone soy broth (TSB)] and 2) a defined synthetic medium [minimal salts supplemented with glucose (MSM + Glucose)]. In addition, these two nutrient types were applied in different concentrations. Biofilm and planktonic community behavior was influenced by the nutrient type and concentration. Species evenness in the planktonic community was influenced by the nutrient conditions, while species richness changed in response to biocide treatment and nutrient conditions. TSB-grown microbial communities were more susceptible directly after biocide treatment than those grown in MSM + Glucose, however, biofilm viability in the latter nutrient condition decreased within 24 h after biocide treatment. Furthermore, a surprising difference in the recovery rate between biofilm and associated planktonic communities was observed. A conceptual model was developed that aimed to explain the observed biofilm-planktonic interactions. This model proposes that the cells found in the outer regions of a biofilm are the primary source of the associated planktonic cells, and that this phenomenon is independent from overall biofilm activity.
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    The application of astaxanthin producing bacteria in poultry feed
    (Stellenbosch : Stellenbosch University, 2017-03) Conradie, Tersia Andrea; Jacobs, Karin; Pieterse, Elsje; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: In the food industry, the colour of the product is important to the consumer as it gives an indication of the freshness and quality of the product. Hens are not able to produce pigments and absorb pigments through their diet. This has led to a rapidly emerging trend in poultry farming to enhance egg yolk colour as the yolk colour is influenced by the diet of the hen. Over the years, natural or synthetic carotenoids have been added to poultry feed. Several studies have focused on using astaxanthin producing microorganisms, such as the microalga, Haematococcus pluvialis, and yeast, Xanthophyllomyces dendrorhous. However, the production costs are expensive and the thick cell walls of the microalga and yeast limits its whole cell application as a feed additive. Some bacterial species are also able to produce astaxanthin, including the bacterium Paracoccus marcusii, and have previously not been used as a feed additive to enhance yolk colour. The purpose of this study was, therefore, to determine the whole cell application of P. marcusii as a feed additive to enhance egg yolk colour, without the need to homogenise the cells or extract the pigment. In the first experimental chapter (Chapter 2), the growth conditions and astaxanthin production of P. marcusii was optimised. Furthermore, the stability of the astaxanthin molecule under different storage conditions, namely lyophilisation and microencapsulation, was determined. The optimum growth conditions for P. marcusii and for astaxanthin production was at 26 °C in a specialised medium containing yeast extract (5 g/L), bacteriological peptone (10 g/L) and NaCl (3%) at a pH between 6 – 7. Astaxanthin is a valuable compound with several health promoting benefits for humans and animals. However, the molecule is unstable when exposed to oxygen, light and temperature. After lyophilisation in sucrose (10% m/v), there was an 85% loss in astaxanthin concentration after 3 weeks. However, the loss in cell viability was low. When P. marcusii was microencapsulated in calcium alginate beads, cell viability significantly decreased when stored at 20 °C compared to 4 °C. However, only 30% of the total astaxanthin concentration was lost after 3 weeks at both storage temperatures. The microencapsulation significantly improved the stability of astaxanthin under storage. The highest concentration of astaxanthin obtained was 24.25 μg/g dry cell weight. Chapter 3 examined the pigmentation effect of P. marcusii when fed to laying hens to enhance egg yolk colour. Paracoccus marcusii was fed to hens daily either in a sucrose solution (10% m/v) or microencapsulated in calcium alginate beads. After the pilot study, it was clear that a diet free of all pigments was needed to effectively determine the pigmentation effect of P. marcusii. In all the experimental trials there was a significant increase in yolk colour and no negative effect on egg quality, laying rate or hen weight was observed. There was also an increase in whole egg and yolk weight when compared to the control groups. Furthermore, the microbial communities of the duodenum and caeca were investigated after a prolonged feeding of P. marcusii to detect any changes that might have occurred (Chapter 4). The microbial community of the hen’s gastro-intestinal tract (GIT) starts out as a simple community in the small intestines which becomes increasingly diverse and complex further down the intestinal tract. The findings in this study revealed a similar pattern when considering the results obtained from the Shannon diversity index and total number of operational taxonomic units (OTUs). Starting in the duodenum, the index ranged between 2.14 – 2.59 and increased in the caeca to between 2.45 – 3.03. OTUs increased from 21.44 – 28.60 in the duodenum to 28.30 – 38.00 in the caeca. A significant difference was only observed for the OTUs of the experimental group compared to the control groups in both the duodenum and caeca. There was no significant difference observed in the microbial community structure of the duodenum. However, distinct patterns and clusters formed in the caeca between the experimental diet group compared to the control diet groups. Since no mortalities were recorded during the trial and all hens appeared in excellent health, it is safe to assume that the change in microbial community structure of the caeca was not negative. Therefore, P. marcusii is safe to use as a feed additive for laying hens. Finally, Chapter 5 evaluated the costs associated with the small-scale production of P. marcusii microencapsulated in calcium alginate beads and its feasibility in the poultry industry. Based on the economic assessment, the total cost for one month’s production of 210 g calcium alginate beads is estimated at R2912.88. This is too expensive and not practical to be used by poultry farmers. Possible solutions can include the use of inexpensive peptones, production on a larger scale and also increasing the concentration of bacterium encapsulated in the bead.
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    Application of solar pasteurization for the treatment of harvested rainwater
    (Stellenbosch : Stellenbosch University, 2017-03) Reyneke, Brandon; Khan, Wesaal; Khan, Sehaam; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Rainwater harvesting has been earmarked by South African governmental authorities as an intervention strategy that could alleviate the pressures on existing centralised water distribution systems, especially in rural areas and urban informal settlements, where insufficient waste removal and potable water infrastructure are available. However, numerous studies have indicated that harvested rainwater may not be safe to use for all daily water requirements, as numerous chemical and microbial contaminants may be associated with stored tank water. Rainwater treatment technologies, including solar pasteurization (SOPAS), have subsequently been investigated (Chapter 1). In order to determine whether decentralised rainwater harvesting SOPAS systems may be a viable alternative in providing the inhabitants of informal settlements with a supplementary water source, two small- (Sites 1 and 2) and one large-scale (Site 3) rainwater harvesting SOPAS systems were installed in Enkanini informal settlement, Stellenbosch, South Africa (Chapter 2). The microbial and chemical quality of the unpasteurized and pasteurized (produced by the respective systems) rainwater was monitored using conventional water quality monitoring techniques, including the culturing of indicator organisms, screening for selected indigenous rainwater pathogens using the polymerase chain reaction (PCR) and quantitative PCR (qPCR) assays and the monitoring of anion and cation concentrations. Additionally, the operational sustainability of the systems and water usage by the participating households were monitored. Chemical analyses indicated that all anions and cations were within the limits stipulated by various national and international drinking water quality guidelines, with the exception of zinc which contravened the respective guidelines before (mean: 3919 μg/L) and after (mean: 3964 μg/L) pasteurization at both Sites 1 and 2. In addition, the arsenic concentrations measured at Site 3 before (mean: 18.69 μg/L) and after (mean: 18.30 μg/L) pasteurization exceeded the respective drinking water guidelines. The increased zinc concentrations were attributed to the galvanised zinc roofing material installed at Sites 1 and 2, while the increased arsenic concentrations may be attributed to a roofing treatment or paint utilised to cover the catchment area at Site 3. Microbial analyses indicated that pasteurization temperatures of 53 °C (small-scale systems) and 55 °C (large-scale system) were required to reduce Escherichia coli and total and faecal coliforms to below the detection limit [< 1 colony forming units (CFU)/100 mL]. However, minimum pasteurization temperatures of 66 °C (small-scale systems) and 71 °C (large-scale system), were required to reduce the heterotrophic plate count (HPC) to within drinking water limits (1.0 × 104 CFU/100 mL). Of the opportunistic pathogens detected using PCR assays, Legionella spp. was the most prevalent pathogen detected in the small-scale systems [unpasteurized (100%) and pasteurized (91%)] and the large-scale system [unpasteurized (83%) and stored pasteurized tank water (100%)]. Quantitative PCR analysis then indicated that while the gene copies of Legionella spp., Pseudomonas spp. and Salmonella spp. were reduced during SOPAS, the organisms were still detected at the highest pasteurization temperatures analysed for each site (Site 1 – 85 °C; Site 2 – 66 °C; Site 3 – 79 °C). Additionally, the application of a metabolic responsiveness adenosine triphosphate (ATP) assay (BacTiter-GloTM Microbial Cell Viability Assay) indicated the presence of metabolically active cells in all pasteurized rainwater samples analysed. Results also indicated that the systems required limited maintenance and the small-scale systems in particular were able to provide the participating households with an alternative warm water source that could be utilised for numerous domestic purposes. As various limitations have been associated with the use of culture-based analyses for the monitoring of water quality, the aim of Chapter 3 was to compare molecular-based viability assays [ethidium monoazide bromide (EMA)-qPCR, propidium monoazide (PMA)-qPCR and DNase treatment in combination with qPCR] as well as the metabolic responsiveness ATP assay to culturing analysis for their ability to accurately determine cell viability in bacterial monocultures following heat treatment. Three Gram-negative (Legionella spp., Pseudomonas spp. and Salmonella spp.) and two Gram-positive (Staphylococcus spp. and Enterococcus spp.) bacteria commonly associated with water sources were selected as test organisms. Of the various concentrations of EMA and PMA analysed, 6 μM EMA and 50 μM PMA were identified as the optimal dye concentrations as low log reductions were recorded (viable and heat treated samples) in comparison to the no viability treatment control. Comparison of the results obtained for all the molecular viability assays (6 μM EMA, 50 μM PMA and DNase treatment) then indicated that the 6 μM EMA concentration was comparable to both the 50 μM PMA and the DNase treatment for the analysis of most of the test organisms (viable and heat treated). In addition, the results for the culturing analysis (CFU) of the viable S. typhimurium as well as the viable and heat treated samples of L. pneumophila and P. aeruginosa were comparable to the gene copies detected using molecular-based viability assays. However, the CFU in the heat treated samples of S. typhimurium were significantly lower than the gene copies detected using DNase in combination with qPCR, with no gene copies or CFU detected in the heat treated samples of S. aureus and E. faecalis. In contrast, while the ATP assays indicated the presence of metabolically active cells in the viable and heat treated samples, the ATP assay also indicated the presence of metabolically active cells in samples that had been autoclaved (negative viability control). It was thus concluded that molecular-based assays may be used to supplement culture based analysis for the comprehensive identification of the viable microbial population in water samples (before and after treatment).
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    Arbuscular mycorrhizal root colonisation and the subsequent host plant response in young grapevines in a South African commercial vineyard
    (Stellenbosch : Stellenbosch University, 2003-03) Meyer, Andre Harold; Botha, Alfred; Valentine, A. J.; Archer, E.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH SUMMARY: Arbuscular mycorrhizal (AM) fungi facilitate the uptake of nutrients, improve growth and alleviate drought stress in grapevines. Consequently, AM fungal root colonisation contributes to the optimum performance of grapevines. It is for this reason that young grapevines are sometimes inoculated with commercial AM fungal strains to reduce environmental stresses during transplant. In the past, soil fumigation has often been considered as a prerequisite for soil conditioning with commercial AM fungal strains. However, grape growers opting to inoculate with these fungal strains will have to do so in unfumigated soils, since the use of fumigants in South African agricultural soils is currently being phased out. Since little is known about the nature and scope of indigenous AM fungi that may be present in SA vineyard soils, it is difficult to predict the grapevine's response to artificial inoculation in soils already containing adequate concentrations of these fungi. In the first part of the study, commercially available AM inocula were tested under field conditions that would prevail on a typical farm. This entailed measuring vine growth, nutrition, drought stress resistance and percentage root colonisation, over two consecutive seasons, from the onset of planting new commercial grapevines. The field trial carried out at Groenland, a commercial farm in the Stellenbosch Region. Merlot grafted onto 101-14 Mgt, 110 Richter (110 R) and 99 Richter (99 R), was planted in December 1998. These rootstocks were selected to accommodate different soil forms: 101-14 Mgt and 110 R on a Westleigh soil form, which was ridged and 99 R on an unridged Fernwood soil form. Vine roots were inoculated during planting with different AM inocula, i.e. Biocult®, Vaminoc" and Glomus sp. 1054. One treatment was left uninoculated and treated with a combination of the fungicides Benlate" (active ingredient: benomyl) and Rovral Flo® (active ingredient: iprodione). The control received neither fungicides nor inocula. Microscopic examination of the vine roots revealed that, apart from a significantly higher level of root colonisation observed in Biocult-treated 99 R vines during the first season, the level of AM root colonisation was similar in both the uninoculated (control) and inoculated vines. Infected control vines indicated that indigenous AM fungi were present in the vineyard soil. This level ranged between 40% and 85%, and 70% and 90% in the first and second season, respectively. Apart from the significant growth improvement observed in 110 R vines inoculated with Glomus sp. 1054 during the first season, no growth improvement was observed for the other rootstocks or treatments. Furthermore, generally no alleviation of water stress and nutritional benefits could be detected for both the seasons. Despite this, less than 1% dieback was recorded for the vines. In the second part of the study, additional information on the diversity of indigenous AM fungal species was obtained, which included the quantification and identification of these fungi present in the soil. The AM fungal spore numbers in the vineyard soil ranged from 1000 to 3779 spores/100 g dry soil. The results confirmed that the majority of AM fungal species found in the soil was not part of the commercial inocula, but originated either from the vineyard and/or the nursery where the vines were obtained. The uncovered AM fungal species belong to the genera Acaulospora, Gigaspora, Glomus, Sclerocystis and Scutellospora. This is similar to the AM fungal genera recorded in vineyards by other workers. To the best of our knowledge, this study provided the first documented evidence on the diversity of indigenous AM fungi present in SA vineyard soils. Although it may be preliminary in nature, the results clearly showed that a wide diversity and abundance of indigenous AM fungal populations may occur in a typical SA vineyard. Depending on the superiority and possible masking effects on the part of the indigenous AM fungal populations, positive responses to inoculation with commercial AM fungal strains in grapevines grown in such vineyard soils may consequently be unlikely. Thus, before reconditioning of vineyard soils with these fungi can commence, it is essential for farmers to first assess the mycorrhizal status of their soils and nursery vines. Since the majority of SA farmers are not yet familiar with inoculation practices and are still unacquainted with the mycorrhizal status of their soils, the findings from this study could be of great benefit to particularly wine grape growers opting to inoculate with commercial AM fungal strains on a large-scale.
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    Architecture and gene repertoire of the flexible genome of the extreme acidophile Acidithiobacillus caldus
    (Public Library of Science, 2013-11-08) Acuna, Lillian G.; Cardenas, Juan Pablo; Covarrubias, Paulo C.; Haristoy, Juan Jose; Flores, Rodrigo; Nunez, Harold; Riadi, Gonzalo; Shmaryahu, Amir; Valdes, Jorge; Dopson, Mark; Rawlings, Douglas E.; Banfield, Jillian F.; Holmes, David S.; Quatrini, Raquel
    Background: Acidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the Acidithiobacillales. As such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. In this study, we explore the genomic diversity of A. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome. Principal Findings: Comparative sequence analysis of A. caldus ATCC 51756 and SM-1 indicate that, despite sharing a conserved and highly syntenic genomic core, both strains have unique gene complements encompassing nearly 20% of their respective genomes. The differential gene complement of each strain is distributed between the chromosomal compartment, one megaplasmid and a variable number of smaller plasmids, and is directly associated to a diverse pool of mobile genetic elements (MGE). These include integrative conjugative and mobilizable elements, genomic islands and insertion sequences. Some of the accessory functions associated to these MGEs have been linked previously to the flexible gene pool in microorganisms inhabiting completely different econiches. Yet, others had not been unambiguously mapped to the flexible gene pool prior to this report and clearly reflect strain-specific adaption to local environmental conditions. Significance: For many years, and because of DNA instability at low pH and recurrent failure to genetically transform acidophilic bacteria, gene transfer in acidic environments was considered negligible. Findings presented herein imply that a more or less conserved pool of actively excising MGEs occurs in the A. caldus population and point to a greater frequency of gene exchange in this econiche than previously recognized. Also, the data suggest that these elements endow the species with capacities to withstand the diverse abiotic and biotic stresses of natural environments, in particular those associated with its extreme econiche.
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    Assessing the capacity for micropollutants to induce changes in the biofilm EPS composition and yield
    (Stellenbosch : Stellenbosch University, 2021-12) Rossouw, Johann Herman; Wolfaardt, Gideon M.; Stone, Wendy; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: A consequence of widespread chemical manufacturing and usage is the increasing presence of a new class of contaminant: micropollutants. Despite the investment of significant resources into the development of novel approaches to wastewater treatment, the removal efficiency of micropollutants has been varied and conflicting between different studies. A notable gap in current research efforts is assessing the capacity for chronic micropollutant exposure to alter the main mechanisms contributing to their removal in secondary wastewater treatment. The aim of this study was three-fold. First, to investigate the comparative homogeneity of the Slime-EPS matrix composition for multi- versus single-species biofilms. Secondly, to attempt to quantify the adsorptive capacity of the Slime-EPS fragment utilizing a published dye-probing analysis protocol. Finally, to assess the capacity for chronic (7-day) micropollutant exposure to influence the composition and yield of the Slime-EPS fragment for a known, biofilm producing Pseudomonas species. Single-species biofilms exhibited a more consistent Slime-EPS composition in terms of their protein: carbohydrate ratio. Dye-probing analysis efforts indicated the capacity for toluidine blue dye to exhibit altered spectral absorbance as a result of increased dimerization – which was found to be influenced by both the introduction of the Slime-EPS itself and increasing concentrations of NaCl. Increasing concentrations of TB dye was shown to induce hypsochromic (blue-) spectral shifts. Ciprofloxacin was found to significantly reduce the biofilm Slime-EPS yield (p < 0.05) following 7 days of continuous exposure, whereas exposure to diclofenac for the same interval had no significant effect on Slime-EPS yield. Neither ciprofloxacin nor diclofenac had a significant effect on the Slime-EPS protein: carbohydrate ratio following 7 days of exposure.
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    Assessing the occurrence and mechanisms of horizontal gene transfer during wine making
    (2009-12) Barnard, Desire; Bauer, Florian; Wolfaardt, Gideon M.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Saccharomyces cerevisiae is the most commonly used organism in many fermentation-based industries including baking and the production of single cell proteins, biofuel and alcoholic beverages. In the wine industry, a consumer driven demand for new and improved products has focussed yeast research on developing strains with new qualities. Tremendous progress in the understanding of yeast genetics has promoted the development of yeast biotechnology and subsequently of genetically modified (GM) wine yeast strains. The potential benefits of such GM wine yeast are numerous, benefitting both wine makers and consumers. However, the safety considerations require intense evaluation before launching such strains into commercial production. Such assessments consider the possibility of the transfer of newly engineered DNA from the originally modified host to an unrelated organism. This process of horizontal gene transfer (HGT) creates a potential hazard in the use of such organisms. Although HGT has been extensively studied within the prokaryotic domain, there is an urgent need for similar studies on their eukaryotic counterparts. This study was therefore undertaken to help improve our understanding of this issue by investigating HGT in a model eukaryotic organism through a step-by-step approach. In a first step, this study attempted to determine whether large DNA fragments are released from fermenting wine yeast strains and, in a second step, to assess the stability of released DNA within such a fermenting background. The third step investigated in this study was to establish whether “free floating” DNA within this fermenting environment could be accepted and functionally expressed by the fermenting yeast cultures. Finally, whole plasmid transfer was also investigated as a unified event. Biofilms were also incorporated into this study as they constitute a possibly conducive environment for the observation of such HGT events. The results obtained during this study help to answer most of the above questions. Firstly, during an investigation into the possible release of large DNA fragments (>500 bp) from a GM commercial wine yeast strain (Parental strain: Vin13), no DNA could be detected within the fermenting background, suggesting that such DNA fragments were not released in large numbers. Secondly, the study revealed remarkable stability of free “floating DNA” under these fermentation conditions, identifying intact DNA of up to ~1kb in fermenting media for up to 62 days after it had been added. Thirdly, the data demonstrate the uptake and functional expression of spiked DNA by fermenting Vin13 cultures in grape must. Here, another interesting discovery was made, since it appears that the fermenting natural grape must favours DNA uptake when compared to synthetic must, suggesting the presence of carrier molecules. Additionally, we found that spiked plasmid DNA was not maintained as a circular unit, but that only the antibiotic resistance marker was maintained through genomic integration. Identification of the sites of integration showed the sites varied from one HGT event to the next, indicating that integration occurred through a process known as illegitimate recombination. Finally, we provide evidence for the direct transfer of whole plasmids between Vin13 strains. The overall outcome of this study is that HGT does indeed occur under the conditions investigated. To our knowledge, this is the first report of direct horizontal DNA transfer between organisms of the same species in eukaryotes. Furthermore, while the occurences of such events appears low in number, it cannot be assumed that HGT will not occur more frequently within an industrial scenario, making industrial scale studies similar to this one paramount before drawing further conclusions.
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    Assessment of wood degradation by Pycnoporus sanguineus when co-cultured with selected fungi
    (Stellenbosch : Stellenbosch University, 2007-03) Van Heerden, Andrea; Botha, Alfred; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: It is commonly known that a diversity of fungi, including yeasts, may occur on plant surfaces. Similarly, on fallen trees an ecological succession of different fungal species is known to occur during wood degradation. Some of these fungi may be pioneer fungi contributing to the initial degradation process, while others may be yeasts associated with the fruiting bodies of macro-fungi which in turn are able to utilize the more recalcitrant polymers in wood. Previously, it was revealed that an increase occurs in the wood degradation rate of certain white-rot fungi when co-cultured with selected yeast species. A well known inhabitant of decomposing trees is the white rot fungus Pycnoporus sanguineus. It was found by some that this fungus is capable of selective delignification while growing on the wood of poplar trees, while other authors found a simultaneous delignification pattern on Eucalyptus grandis trees. In the latter case cellulose and lignin are degraded simultaneously. We were interested in how yeasts occurring on the surface of P. sanguineus fruiting bodies, and the pioneer fungus Aspergillus flavipes, impact on wood degradation by this white-rot fungus. Restriction Fragment Length Polymorphisms (RFLP) analyses were used to obtain an indication of the species composition of the culturable yeast community associated with fruiting bodies of P. sanguineus. The impact of the most dominant of these yeasts species, i.e. Pichia guilliermondii and Rhodotorula glutinis, as well as A. flavipes, on wood degradation by P. sanguineus was then determined by analyzing the major wood components after growth of co-cultures on hot water washed E. grandis wood chips. Co-cultures of P. sanguineus with the other fungi were prepared by inoculating the wood chips, contained in solid state bioreactors and supplemented with molasses and urea, with the an appropriate volume of fungal inoculum, resulting in an initial moisture content of 60%. After two weeks of incubation at 30°C with constant aeration, the chips were harvested. Standard protocol (TAPPI Standard Methods), commonly used by the paper and pulp industry, were then employed to determine the percentage cellulose, Klason Lignin, as well as polar and solvent-borne extractives in the chips. The resulting data were analyzed using box plots, as well as biplots. No degradation of Klason lignin was observed, while the percentage cellulose did decrease during fungal degradation. Taking into account the inherent shortcomings of the Klason Lignin determination, the results supported the findings of others that P. sanguineus shows a simultaneous delignification pattern while growing on E. grandis wood. In addition, it was found that the yeasts played no significant role in the degradation ability of P. sanguineus, while A. flavipes showed an antagonistic effect on P. sanguineus with respect to cellulose degradation. However, it was clear that the analytical methods used in this study were inadequate to accurately determine fungal degradation of wood. In addition, it was obvious that the methods used did not distinguish between fungal biomass and wood components. Nevertheless, the methods provided us with a fingerprint of each culture growing on E. grandis wood, allowing us to compare the chemical composition of the different cultures and the un-inoculated hot water washed wood chips. The question, therefore, arose whether the effect of a particular coculture, on the chemical composition of wood, differs between tree species. Consequently, chemical alterations in different tree species, induced by a P. sanguineus / A. flavipes co-culture, were investigated in the next part of the study. Wood chips originating from four tree species, i.e. Acacia mearnsii, Eucalyptus dunnii, E. grandis, and Eucalyptus macarthurii, were inoculated with this co-culture. The culture conditions and subsequent analyses of the wood components were the same as in the first part of the study. From the box- and biplots constructed from the resulting data, it was clear that the chemical composition of each tree species were altered in a different manner by the coculture. Lignin content showed an apparent increase in A. mearnsii, while E. dunnii showed a decrease in cellulose content. The results indicate that wood of different tree species are degraded in a different manner and this phenomenon should be taken into account in selecting fungi for biopulping.
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    Bacteria of the genus xenorhabdus, a novel source of bioactive compounds
    (Frontiers Media, 2018-12-19) Dreyer, Jonike; Malan, Antoinette P.; Dicks, Leon Milner Theodore, 1961-
    The genus Xenorhabdus of the family Enterobacteriaceae, are mutualistically associated with entomopathogenic nematodes of the genus Steinernema. Although most of the associations are species-specific, a specific Xenorhabdus sp. may infect more than one Steinernema sp. During the Xenorhabdus–Steinernema life cycle, insect larvae are infected and killed, while both mutualists produce bioactive compounds. These compounds act synergistically to ensure reproduction and proliferation of the nematodes and bacteria. A single strain of Xenorhabdus may produce a variety of antibacterial and antifungal compounds, some of which are also active against insects, nematodes, protozoa, and cancer cells. Antimicrobial compounds produced by Xenorhabdus spp. have not been researched to the same extent as other soil bacteria and they may hold the answer to novel antibacterial and antifungal compounds. This review summarizes the bioactive secondary metabolites produced by Xenorhabdus spp. and their application in disease control. Gene regulation and increasing the production of a few of these antimicrobial compounds are discussed. Aspects limiting future development of these novel bioactive compounds are also pointed out.
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    The bacteria, biology and biotechnology of biomining
    (Stellenbosch : Stellenbosch University, 1999) Rawlings, Douglas Eric
    Inaugural address delivered by Prof Douglas Eric Rawlings during April 1999, Stellenbosch University.