A collection of 29 clinical streptococcal isolates obtained from the University of KwaZulu-Natal, Medical School, Durban Metro area (South Africa) were studied to establish their penicillin G susceptibility patterns often refered to as minimal inhibitory concentration (MIC) and to determine the genetic diversity among them using two genotyping methods, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) analysis. All isolates with MIC less than or equal to 0.12 µg/ml were considered susceptible, intermediate resistant if MIC was between 0.25 µg/ml and 4 µg/ml and resistant if greater than 4 µg/ml, The percentage of isolates with resistance was relatively high (75.9%), only 10.3% of isolates showed intermediate resistance and 13.8% of the isolates were completely susceptible to penicillin G. Some of the resistant isolates were highly resistant reaching penicillin G MIC levels of 5000 µ/ml. They were speculated to contain Path altered penicillin binding proteins and high level of crosslinking cell wall induced by the gene products of the MurMN operon. RAPD analysis was performed using three primers, MBPZ-1, MBPZ-2, and MBPZ-3, respectively. RAPD analysis allowed for the identification of 27 RAPD types with MBPZ-1 and MBPZ-3 and 26 RAPD types with MBPZ-2. Ninety-eight percent of these isolates were clustered into two groups, group I and group II, with 90% to 100% dissimilarity among them. Fifty two percent of the isolates of MBPZ-1 group I were in MBPZ-2 group I, 72% isolates of MBPZ-1 group I were in MBPZ-3 group I, and 72% of the isolates of MBPZ-2 group I were in MBPZ-3 group 1. This shows the discriminatory ability of the primers used in this study. Despite clustering of isolates, relatively high diversity was seen. PFGE analysis of macrorestriction fragments obtained after digestion of chromosomal DNA by restriction enzyme, SmaI showed 24 PFGE patterns. The 24 PFGE patterns were divided into three groups (I, II and III) of isolates, with an average of 85% dissimilarity (15% homology) among them. At 25% homology, four clusters, A (13 isolates), B (9 isolates), C (4 isolates), and D (4 isolates) were observed. Two pairs of isolates in group I, cluster A, showed 100% homology. This suggested that each represent the same strain. Four isolates of group I, cluster B, also exhibited 100% homology. This study showed that most of streptococcal isolates with the same penicillin G susceptibility patterns grouped together in a phylogenetic tree by both RAPD and PFGE analysis. There was also some similarity between the results obtained by RAPD analysis and PFGE analysis. Seventeen and nine of the 29 isolates grouped into group I and group II, respectively, two pairs of isolates were indistinguishable, and two pairs of islates were closely related by both RAPD (using MBPZ-3) and PFGE analysis. Although, RAPD analysis is sensitive, specific, faster and cost effective, the ease with which PFGE analysis can be performed, high discriminatory power, reproducibility of the results, and the polymorphism seen in the patterns, suggests that PFGE method has the potential to be very useful for epidemiological evaluations of nosocomial streptococcal infections in KwaZulu-Natal. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

The genus Eucalyptus has been planted extensively throughout the world in tropical and subtropical regions, primarily because of its economic importance and use in wood and pulp production. Due to the growing demands for timber, forestry companies need to increase the productivity of available forest land. The genetic improvement of forest trees through selection and breeding involves a lengthy process of scientifically controlled trials focused on short-term and long-term goals using breeding and production populations. This investigation focused on the use of Best Linear Prediction (BLP) and its application to: (1) the prediction of genetic gains for a breeding population and, (2) the selection of superior individuals for clonal production of E. grandis. A CSIR dataset for a 20-year-old progeny trial involving 90 open-pollinated families was obtained. Four traits, namely, diameter at breast height (DBH), stem form, splitting and density were identified for use in this investigation. Relevant data were extracted and a file termed, Dataset created. Dataset was edited, standardized and corrected for the fixed effect of replication using SAS® procedures. Precise and accurate population parameter estimates are fundamental in determining breeding strategies and thus, heritabilities of each trait and phenotypic correlations between traits in Dataset were estimated using SAS® procedures. DBH was found to have the highest heritability (0.600), followed by density (0.492). The estimated heritability for stem form was 0.401 and splitting had the lowest heritability at 0.214. A high positive phenotypic correlation of 0.83 was estimated between DBH and stem form. The phenotypic correlations between other traits were close to zero. An index provides a weighted score for individuals, which takes all relevant information into account and allows individuals or families to be chosen for breeding and production purposes. Consequently, Best Linear Prediction (BLP) of individual breeding values were calculated using MATGEN® (2003). Thereafter, BLP values were used to determine the rankings of individual trees for 15 different selection indices. In order to determine the effect of selection on the change in the population mean of a trait, the breeding population's response to selection was predicted and compared across three selection strategies, namely: (1) individual selection, (2) single-trait index selection, and (3) multiple-trait index selection. The top 8% of individuals in the breeding population were selected for and the genetic gains were predicted. It was found that the response to selection was greatest when using individual selection. Furthermore, DBH had the best selection response for all three strategies as compared to the other traits under investigation. Fifteen indices, considering different numbers and choice of traits, were compared for commonality among rankings of the top 30 individuals. Two methods, namely, (1) a rank-correlation matrix and (2) a manual assessment, were used. The commonality between indices showed that a simple index, considering two traits (DBH and density) was equally effective (93%) in identifying genetically-superior individuals as the more complex index that considered four traits. Furthermore, it was possible to select for only three traits (DBH, splitting, density) and identify the same top 30 individuals as using the index that considered four traits. The researcher's goal was to find the most desirable individuals in the population to be used for production purposes, such as clonal forestry. Consequently, various selection options, specifying certain trait requirements, were used to select superior individuals for use in production and deployment. The "commercial selection" option was the only option successful in obtaining an individual that met the required criteria for the four traits in the population of 475 individuals. The results suggested that breeders should consider large populations and only a few important traits in order to obtain a greater number of individuals suitable for mass propagation in clonal forestry. In order to further investigate the effect of population size on the number of individuals suitable for clonal forestry, a hypothetical population was generated. This was accomplished using between family and within family standard deviation values obtained from Dataset. The large hypothetical population of 1000 individuals produced twelve individuals suitable for production purposes, as opposed to only one in the real population of 475 individuals. This result further indicates that a larger population provides a greater number of individuals appropriate for use in production and deployment. This investigation successfully addressed the aims by: (1) calculating individual breeding values (BLP) and ranking individuals, (2) predicting the breeding population's response to selection, according to three strategies, for the four traits under investigation, and (3) identifying superior individuals for use in commercial clonal forestry. As the work of tree breeders is aimed at improving the growth and quality of trees by increasing the frequency of desirable genotypes in the population, further research could focus on (1) the effect of different sets of economic weightings on index rankings in a population and (2) the influence that population structure has on the optimal genetic gains obtained. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Inhibition of Fusarium oxysporum f. sp. cubense race 4 by Burkholderia cepacia was evident when grown on various media (TSA, PDA, PSA, YM, KMB, PPM, NYGA, LA) with different carbon sources and under various pH and temperature conditions. In addition, B. cepacia was able to inhibit several fungal pathogens in vitro. Antagonism of B. cepacia against F. oxysporum f. sp. cubense occured at high levels of Fe³+, which may suggest that antagonism by B. cepacia did not involve siderophore production. Thin layer chromatogram (TLC) examination showed that B. cepacia produced several substances, one of which had similar R[f] value to that described for pyrrolnitrin. Cell-free supernatant of a 4-day culture of 6. cepacia was applied to an Amberlite XAD-2 column and inhibitory activity co-eluted with the 95% methanol (pH 9.5) fraction. The concentrated activated fractions showed inhibitory activity against F. oxysporum f. sp. cubense. A GC-MS chromatogram indicated numerous components in the antifungal extracts. The only compound identified in the Wiley 138 library, was 1,2- Benzenedicarboxylic acid, bis (2-Ethylhexyl) ester. Observations by light microscopy indicated that B. cepacia inhibited spore germination in F. oxysporum f. sp. cubense race 4 and retarded the mycelial growth. The interaction between the endophytic bacterium, B. cepacia and F. oxysporum f. sp. cubense race 4 was investigated with aid of scanning and transmission electron microscopy. This demonstrated that the bacterium was able to colonize the surface of hypha and macrospore of F. oxysporum f. sp. cubense. Mycelial deformation, terminal and/or intercalary swelling were evident. At later stages, hyphae of F. oxysporum f.sp. cubense, colonized by B. cepacia, were found to have collapsed. Further studies in vivo confirmed that B. cepacia colonized the hypha of F. oxysporum f. sp. cubense which had invaded banana roots. TEM observation showed that in the banana plant B. cepacia was closely associated with the healthy banana roots and a matrix was frequently found to be present between the bacterium and the plant surface. In addition, B. cepacia exists mainly in the intercellular space of the banana roots. UV irradiation treatment of B. cepacia resulted in a mutant that had lost inhibitory activity against F. oxysporum f. sp. cubense on TSA agar. Transposon mutagenesis of B. cepacia was performed by Tn5 insertion. Six mutants which had lost or had reduced inhibitory activity against F. oxysporum f. sp. cubense were generated. These mutants showed no inhibitory zones on TSA medium in the presence of the fungus. It was observed that one mutants. cepacia :: Tn5-188 appeared to lose the ability to colonize the fungal hypha, whilst a different mutant B. cepacia ::Tn5 - 217 was still able to colonize the fungal hyphae. TLC analyses showed that there was a decrease in antibiotic production in mutants B. cepacia :: Tn5 - 217 and B. cepacia - UV - 34, compared with the wild type. GC- MS analyses showed that there was no evidence of the peaks at 14.62 minutes, 20.0 minutes and 20.46 minutes in both chromatograms of mutants B. cepacia :: Tn5 -217 and 8. cepacia -UV - 34, compared with the wild type B. cepacia. No PCR products were detected using primers that were developed from sequences within the biosynthetic loci for Phi of P.fluorescens Q2-87(GenBank accession no. U41818) and PCA of P. fluorescens 2-79 (GeneBank no. L48616). Colony hybridization suggested that genomic DNA from B. cepacia could contain both Phi- and PCA probes. It was found that hybridization of genomic DNA digested with Cla-I of B. cepaca with Phl2a probe only occurred at low stringency. A hybridization signal was detected from a Cla-l fragment of approximately 2800bp. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.

A large sector of the forestry industry of South Africa comprises Eucalyptus species, covering approximately 49% of the forestry plantation area. Polyploidy induction has become an attractive tool to increase yield and reduce invasiveness in forestry species. Polyploidy induction in Eucalyptus using colchicine treatments on seed and axillary buds was undertaken to produce tetraploids that could be used in breeding programmes; specifically to increase yield and decrease species invasiveness through the production of triploids after crossing with diploid parents. Eight seedlots of E. urophylla and seven of E. grandis were treated with four colchicine concentrations (0.00, 0.01, 0.03, 0.05%) at two exposure times (18 h and 24 h), treating two seeds per treatment, repeated eight times. For axillary bud induction, 20 buds of two E. grandis clones and three E. grandis × E. urophylla hybrids and one E. grandis × E. nitens hybrid were treated with four colchicine concentrations (0.0, 0.5, 1.0, 1.5%) for three consecutive days. A known tetraploid hybrid E. grandis E. camaldulensis and its corresponding diploid were included as reference material. Seedlings and bud sports were pre-screened by determining stomatal guard cell lengths. Seedlings and bud sports displaying cell lengths significantly (p<0.0001) larger than the diploid were selected as putative polyploids. Polyploidy was then confirmed by quantifying the DNA content using flow cytometry. Stomatal frequencies and guard cell chloroplast frequencies were also determined in the induced tetraploid seedlings to evaluate their suitability to discern between ploids. All putative polyploidy seedlings, identified in the pre-screening process, were confirmed, using flow cytometry, as either tetraploids or mixoploids. Of the 17 E. urophylla putative polyploids, from various seedlots, six were tetraploid and 11 mixoploid. In E. grandis one of the five putative polyploids, from various seedlots, was tetraploid and four mixoploid. Pre-screening of bud sports was less accurate; only four of the 12 E. grandis hybrid putative polyploids were mixoploid and only three of the six E. grandis putative polyploids were mixoploid. E. urophylla seedlings were more sensitive to colchicine than E. grandis seedlings displaying a lower survival rate (52%) than E. grandis (63%). Extreme treatments that caused the lowest survival rates were also responsible for most of the polyploidy successful inductions; 0.05%/18 h and 0.05%/24 h for E. urophylla and 0.03%/24 h and 0.05%/24 h for E. grandis. Phenotypic effects of colchicine included shorter, thicker roots and hypocotyls; darker leaves; longer and narrower leaves in some tetraploids; and asymmetrical leaf margins in many mixoploids and tetraploids compared with the controls. In the tetraploids, stomata were significantly larger (p<0.0001) and less frequent (p<0.001). A significant (p<0.001) increase in the number stomatal chloroplasts was also ascertained. Confirmed mixoploid seedlings all displayed tetraploid leaves based on stomatal size and thus classified as periclinal chimeras. In bud sports, only leaves with islands of diploid and tetraploid stomata in the confirmed mixoploids were encountered. Mixoploid bud sports were thus either sectional or mericlinal chimeras. Stomatal size proved to be a suitable pre-screening method, especially in polyploidy induction in seedlings. Additionally confirmed tetraploids exhibited significantly different stomatal frequencies and stomatal chloroplast frequencies compared with the diploids, thus proving to be suitable detection methods for polyploidy screenings. Polyploidy induction in seed was effective, however, less effective in axillary buds which requires further research to refine methods. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Three Streptococcal strains: S. milleri P213, S. milleri P35 and S. milleri B200 and three enterococcal strains: E. faecalis 123, E. faecalis 126 and E. faecium were used to test for vancomycin resistance. Two strains were used as reference strains that were already characterized as vancomycin resistant. E. faecium BM4147 was used as a VanA control and E. faecalis ATCC was used as a VanB control. Susceptibility of each strain to this antibiotic was tested by disk-diffusion assay and the MIC values for the strains were found to be between 5 - 10 ug/ml and for the VanA control, the MIC was > 64 ug/ml and for the VanB control was 32 ug/ml. These MIC values indicate that S. milleri P213, S. milleri P35, S. milleri B200, E. faecalis 123, E. faecalis 126, and E. faecium are all of the VanC phenotype. All strains were tested for lysis by means of addition of vancomycin (10 ug/ml) to the bacterial cultures. Lytic curves were constructed and the VanB control was found to be most autolytic upon addition of vancomycin and E. faecalis 123 was the least autolytic. However, under normal conditions in phosphate buffer, lytic curves showed that S. milleri P213 was the most autolytic and the VanA control, the least autolytic. PCR assays were performed to detect specific antibiotic resistant genes. Primers were selected from Dukta-Malen et al., 1995. The VanA primer yielded amplification of 732 bp for only the VanA control DNA and the VanB primer set yielded products for the VanB control DNA. S. milleri P213, P35, B200 and E. faecalis 123 and 126, and E. faecium DNA were amplified with the VanC primers. This supports the results obtained in MIC that these strains are possibly VanC resistant strains. Amplified VanA control and that of E. faecalis 126 were thereafter sequenced. VanA control amplicon was correctly amplified since it showed homology to E. faecium BM4147 as well as the VanB amplicons which was found to be homologous to the transposon Tn1549 found on the well-characterized E. faecalis strain which is known to harbour the VanB vancomycin-resistant genes. Whilst E. faecalis 126 which represented the VanC phenotype showed 96% homology to E. gallinarum BM4147 which is a well-characterized glycopeptide-resistant enterococci belonging to the VanC phenotype. Southern blots were performed using specific primers as a probe to verify whether the gene sequences for the specific genotype were present in these strains and results confirmed those found in the PCR assays and in DNA sequencing. The peptidoglycan precursors of each strain were arrested in vancomycin (20 ug/ml) to block transpeptidation and transglycosylation steps of peptidoglycan synthesis and bacitracin (100 ug/ml) was used to amplify precursors at the transglycosylation step. Precursors were extracted and analysed by reverse-phase HPLC. UDP-MurNAc-tetrapeptides cell wall precursors, which are found abundantly in vancomycin-resistant strains, were found in large proportions in all strains, except in E. faecalis 123 when arrested with vancomycin. This precursor has a noticeably decreased affinity for vancomycin, hence contributing to its resistance. The precursor accumulated when arrested with bacitracin, was, UDPMurNAc-tetrapeptide in all strains except in E. faecalis 126. UDP-MurNAc-pentapeptides were also found in moderate amounts in most strains. The molecular masses of the peptidoglycan precursors obtained from mass spectrometry correctly identified them. This confirmed that the bacterial strains investigated were in fact resistant to the antibiotic vancomycin and this study shows that results obtained from conventional phenotypical screening methods reliably correlated with the genotypes classified using more advanced techniques such as PCR, southern blot/hybridisation and DNA sequencing. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Leucocin A is a bacteriocin produced by Leucoconostoc gelidium UAL 187-22. Bacteriocin producer strains possess an immunity protein, which enables the strain to protect itself against its own bacteriocin. The immunity gene from Leucoconostoc gelidium was isolated via PCR from a recombinant clone pJF5.5. This fragment was cloned by amplifying the immunity gene from pJF5.5 and ligating it into pMALc2. The resulting recombinant plasmid pKP1 was then transformed into Escherichia coli strain JM103. The clone putative, was confirmed by DNA sequencing and southern blot hybridization using the primers EAL-2 and EAL-3. It was shown to contain an insert of 3.6 kb. Expression analysis showed the construct as an in frame malE fusion protein expressed within E. coli. The fusion construct was isolated by affinity chromatography. Leucocin A was purified to 90% purity, from the supernatant of Leucocnostoc gelidium UAL 187-22 by ion-exchange chromatography and HPLC. It was found to elute from a C18 reverse phase column at 55% actetonitrile, 0.1% TFA. Binding interaction and the stability of the immunity gene fusion protein were compared using a Biacore 2000. The supernatant and cytoplasmic extract isolated from Leucocnostoc gelidium UAL 187-22 were tested for interaction with the fusion construct. Surface Plasmon resonance studies indicated that there was no binding partner present in the supernatant which would influence the immunity process. However, a stable interaction was found between the immunity protein and an orphan ligand within the cytoplasm. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

The characteristic trait of the Borna disease virus is that it is a complicated single negative stranded RNA virus that is capable of infecting a wide array of mammalian species including human beings. It has been implicated in a diverse variety of human neuropsychiatric diseases. The infection capability, mechanism of infection and range of protein action of this virus remain to be identified. The purpose of the present study was to determine (1) whether the previous Bornaviridae family classification is indeed accurate as the action of BDV indicates that it is related to other viruses and (2) to estimate the number of synonymous (nucleotide substitution) and non-synonymous (amino acid change) evolutionary mutation rates of proteins (nucleoprotein, phosphoprotein, glycoprotein, matrix protein) exhibited by various Borna disease virus host species and the proteins (nucleoprotein, phosphoprotein, glycoprotein, matrix and X protein) of three Borna disease virus strains. The latter study would give an indication as to which proteins are subjected to positive selection. Phylogenetic methods were used to determine the accuracy of the Bornaviridae classification. Phylogenetic trees obtained through an alignment and analysis of the polymerase protein, which displays a uniquely conserved GDN motif, of various RNA negative single stranded viruses using neighbourhood and parsimony methods enabled comparison with other RNA virus families. A method adapted from Ina, (1995) for estimating the synonymous and non-synonymous evolutionary mutation rate was applied to various BDV proteins in order to provide more information on inter (host virus) and intra (virus) mutation rate. This information in turn was used to create an evolutionary model to clarify the positive and neutral evolutionary trend of the inter- and intra-virus proteins examined, which may help clarify and enhance the lack of current knowledge relating to species infection and the epidemiological nature of the virus. The results obtained by the polymerase alignment analysis indicates the presence of two newly discovered BDV motifs, v and vi, confirmed by three diverse alignment programmes. An analysis of the alignment of BDV proteins indicated that the BDV nucleoprotein nuclear localization signal aligns the BDV nucleoprotein between motifs IV and vi of the BDV polymerase. The results obtained by the phylogenetic analysis indicate that the Rabies virus and the Vesicular stomatitis virus are the most closely related animal viruses to BDV, whereas the Rice transitory yellowing (unclassified Rhabdovirus) and Sonchus yellow net plant virus are closet to BDV than other animal Rhabdoviridae raising intriguing questions on the evolutionary origins of the Borna disease virus. The phylogenetic analysis indicates that the Borna disease virus does not fall into a separate Bornaviridae family classification, and suggests that BDV may be more appropriately placed into a separate subfamily in the family Rhabdoviridae. The results of the evolutionary analysis indicate considerable diversity between BDV host virus (inter-species) and BDV virus (intra-species) protein sequences. In the host virus sequence comparison analysis all of the proteins examined displayed a high pattern of non random evolution, which is in contrast to the intra species comparison in which only three proteins; the BDV glycoprotein, nucleoprotein and X protein; displayed a non random pattern of evolution. The positive selection effect displayed by the inter-species (host) proteins may be attributed to antigenic variation displayed by the inter-species sequences and a super infection hypothesis, which indicates that positive selection on host variants could arise during the course of an infection as a result of specific immune responses. The positive and neutral selection trend of the proteins displayed by the intra-species (virus) sequences may be a result of a pattern of nucleotide substitution that is physio-chemically conservative. Conservation may be evident in volume, polarity, hydrophilicity, or molecular weight of amino acids of the proteins. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2002.

Escherichia (E) coli infections result in diarrhoea and oedema in growing pigs. Enterotoxigenic (ETEC), shigatoxin producing (STEC) and enteroaggregative (EAEC) E. coli have been identified as the principal causes of colibacillosis in most pig production systems. These E. coli use fimbrial and non-fimbrial adhesins to adhere to the intestines and cause infection. Absence or presence of the receptors on the intestinal walls determines the resistance or susceptibility of the host to the E. coli. In other populations, candidate genes linked to the receptors have been found to be associated with resistance/susceptibility to infection and are used in marker-assisted selection programs. This study investigated the presence and prevalence of ETEC, STEC and EAEC and the associated virulence genes in 263 E. coli isolates sampled from Landrace, Large White, Duroc and Indigenous piglets from the Animal Production Institute of the Agricultural Research Council (ARC) in Irene and Middledrift farm in Eastern Cape Province. The study also investigated polymorphisms at six candidate genes associated with two E. coli receptors in the same pig populations. Over 39 % of the isolates tested positive for the E. coli virulent genes investigated. None of the samples had fimbrial adhesins. The mode of attachment of the investigated E. coli was through non-fimbrial adhesins which were found in 49.06% of the isolates. The 106 E. coli isolates were categorized into 25 pathotypes carrying definable and unique combinations of E. coli virulence factors. The resistant allele for Alfa (1) fucosyltransferase 1 (FUT1) M307, a candidate gene for FI8R, was present in less than 1 % of the population. Various mutations of mucin genes MUC4 g.8227, MUC20 c1600 and g.191 were found in the population. Their respective alleles for controlling F4ab/ac E. coli adhesion in pigs were predominant in both breeds. Three loci (FUT1, MUC20 g.191 and MUC20 c.1600) deviated from Hardy Weinberg equilibrium (HWE) in the Indigenous and the Large White breeds. Heterozygotes deficiency and high levels of within breed diversity was observed in these two breeds at the mentioned loci. Overall, the study observed a wide range of toxin and colonisation factors (CFs) giving rise to diverse pathotypes in South African pigs. The absence of fimbrial adhesins suggests a different colibacillosis control program from that previously used. The presence of the resistant alleles in most of the loci investigated was low, however their presence suggest it is possible to use them to generate a resistant population using marker assisted selection. This study serves as a foundation for future pig colibacillosis control and immunity studies in the South African pig herds. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Bacteriocin leader pepides are currently receiving much attention due to their possible functions. It is predicted that these leaders prevent cytoplasmic toxicity within the producer organism by rendering the bacteriocin inactive. Leucocin A, a class IIa bacteriocin produced by Leuconostoc gelidum UAL187-22 is synthesized with a 24 amino acid leader pepide which is cleaved during extracellular translocation. The antimicrobial activity of the leucocin A precursor, pre-leucocin A, was determined to gain insight into whether, the presence of a leader peptide has an impact on anti-listerial activity. The leucocin A and pre-leucocin A genes were generated by PCR of L. gelidum UAL187-22 plasmid DNA. Recombinant plasmids, pLcaA and pPreLcaA were isolated by cloning the amplified genes into the Escherichia coli pMAL.c2 vector, and by screening transformant colonies using blue white selection methods. The malE-LcaA and malE-preLcaA fusion genes were expressed, and resulting maltose binding fusion proteins, were purified using amylose affinity chromatography. Fractions collected, contained partially pure forms of MBP-LcaA (46.433 kDa) and MBP-preLcaA (49.088 kDa) fusion proteins. Following Factor Xa digestion, the MBP affinity tag was removed; and recombinant peptides, leucocin A and pre-leucocin A were further purified by reverese phase high performance liquid chromatography. It was determined that leucocin A was eluted with a retention time of 24.893, while pre-leucocin A was eluted with a retention time of 31.447. Fractions of pure leucocin A and pre-leucocin A were thereafter assayed for activity using a deferred antagonism assay, with Listeria monocytogenes being the indicator strain. Pre-leucocin A tested positive for antimicrobial activity. However, when compared to leucocin A it was found that the leucocin A precursor inhibits Listeria to a lesser degree than leucocin A. The relative bactericidal activities of leucocin A and pre-leucocin A was calculated at 6.0 x 10⁵ AU and 4.0 x 10⁵ AU. Taking this into consideration, it was estimated that the leucocin A precursor is ~66.667 % active as mature leucocin A. Hence the presence of a leader peptide does not have an influence on leucocin A antimicrobial activity. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

The disease listeriosis is caused by Listeria monocytogenes. This common food-borne disease has been responsible for about 0.1 to 10 cases per million inhabitants per year. However, this disease is serious with its high fatality rates of 20% - 30%, and 40% of all cases reported have been in pregnant women suffered from a foetal abortion. Recently the organism has acquired resistance to antibiotic treatment and the development of an alternative treatment is necessary. Class IIa bacteriocins such as leucocin A have been shown to be active against L. monocytogenes. However, the leucocin A receptor molecule responsible for growth inhibition within L. monocytogenes remains unclear. Various studies have implicated the mannose PTS permease (EIIt Man) of L. monocytogenes as the putative receptor for class IIa bacteriocins. The results from studies reviewed indicate that the EIIt Man of L. monocytogenes could be the chiral receptor needed for bacteriocin interaction at the surface of targeted cells. Specifically, the membrane associated IIDMan and IICMan subunits were implicated in direct interaction with class IIa bacteriocins. Our study focused on cloning, expression and purification of the subunits of the mannose PTS permease of L. monocytogenes EGD. Primers were designed to amplify the subunit genes of the mptACD operon. The mptC, mptD and mptAB genes which were then successfully cloned into pET28a expression vector and transformed into E. coli JM109(DE3) host strain. Recombinant plasmids were screened using colony PCR. Subsequently recombinant pET28-C, pET28-D and pET28-AB was once again transformed and expressed in the E. coli BL21(DE3) pLysS expression host strain. After an induction at 30°C for 5 hours, IICMan and IIDMan were found to be expressed in the cell membrane, whilst IIABMan was expressed in the cytosol of the host expression strain. Membrane proteins His-IICMan, His- IIDMan, and cytosol associated His-IIABMan were purified using Ni2+-NTA affinity chromatography. Results for His-IICMan yielded a 28 kDa protein and a 55 kDa co-purified protein. Results for His-IIDMan yielded a 31 kDa protein and a 60 kDa co-purified protein. Results for His-IIABMan yielded a 35 kDa protein and a 68 kDa co-purified protein. A western blot analysis revealed that all proteins purified carried an attached His-tag as detected by an anti-mouse peroxidase conjugate anti-His-tag antibody. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.