Browsing by Author "Steyn, Lafras M"

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    Characterisation of the cold-shock response in Mycobacterium smegmatis
    (1999) Shires, Karen Lesley; Steyn, Lafras M; Zappe, Harold
    The response of Mycobacterium smegmatis to a cold shock was investigated in order to gain insight into the stress responses of members of the genus Mycobacterium. Mycobacterium smegmatis cultures were shocked from 37°C to 30°C, 25°C, 15°C, and 10°C and the effects on both growth (ATP concentration, culture turbidity, colony-forming units) and metabolism (incorporation of ¹⁴C-leucine and ³H-uracil) were investigated. The magnitude of the cold-shock response was found to be dependent upon the degree of the cold shock. A cold shock to 10°C had the greatest effect and resulted in a "lag period" of 24 hours in both the growth and metabolism of the culture. The synthesis of proteins was reduced 20-fold during this period, indicating at block in translation. The cold-shock response in Mycobacterium smegmatis was an adaptive response with growth eventually being resumed at the colder temperature, but at a reduced rate. Using the techniques of one-dimensional sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and two-dimensional protein gel electrophoresis, ³⁵S-methiononine-labelled proteins that were synthesised during the cold shock were analysed. At least fourteen radio-labelled proteins were induced during the first 24-hour period and these demonstrated two distinct patterns of cold-shock induced expression: transient and continuous. Depending upon the pattern of expression and size, the cold-shock proteins were classified as "cold-induced proteins", "cold-shock proteins" or "cold-acclimation proteins". CipM, a 27kDa protein, was identified as the major cold-shock protein through one-dimensional protein electrophoresis. From N-terminal sequence data generated from a protein (CipM.1) within this band, a corresponding degenerate DNA probe was used to isolate cipM.1. This gene was cold-inducible, with mRNA levels transiently increasing 5-7 fold after a 37°C to 10°c cold-shock. Homologues of this cold-shock gene are found in the genomes of Mycobacterium tuberculosis and Mycobacterium leprae. The corresponding mycobacterial proteins showed homology at the N-terminus to the HU~ subunit of HU of Escherichia coli and possessed similar C-terminal praline, lysine and alanine degenerate repeats to the mycobacterial heparin-binding hemagglutinin. The response of several mycobacterial cold-shock gene homologues to a cold shock was also investigated, by northern-hybridisation and S1 nuclease analysis. The cspA homologue of Mycobacterium smegmatis demonstrated a 16-24 fold transient induction in mRNA levels following a 37°C to 10°C temperature-shift, while gyrA mRNA levels were maintained at a constant level throughout the cold shock. Although some similarities were demonstrated between the cold-shock response of Escherichia coli and Mycobacterium smegmatis, definite differences occur in the proteins that are involved in the adaptive stages of the response.
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    Conditions affecting ergothioneine levels in Mycobacterium smegmatis & the attempted isolation of α-N, N, N-Histidine methyltransferase, the first enzyme in ergothioneine biosynthesis
    (2007) Williams, Monique J; Steyn, Lafras M; Steenkamp, D J
    Ergothioneine and mycothiol are the two major low molecular weight thiols present in mycobacteria. The generation of mycothiol-deficient mutants has demonstrated its role in protecting M tuberculosis against oxidative and nitrosative stress. To date, no ergothioneine-deficient mutants have been identified and the role of ergothioneine in mycobacteria remains unknown. The work in this thesis was performed with the aim of better understanding the function of ergothioneine in mycobacteria, by studying its biosynthesis and the conditions affecting its production.
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    Demonstration of new subtypes of adenovirus 7 in South Africa, and probing oesophageal carcinoma cell lines for evidence of adenovirus or of other oncogenic viruses
    (1987) Brooks, Louise Alexandra; Steyn, Lafras M; Renan, M J; Dumbell, K R
    This study was carried out in 2 parts: 1. Genome analysis of human adenovirus species 7; 2. Search for a possible viral aetiology in oesophageal carcinoma. Sixteen laboratory isolates of adenovirus species 7, isolated in South Africa between 1975 and 1986, were characterized by restriction endonuclease analysis of their DNA genomes. Virus was propagated in human embryo fibroblast cells; genomic DNA, extracted and purified from cellular DNA extracts, was analyzed using 9 different restriction enzymes. Results of this study have demonstrated 2 new genome types of adenovirus 7c which have not previously been identified. The 2 novel strains, designated as genome types Ad7c1 and Ad7c2, were shown to differ from prototype Ad7 c according to restriction profiles with EcoRI; 2 new EcoRI sites were demonstrated in Ad7c1 and 1 in Ad7c2. The restriction sites were mapped on the viral genomes (at 3.68kb and 5.32kb from the left terminus) by double enzyme digestions, cloning of viral DNA, and nucleic acid hybridization using a cloned Ad7 probe. Strains resembling the prototype Ad7c and Ad7p (Gomen) genome types were also identified in the 1985 and 1986 Ad7 isolates. In order to investigate the possible role of a viral co-factor in the aetiology of oesophageal carcinoma, 18 probes, derived from potentially oncogenic viruses, were used to screen 3 human oesophageal carcinoma cell lines for the possible presence of integrated viral DNA. One of these, an Ad7 recombinant plasmid probe, was developed by cloning DNA from the transforming region of the Ad7cl strain into the plasmid vector pUC19. Cellular DNA, extracted from the 3 tumor lines HCU18, HCU33 and HCU39, was tested by means of both DNA dot hybridization and Southern blot hybridization for the presence of Epstein-Barr virus, human papillomavirus (types 1, 5, 6, 8, 11, 16, 18), human adenovirus (strains 5, 7, 12, 31) and human T-lymphotropic virus type I DNA. Both assays were demonstrated to be sensitive enough to detect 1 copy of viral DNA per cell. No hybridization between HPV, EBV, HTLV-I or adenovirus DNA probes, and the cellular DNA was detected. These findings indicate that the stable integration of these tumor viruses in host chromosomes did not play a role in the maintenance of the malignant phenotype of the 3 extensively passaged cell lines. Cells of the 3 oesophageal tumor lines were further examined by transmission electron microscopy, but the presence of virus particles in these cells was not observed.
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    Development of novel anti-tuberculosis drugs from African medicinal plants
    (2006) Ngwane, Andile H; Steyn, Lafras M; Skepu, Zoleka
    Includes bibliographical references (leaves 55-63).
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    Identification and isolation of growth-phase specific proteins of mycobacteria
    (1999) Bettoni, Jane Clementina; Steyn, Lafras M; Zappe, Harold
    The aim of this project was to identify growth phase-specific heat shock proteins of Mycobacterium smegmatis LR222. A growth curve was constructed using the ATP assay. This method was shown by B. A. Ntolosi to be the most accurate indicator of when the organism entered the various phases of growth. It was possible to determine that M. smegmatis LR222 entered the exponential phase of growth after a short lag phase of 4 to 8 hours and persisted in this phase for 20 to 22 hours. It then reached the stationary phase, which lasted for 40 to 46 hours. Protein heat shock assays were performed on growth phase-specific samples. This allowed the identification of a 43-46 kDa in molecular weight stationary phase protein on one-dimensional SOS-PAGE. The protein was induced in cells entering the stationary phase of growth and not by heat shock as it was induced under both the control and the heat shock temperatures. The protein was further characterised by two-dimensional gel electrophoresis, which demonstrated resolution into two, strongly age-associated, proteins. Subtractive RNA hybridisation was attempted in order to obtain a subtraction cDNA probe from a stationary phase RNA sample depleted of sequences common in both the exponential and the stationary phase samples. Ribosomal RNA was removed from the total RNA by the process of photobiotinilation. The mRNA was then used as a template to synthesise with reverse transcriptase single stranded cDNA. cDNA/mRNA denatured hybrids were hybridised to the exponential phase RNA sample. The new hybrids were "subtracted" by chemical cross-linking with DZQ and the unique cDNA used to produce by random primers a radioactively labelled probe. A M. smegmatis library was probed but unfortunately no signal was observed. Further adjustments and improvements to this technique are required before it can be used effectively.
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    A molecular biological study on Campylobacter pylori
    (1989) Penfold, Sonya; Lastovica, A J; Steyn, Lafras M
    C. pylori have been shown to be associated with gastritis and peptic ulceration, but the mechanism of their pathogenicity is unknown. Since a number of virulence factors are known to be plasmid mediated, it was decided to study the plasmids of C. pylori. A variety of techniques were used to establish the best method of plasmid extraction from C. pylori. The method of alkaline lysis as described by Birnboim and Doly was shown to give the most consistent results and the greatest plasmid yield. Plasmid DNA was found in 54% (26 out of 48) of the isolates examined and the plasmids varied in size from 3,4kb to greater than 137kb. The majority (21 out of 26) of isolates had unique plasmid profiles, but 5 isolates showed common ones. Three of these 5 isolates were studied in more detail. The evidence presented here suggests that all 3 plasmid bands visible in these three isolates were different conformations of the same plasmid which has a molecular weight of 6, 2 kilo bases. The plasmids appeared labile and covalently closed circular DNA was rarely isolated. Restriction enzyme digestion was done with a variety of enzymes, but only 3 of the enzymes used digested the DNA. EcoRI and HindIII partially digested the DNA, while Sau3A digested the plasmids completely, generating 2 fragments of 2,2kb and 2,4kb, and a number of smaller fragments. The DNA was shown to be methylated and the fragments generated by Sau3A digestion suggest that the plasmids may contain a repetitive element. Chromosomal DNA was also isolated and digested with a variety of enzymes. The chromosomal DNA restriction pattern was shown to be affected by methylation, which may be important when using restriction enzyme patterns to differentiate between strains. Plasmid restriction fragments were end-labelled to detect bands which were poorly visible by ethidium bromide staining. This technique was shown to be more sensitive than ethidium bromide staining of DNA, but the inability to obtain complete digestion of C. pylori DNA made it impossible to construct a restriction enzyme map of the plasmids. Hybridization experiments showed the plasmids of C. pylori to be related and was also used to detect bands which were not easily visible after ethidium bromide staining. Attempts were made to clone C. pylori DNA into pUC18 and pUC19, but no recombinant plasmids containing C. pylori DNA were obtained.
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    The Mycobacterium smegmatis "Proteome" : effects of growth phase on total protein synthesis and on the response to heat shock
    (1998) Ntolosi, Bongi Audrey; Steyn, Lafras M; Zappe, Harold
    As an initial step towards characterisation of the molecular processes that define the phenotype of the mycobacterial stationary phase, the effect of growth phase of Mycobacterium smegmatis on total protein synthesis and on the heat shock response was investigated. De novo protein synthesis was monitored by labelling with 35 [S]methionine and the protein expression profiles analysed using one- and/two-dimensional polyacrylamide gel electrophoresis, autoradiography, and/or immunoblot analysis. The ATP content of the culture was found to be a more accurate indicator that cells were entering stationary phase than the number of colony forming units (CFU). A plateau in the ATP growth curve preceded several stationary phase-induced events : a transitory cessation in the increase in number of CFU ; a decrease in the rate of accumulation of the cell division protein FtsZ; inhibition of the synthesis of 58, 30.5, and 20 kDa exponential phase proteins; induction of the 48, 46, 32, 31, 25, and 20 kDa stationary phase (postexponential phase) proteins ; and the highest induction of the 95 kDa, 75 kDa (DnaK), 66 kDa ( GroEL ), and - 17 kDa (doublet) proteins in response to heat shock. Identification of the stationary phase-induced proteins should enable their roles in the multigenic processes that occur during transition into stationary phase to be determined. The amino acid sequence of one of the - 17 kDa heat shock proteins (with an apparent molecular weight of 16.8 kDa, named Hspl7-2) showed significant homology to open reading frame 28 of M tuberculosis cosmid MTCY01B2. This is the first time a functional characteristic has been assigned to this open reading frame, and it remains to be seen if Hspl 7-2 represents a new family of heat shock proteins. Synthesis and secretion of the antigen (Ag)-85 complex proteins was demonstrated for the first time in M smegmatis. Heat shock resulted in increased release of Ag85A and Ag85B but not of Ag85C in M smegmatis. No heat-induction of the Ag85 complex could be demonstrated in My cobacterium bovis BCG. Whereas heat shock resulted in increased release of the 19 kDa lipoprotein antigen in both M bovis BCG and M tuberculosis H37Rv, its presence in M smegmatis could not be demonstrated. This study presents an experimental approach which may prove useful in investigating the effect of various environmental stresses on the profile, and hence the function of secreted proteins.
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    The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression
    (1998) Mulder, Michelle Anne; Steyn, Lafras M; Zappe, Harold
    A clone containing the terminal third of the Mycobacterium tuberculosis katG gene was previously shown to confer resistance to ethyl methane sulfonate on DNA repair-deficient Escherichia coli cells. The first aim of this study, therefore, was to examine the role played by the M tuberculosis katG gene in DNA repair. The strategy used was overexpression of different regions of the gene in DNA repair-deficient mutants of E. coli, and examination of the sensitivities of the transformants to DNA damaging agents. Overexpression of the gene resulted in an increase in the survival of recA mutants exposed to ultraviolet (UV) light irradiation (254 run) and hydrogen peroxide, and uvr mutants exposed to mitomycin C. Both the 5' and 3' regions of the M tuberculosis KatG protein conferred the above effects, and this was independent of the catalase or peroxidase activity of the enzyme. The results suggest that the M tuberculosis katG gene may encode a novel function related to the repair of DNA damage, and this may have implications for the survival of M tuberculosis in the presence of DNA damaging agents, for example, in the macrophage. UV sensitivity tests on M intracellulare and M tuberculosis strains mutant in katG revealed that the katG gene product does not play a demonstrable role in the survival of repair-competent mycobacterial cells after exposure to UV irradiation. The second aim of this study was to examine the regulation of expression of the M tuberculosis katG gene. An E. coli-mycobacterial shuttle vector, pJCluc, containing the luciferase reporter gene, was constructed and used to examine the katG promoter sequences. The region required for optimal expression in M. smegmatis was localized to a 559 hp fragment immediately upstream of the gene. Two transcription start sites were mapped and putative -10 and -35 promoter sequences identified. It was demonstrated that expression from the promoter peaks during late exponential phase, and declines during stationary phase, and that the promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. An upstream element that increased expression from the M. tuberculosis katG and the M. paratuberculosis PAN promoters was identified, and shown to bind to one or more M smegma/is proteins. Similar results were obtained in M bovis BCG. Understanding the regulation of gene expression in mycobacteria is essential for determining the processes that govern interaction with the host. This study provides information on both the mycobacterial transcription signals and gene regulatory mechanisms.
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    Species-specific DNA probes for the identification of Actinobacillus actinomycetemcomitans
    (1990) Emanuel, Margot; Steyn, Lafras M
    A DNA probe was developed for the identification of the periodontal pathogen, Actinobacillus actinomycetemcomitans. Chromosomal DNA was extracted from A. actinomycetemcomitans, digested with a restriction enzyme, Sau3A, ligated to plasmid DNA (pUC18) and transformed into JM109 cells to give a partial A. actinomycetemcomitans library. The library was screened using Southern blot analysis. Out of the nine inserts tested, one was found to be species specific as it did not cross-hybridise to Haemophilus aphrophilus, a closely related organism which occurs in the normal oral microflora, nor did it cross-hybridise with 7 species of Bacteroides tested. A level of detection of 104 cells or 50ng of A. actinomycetemcomitans was obtained. The probe has a length of 779bp and out of 30 restriction enzymes tested, only SspI was found to have a restriction site in the insert. The probe was tested on clinical specimens obtained from five different periodontitis patient groups and was shown to correlate with culture results in eighteen out of twenty-two cases in detecting A. actinomycetemcomitans.