OpenUCT :: Browsing by Author "Reid, Sharon J"

Browsing by Author "Reid, Sharon J"

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Open Access
Characterization of a transposon-induced pleiotropic metronidazole resistant mutant of Clostridium acetobutylicum P262
(1996) Collett, Helen Jeanne; Reid, Sharon J
Metronidazole is a pro-drug which must be reduced to elicit a bactericidal effect. In the clostridia, some of the electron transport proteins that provide the source of electrons for the reductive activation of metronidazole play a key role in electron distribution, which in turn regulates the direction of carbon flow in the cell. The aim of this research project was to isolate electron transport gene(s) from the solvent-producing Clostridium acetobutylicum strain P262, using transposon-induced metronidazole resistance as a selection system. In the process, the feasibility of transposon mutagenesis in this strain, which lacks conventional systems for DNA delivery, was assessed, and the nature of metronidazole susceptibility in the C. acetobutylicum wild type was investigated. The metronidazole resistant transconjugant of interest, referred to as mutant 3R, was shown to harbour a single insertion of the Tn925: :Tn917 transposon cointegrate within a structural gene, designated sum (susceptibility to metronidazole).
Open Access
Characterization of an alkalophilic Bacillus brevis isolate with respect to its endo-(1,3-1,4)-β-glucanase gene, protein hyperproduction and the degS-degU operon
(1994) Louw, Maureen Elizabeth; Reid, Sharon J
Bacillus brevis Alk 36 was isolated from soil during a screening programme for the selection of extracellular enzyme producing strains. A gene coding for an endo(1,3- 1,4 )-.8-glucanase (or lichenase) was cloned from B. brevis Alk 36 and expressed in Escherichia coli. The nucleotide sequence of this gene was determined and found to encode a protein of 252 amino acid residues. The amino acid sequence of the B. brevis lichenase gene showed only a 50% similarity to previously published data for Bacillus endo-(1,3-1,4)-β-glucanases. The enzyme exhibited some unique properties. The optimum temperature and pH for enzyme activity were 65-70°C and 8-10, respectively. When held at 75°C for 1 h, 75% residual activity was measured. The molecular mass was estimated to be 29 kDa and the enzyme was found to be resistant to sodium dodecyl sulphate (SDS). B. brevis Alk 36 was evaluated as a potential host strain for the efficient production and secretion of foreign proteins and was found to grow optimally between pH 8.0 and pH 9.5 and between 42°C and 52°C. B. brevis was successfully transformed using vector DNA and was found to produce relatively low levels of protease. In addition, it was evaluated as a possible protein hyper-secreting strain. However, using PCR technology, the highly conserved cell wall protein genes could not be positively identified in B. brevis Alk 36.
Open Access
A clinical and molecular analysis of Clostridium difficile strains isolated from Groote Schuur Hospital
(2015) Brock, Tunehafo Elisabeth; Abratt, Valerie Rose; Reid, Sharon J
A clinical and molecular analysis of Clostridium difficile isolated from symptomatic patients at Groote Schuur Hospital was conducted in order to gain insight into the identity, epidemiology and pathogenesis of the various strains. C. difficile was detected and isolated by selective culture from stool specimens from 34 of the 162 symptomatic patients (20%). Three toxigenic-types were distinguished by PCR: A+B+ (47%), A-B+ (47%) and A-B- (6%), none of which harboured the binary toxin genes. Compared to the direct culture method, enzyme immunoassay-based detection tests (Meridian ImmunoCard and bioMérieux MiniVidas) were found to be lacking clinical sensitivity, while nucleic acid amplification tests (Hain Lifescience CDiff and Cepheid GeneXpert) were far more sensitive in the local clinical setting. PCR ribotyping identified all the A-B+ strains as PCR ribotype 017, which was the prevalent strain type (47%). Genotyping based on the tcdC gene and MLVA both grouped the ribotype 017 strains in a single clade. The antimicrobial susceptibility of all the isolates to metronidazole (MET), vancomycin (VAN), moxifloxacin (MOX) and erythromycin (ERY) were determined by the Etest method. All were sensitive to MET and VAN; however, four ribotype 017 strains displayed reduced susceptibility to MET. With regard to MOX, reduced susceptibly and full resistance were observed in 32% and 12% of the isolates, respectively, with all of these belonging to ribotype 017. MOX-resistant strains had a Thr82->Ile amino acid substitution in the GyrA enzyme and strains displaying reduced susceptibility to MOX had an Asp426->Asn amino acid substitution in GyrB. High-level resistance to ERY was observed in 47% of the isolates, which were primarily ribotype 017. ERY-resistant strains all harboured the ermB gene, suggesting that this was the genetic basis of the observed phenotype. Auto-aggregation analysis revealed that the ribotype 017 strains were significantly stronger auto-aggregators than the other ribotypes examined. Results of semi-quantitative RT-PCR analysis suggest that the expression level of the cwpV gene, encoding the CwpV protein, may play a role in auto-aggregation. In conclusion, this pilot study revealed that the GeneXpert method was the most accurate and sensitive technique for diagnosing CDI in the clinical setting at Groote Schuur Hospital. Ribotype 017 was the most prevalent strain type, and the antimicrobial resistance profile and increased auto-aggregation capacity of this ribotype may contribute to its high prevalence.
Open Access
A cluster of two serine transfer RNA genes from Clostridium acetobutylicum P262
(1994) Sealy, Victoria Rosamond; Woods, David R; Reid, Sharon J
A cloning system, using metronidazole as a screening tool and E. coli FI9 as a selection host, was previously established to clone C. acetobutylicum P262 electron transport genes which may play a role in solvent metabolism in this bacterium. In theory, metronidazole would be reduced under anaerobic conditions to a cytotoxic intermediate by C. acetobutylicum P262 electron transport genes or reductive enzymes cloned into a recombinant plasmid. This intermediate would kill the host E. coli FI9. One C. acetobutylicum P262 clone, pMETIOB, was found to render the E. coli strain FI9 sensitive to metronidazole, under anaerobic conditions. A number of subclones of the 2.56kb C. acetobutylicum P262 insert DNA were generated in Bluescript pKS and pSK. A range of exonuclease III deletions of this C. acetobutylicum P262 insert DNA were also generated which were shown to lose the metronidazole sensitivity phenotype on progressive deletion of the insert DNA. In vitro and in vivo protein transcription/translation experiments failed to reveal a protein product that was related to the metronidazole sensitivity phenotype. DNA hybridization confrrmed that the insert DNA of pMETIOB hybridized to C. acetobutylicum P262 chromosomal DNA, but not to E. coli chromosomal DNA. The nucleotide sequence of a 933-bp fragment of the C. acetobutylicum P262 insert DNA was determined.
Open Access
The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36
(2007) Crampton, Michael Craig; Reid, Sharon J; Louw, Maureen E; Berger, E
This study relates to the development of an alkaliphilic, thermo-tolerant, Gram-positive isolate, Bacillus halodurans Alk36, for the over-production and surface display of chimeric gene products. This bacterium harbors the endogenous genetic background to over-produce flagellin protein continuously. In order to harness this ability, key genetic tools, such as gene targeted inactivation, were developed for this strain. The hag gene which codes for flagellin was inactivated on the chromosome giving rise to the B. halodurans BhFC0l mutant. This strain was non-motile as determined on motility plates and confirmed by PCR analysis. Motility was, however, restored through complementation of the expression vector carrying a functional hag gene. Polylinkers were inserted as in-frame, chimeric, flagellin sandwich fusions in order to identify the permissive insertion sites corresponding to the variable regions of the flagellin protein. Flagellin expression and motility were evaluated for these constructs. Two sites were identified for possible peptide insertion in the flagellin gene, one of which produced functional flagella and was able to restore the motility phenotype to a non-motile mutant. Peptides encoding a poly-histidine peptide and the HIV-l clade C gpl20 epitope were respectively incorporated into both of the permissive sites as in-frame fusions and found to be successfully displayed on the cell surface. The poly-His peptide was shown to be functional through metal binding and affinity purification studies. The display of the HIV-1 subtype C gp 120 V3 loop was also shown to be functional through immunological studies using peptide specific antibodies. Surface display of the poly-His and HIV-l epitope was shown to have improved metal binding and enhanced expression levels of the chimeric flagellin when the peptides were insel1ed at amino acid position 180 (pSECNC6). This specific site is the only insertion point that falls within the re-defined variable domain of the FliC protein from B. halodurans Alk36.
Open Access
The development of probiotics for use in the ostrich farming industry in South Africa
(2011) Du Toit, Elloise; Reid, Sharon J
Ostrich farming in South Africa is an important industry but it often suffers from high mortality rates among the ostrich chicks. This is thought to be due in part to environmental stress, leading to the delay in the development of the microbiota, thus making invasion by pathogens likely. Very little is known about the microorganisms inhabiting the ostrich gastrointestinal tract, however they do play an important role in humans and animals and disturbance of this community can be fatal. It has been established that probiotic bacteria such as Lactobacillus and Bifidobacterium spp. play an important role in the health of the host. In order to discover novel probiotic strains, bacteria should be isolated from the host, identified, characterised in-vitro to screen their potential suitability, and finally these results should be confirmed in-vivo. The aim of this study was to follow these guidelines in order to find a successful probiotic mix to decrease the mortality observed on ostrich farms, and to compare the effects of the probiotic mix to that of the antibiotic, tylosin, on the microbiota of the gastrointestinal tract (GIT).
Open Access
Elucidation of the reaction mechanisms involved in the catalysis mediated by glutamine synthetase in Escherichia coli
(2005) Oldfield, Lyndon Carey; Reid, Sharon J; Kenyon, Colin
Structural and molecular dynamics analysis of the glutamine synthetase from E. coli indicates that a possible mechanism by which the adenylylation/deadenylylation of the enzyme affects the enzyme specificity for either MgATP or Mn2ATP and NH4+ or NH3, is by switching between two putative serine protease-like catalytic triads. Site-directed mutagenesis of a number of residues identiï¬ ed as playing a role in these catalytic triads, led to the following observations. Both Ser52 and Ser53 were important for the catalytic activity of the enzyme. It was determined that the Ser52 residue appeared to have adenylylated functionality and the Ser53 residue, deadenylylated functionality. Evaluating these serine mutant enzymes in the presence of the serine protease inhibitors, AEBSF and PMSF, led to the conclusion that Ser52 and Ser53, did, indeed, appear to form part of a catalytic triad, as the activity of the enzymes were inhibited in the presence of the inhibitors. When both serine residues were removed in a single mutant, activity was not signiï¬ cantly inhibited by either inhibitor. His210 and His211 were found to be equally important to the functionality of the enzyme, and the results, in consultation with the enzyme model, led to the conclusion that the His210 residue had deadenylylated activity and the His211 residue had adenylylated activity. All the potential acid residues, when removed, had an effect on activity, but this was to be expected as all had previously been identified in the literature as important in the active site of the glutamine synthetase from E. coli. Again, consultation of the model led to the conclusion that the two acid residues that filled the function of the acid residue in each catalytic triad, were Glu129 for the adenylylated from of the enzyme, and Glu357 for the deadenylylated form of the enzyme. These catalytic triads are believed to be comprised of Ser52', His211 and Glu129 for the adenylylated form of the enzyme, and Ser53', His210 and Glu357 for the deadenylylated form of the enzyme. Possible model mechanisms for the both the adenylylated and deadenylylated forms of the enzyme are proposed.
Open Access
Evaluating oxalate-degrading Lactobacillus spp. for their ability to be used as probiotics in the treatment of kidney stone disease
(2010) Kabanda, Siti M; Abratt, Valerie Rose; Reid, Sharon J
Although the direct cause of kidney stone formation is not known, reports have suggested it is probably a multifactorial disease. Lactobacillus strains which potentially had increased ability to degrade oxalate were previously isolated from a healthy low kidney stone risk group. The aim of this study was to identify these natural Lactobacillus strains and evaluate their potential for use as probiotics in reducing the risk of kidney stone disease. Identification was achieved by PCR amplification and sequencing of the 16S rRNA gene and the 16S-23S rRNA internal transcribed spacer (ITS) region. The strains were identified as follows; Lactobacillus gasseri 7(3), L. gasseri 17(4), Lactobacillus reuteri 17(7) and L. reuteri 16(9). Their probiotic characteristics were also evaluated.
Open Access
Evaluation of five bifidobacterium isolates as potential probiotics and genetic analysis of their ability to withstand oxidative stress
(2010) Nonyane, Molati Albert; Reid, Sharon J; Abratt, Valerie Rose
The diverse microbiota of the human gastrointestinal tract plays a major role in the general health of humans. A number of bacterial strains with distinctive properties have been isolated and used commercially as probiotics in order to harness these health benefits and offer them to unhealthy hosts. There are set criteria that have to be followed before new probiotics can be introduced into the market. Five Bifidobacterium isolates with extremely high sucrase activity were randomly selected from a faecal sample from a healthy donor for further characterisation as potential probiotics with the ability to utilize fructo- oligosaccharide substrates in the gut. Phylogenic identification of the isolates to the species level was carried out using sequences of the 16S rRNA gene, 16S-23S rRNA gene spacer region, the heat shock protein (hsp60) and Elongation factor Tu (tuf).
Open Access
Evaluation of probiotics as feed supplements for ostrich chicks
(2010) Greenhill, Nikita; Reid, Sharon J
Production in farming of ostriches (Struthio camelus) is limited by the high mortality rate of ostrich chicks. Chicks which lack a well established microbiota are more susceptible to potentially fatal pathogenic infections. Therefore, the mortality rate may be decreased by establishing the correct gut microbiota by the use of ostrich specific probiotic strains. Five selected strains were conclusively identified and their mucin adhesion abilities characterised: Strain P1.2 was identified as Enterococcus faecalis; the identity of strain 5934.3.1 was confirmed to be Lactobacillus oris; Strains Lactobacillus brevis 512.3.1 and Lactobacillus oris 5934.3.1. The five selected strains were included in an in vivo probiotic feeding trial, where ostriches were treated with an encapsulated mixture of the five strains and/or the antibiotic tylosin.
Open Access
Genetic studies on Clostridium acetobutylicum
(1982) Allcock, Errol Ralph; Woods, David; Reid, Sharon J
The aim of this study involved the characterisation of the cellulolytic properties of Clostridium acetobutylicum and the development of a genetic transfer system for this organism. The production of a carboxymethyl cellulose and a cellobiase by C acetobutylicum was demonstrated. In liquid medium the carboxymethyl cellulase was induced by molasses, and it was not repressed by glucose. Optimum carboxymethyl cellulase activity occurred at pH 4.6 and 37°C. Optimum conditions for autolysis and autoplast formation in C. acetobutylicum were defined. Autolysis-deficient mutants which produced less autolysin than the parent strain were isolated. Growth of the P262 strain and the lyt-1 mutant was inhibited by the same concentrations of wall-inhibiting antibiotics.
Open Access
Glycogen metabolism in Corynebacterium glutamicum ATCC 13032
(2004) Kensley, Joy A; Reid, Sharon J
Corynebacterium glutamicum is a Gram-positive facultative aerobe particularly known for its industrial application in the synthesis of amino acids, such as L-glutamate and Llysine. The central metabolic pathways of this organism has been an area of much research by many groups. Linked to glycolysis is the synthesis of glycogen, previously considered a storage molecule of excess glucose. No information concerning the role of glycogen or its metabolism in C. glutamicum was known, and the aim of this work was to elucidate glycogen metabolism in this industrially important organism.
Open Access
Investigation of carbon catabolite repression in Clostridium beijerinckii NCIMB 8052
(2001) Rafudeen, M S; Reid, Sharon J
The substrate basis for the industrial acetone-butanol-ethanol (ABE) fermentations, has been agricultural products rich in starch or sucrose, and employed taxonomically distinct amylolytic and saccharolytic solventogenic clostridial strains respectively. There is evidence to suggest that the utilization of these substrates is subject to carbon catabolite repression. In Gram-positive bacteria, carbon catabolite repression is controlled by a global regulatory mechanism, central to which is an imperfect palindromic sequence, the cre element, which is recognized by a protein of the GalR-LacI family, the CcpA protein. A ccpA homologue, regA, has been previously identified in C. acetobutylicum NCP262 and successfully complemented a B. subtilis ccpA mutant strain. The sucrose operon from C. beijerinckii NCIMB 8052, scrARBK, has been characterised at the physiological and genetic levels with the ScrR repressor found to negatively auto-regulate the operon.
Open Access
Isolation and characterization of n-alkane utilizing bacteria, which produce biomulsifiers
(2003) Ghilamicael, Amanuel Menghs; Reid, Sharon J; Stutz, Helen
Bacterial strains were isolated by enrichment cultures from oil-contaminated soil samples. In the present study, several strains, capable of growing on crude oil, were isolated. Isolates were screened for their inherent abilities to produce bioemulsifiers when they were grown on hydrocarbon substrates.
Open Access
Isolation of bacterial strains capable of efficient conversion of n-alkanes into value added products
(2004) Mathopa, Byatlela Abel; Reid, Sharon J
Bacteria that are capable of degrading alkane fractions, C12-C13 and C14-C17, were isolated from oil-contaminated soil collected from the petrochemical company, CALTEX Refineries, South Africa. A total of twenty-three environmental strains were isolated. A preliminary procedure, Nile Blue A straining suggested that twelve of the twenty-three environmental strains might accumulate polyhydroxyalkanoates (PHAs) in their cytoplasm, which is a good candidate for biodegradable plastics. The gene that catalyzes PHA polymerization, phaC, was detected using PCR in some of the environmental strains. The strains of interest were identified and characterized biochemically using various techniques and later sequenced by 16S rONA PCR. The environmental isolate 2 showed a 99 % identity to Pseudomonas aeruginosa BHP7 -6 and was for that reason given a name, Pseudomonas aeruginosa MB2SA. P. aeruginosa MB2SA was shown to possess a 0.5 kb internal fragment corresponding to the phaC gene and capable of degrading the alkane fractions, Cn-CI3 and CI4-CI7, effectively. P. aeruginosa MB2SA was shown to grow optimally in the long alkane fraction, Cw C17, and was further grown in pure alkanes, n-dodecane, n-tetradecane, and hexadecane for comparison. In addition, the strain, P. aeruginosa MB2SA, was grown in an appropriate medium for PHA synthesis and high yields of PHA were observed when both the long alkane fraction, Cw CI7, and pure alkane, hexadecane, were employed as sole carbon sources respectively.
Open Access
L-Arginine Overproduction in Corynebacterium glutamicum ATCC 13032
(2009) de V Theron, Grant; Reid, Sharon J
Open Access
L-arginine overproduction in Corynebacterium glutamicum ATCC 13032
(2009) Theron, Grant de V; Reid, Sharon J
Corynebacterium glutamicum is widely used for the commercial production of a variety of amino acids, including L-lysine, L-glutamate and L-threonine. With the exception of Larginine, the biosynthesis and regulation of most of these compounds in this bacterium are relatively well characterised in the literature. The research presented here focuses on improving our understanding of the regulation of L-arginine biosynthesis in C. glutamicum. This was performed with the ultimate goal of creating strains capable of producing L-arginine commercially. A novel gene replacement system was initially used for the directed mutation of the Larginine biosynthetic gene cluster in C. glutamicum ATCC 13032. This was met with limited success, however, and the pK19mobsacB vector was thus adopted for further mutagenesis of this region.
Open Access
Microbial solvent formation revisited by comparative genome analysis
(BioMed Central, 2017-03-09) Poehlein, Anja; Solano, José D M; Flitsch, Stefanie K; Krabben, Preben; Winzer, Klaus; Reid, Sharon J; Jones, David T; Green, Edward; Minton, Nigel P; Daniel, Rolf; Dürre, Peter
Background: Microbial formation of acetone, isopropanol, and butanol is largely restricted to bacteria belonging to the genus Clostridium. This ability has been industrially exploited over the last 100 years. The solvents are important feedstocks for the chemical and biofuel industry. However, biological synthesis suffers from high substrate costs and competition from chemical synthesis supported by the low price of crude oil. To render the biotechnological production economically viable again, improvements in microbial and fermentation performance are necessary. However, no comprehensive comparisons of respective species and strains used and their specific abilities exist today. Results: The genomes of a total 30 saccharolytic Clostridium strains, representative of the species Clostridium acetobutylicum, C. aurantibutyricum, C. beijerinckii, C. diolis, C. felsineum, C. pasteurianum, C. puniceum, C. roseum, C. saccharobutylicum, and C. saccharoperbutylacetonicum, have been determined; 10 of them completely, and compared to 14 published genomes of other solvent-forming clostridia. Two major groups could be differentiated and several misclassified species were detected. Conclusions: Our findings represent a comprehensive study of phylogeny and taxonomy of clostridial solvent producers that highlights differences in energy conservation mechanisms and substrate utilization between strains, and allow for the first time a direct comparison of sequentially selected industrial strains at the genetic level. Detailed data mining is now possible, supporting the identification of new engineering targets for improved solvent production.
Open Access
Molecular characterisation of selected gastrointestinal microbiota in South African HIV-positive patients during HAART
(2012) Du Plessis, Sarah Jane; Abratt, Valerie Rose; Reid, Sharon J
Progression of the HIV disease is characterised by a massive depletion of CD4+ T cells and it has been shown that patients living with a more advanced HIV infection have a higher risk of developing diarrhoea due to the disruption of the gastrointestinal microbiota caused by either the HIV-infection or the use of antibiotics and drugs such as highly active antiretroviral therapy (HAART). An imbalance in the microbial composition, attributable to a disturbed mucosal barrier, as well as increased permeability and inflammation caused by HIV, can influence the metabolic (carbohydrate fermentation) and protective functions provided by the microbiota. The effect of HIV on the intestinal microbiota has not been widely examined and those studies that have focused on HIV and the gastrointestinal tract, have investigated it mainly from a virological perspective. Consequently, the aim of the study was to ascertain whether the diversity and/or abundance of the endogenous intestinal microbiota of South African HIV-positive patients was disrupted on account of HIV within the gastrointestinal tract. An additional aim was to determine whether the administration of HAART affected the microbiota during a 6 month longitudinal study. The diversity of the intestinal microbial composition was characterised with respect to the total bacteria, Bifidobacterium and Lactobacillus species using PCR-DGGE. qPCR was used to determine the abundance of total bacteria, Bifidobacterium, Lactobacillus, Escherichia coli, the Bacteroides/Prevotella, Clostridium coccoides and Clostridium leptum groups. ... In addition, three potential intestinal pathogens (Clostridium difficile, Campylobacter jejuni and Salmonella enterica) were monitored by qPCR during this period, to determine their prevalence in the HIV-positive patients.
Open Access
Molecular characterisation of the fibre-digesting bacteria isolated from the ostrich (Struthio Camelus var. Domesticus) hindgut
(2000) Russouw, Tracy Karen Joe; Thomson, Jennifer Ann; Reid, Sharon J
Eleven bacterial strains that have been isolated from the ostrich hindgut, have successfully been identified with the aid of PCR amplification of the 16S rDNA genes and sequence analysis using the BLASTN search of the GenBank database. The nucleotide sequences were determined either through direct sequencing of the PCR products or after cloning the products into the plasmid vector pBluescript (SK). The results obtained from the 16S rDNA sequence homologies strongly suggests that the newly identified bacteria should be assigned to three major bacterial genera, namely, Ruminococcus, Buryrivibrio and Bacteroides. Four highly cellulolytic strains were identiﬁed as ruminococci while two weakly cellulolytic strains, showed highest homology to members of the genus Butyrivibrio. Another weakly cellulolytic strain showed closest identity to the sulfur reducing bacterial genus Desulfovibrio. Four non-cellulolytic strains showed highest homology to the genus Bacteroides.